In vitro and in vivo functions of SARS-CoV-2 infection-enhancing and neutralizing antibodies.

SARS-CoV-2-neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) or the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Three of 46 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can rarely occur in SARS-CoV-2 antibody-infused macaques.

Windows of Susceptibility and Consequences of Early Life Exposures to 17ß-estradiol on Medaka (Oryzias latipes) Reproductive Success.

Estrogens and estrogen mimics are commonly found in surface waters and are associated with deleterious effects in fish populations. Impaired fertility and fecundity in fish following chronic exposures to estrogens and estrogen mimics during critical windows in development are well documented. However, information regarding differential reproductive effects of exposure within defined developmental stages remains sparse. In this study, reproductive capacity was assessed in Japanese medaka (Oryzias latipes) after exposure to two concentrations of 17ß-estradiol (E2ß; 2 ng/L and 50 ng/L) during four distinct stages of development: gonad development, gonad differentiation, development of secondary sex characteristics (SSC) and gametogenesis. Exposure to E2ß did not adversely impact survival, hatch success, growth, or genotypic ratios. In contrast, exposure to 50 ng/L E2ß during SSC development altered phenotypic ratios and SSC. Exposure to both E2ß treatments reduced reproductive capacity (fertility, fecundity) by 7.3-57.4% in adult medaka breeding pairs, with hindrance of SSC development resulting in the largest disruption in breeding capacity (51.6-57.4% decrease) in the high concentration. This study documents differential effects among four critical stages of development and provides insight into factors (window of exposure, exposure concentration and duration of exposure period) contributing to reproductive disruption in fish.

TDP-43 chronic deficiency leads to dysregulation of transposable elements and gene expression by affecting R-loop and 5hmC crosstalk.

TDP-43 is an RNA/DNA-binding protein that forms aggregates in various brain disorders. TDP-43 engages in many aspects of RNA metabolism, but its molecular roles in regulating genes and transposable elements (TEs) have not been extensively explored. Chronic TDP-43 knockdown impairs cell proliferation and cellular responses to DNA damage. At the molecular level, TDP-43 chronic deficiency affects gene expression either locally or distally by concomitantly altering the crosstalk between R-loops and 5-hydroxymethylcytosine (5hmC) in gene bodies and long-range enhancer/promoter interactions. Furthermore, TDP-43 knockdown induces substantial disease-relevant TE activation by influencing their R-loop and 5hmC homeostasis in a locus-specific manner. Together, our findings highlight the genomic roles of TDP-43 in modulating R-loop-5hmC coordination in coding genes, distal regulatory elements, and TEs, presenting a general and broad molecular mechanism underlying the contributions of proteinopathies to the etiology of neurodegenerative disorders.

Antisense, but not sense, repeat expanded RNAs activate PKR/eIF2α-dependent ISR in C9ORF72 FTD/ALS.

GGGGCC (G4C2) hexanucleotide repeat expansion in the C9ORF72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The repeat is bidirectionally transcribed and confers gain of toxicity. However, the underlying toxic species is debated, and it is not clear whether antisense CCCCGG (C4G2) repeat expanded RNAs contribute to disease pathogenesis. Our study shows that C9ORF72 antisense C4G2 repeat expanded RNAs trigger the activation of the PKR/eIF2α-dependent integrated stress response independent of dipeptide repeat proteins that are produced through repeat-associated non-AUG-initiated translation, leading to global translation inhibition and stress granule formation. Reducing PKR levels with either siRNA or morpholinos mitigates integrated stress response and toxicity caused by the antisense C4G2 RNAs in cell lines, primary neurons, and zebrafish. Increased phosphorylation of PKR/eIF2α is also observed in the frontal cortex of C9ORF72 FTD/ALS patients. Finally, only antisense C4G2, but not sense G4C2, repeat expanded RNAs robustly activate the PKR/eIF2α pathway and induce aberrant stress granule formation. These results provide a mechanism by which antisense C4G2 repeat expanded RNAs elicit neuronal toxicity in FTD/ALS caused by C9ORF72 repeat expansions.

Cross-species transcriptomic analysis identifies mitochondrial dysregulation as a functional consequence of the schizophrenia-associated 3q29 deletion.

Recent advances in the genetics of schizophrenia (SCZ) have identified rare variants that confer high disease risk, including a 1.6 Mb deletion at chromosome 3q29 with a staggeringly large effect size (O.R. > 40). Understanding the impact of the 3q29 deletion (3q29Del) on the developing CNS may therefore lead to insights about the pathobiology of schizophrenia. To gain clues about the molecular and cellular perturbations caused by the 3q29 deletion, we interrogated transcriptomic effects in two experimental model systems with complementary advantages: isogenic human forebrain cortical organoids and isocortex from the 3q29Del mouse model. We first created isogenic lines by engineering the full 3q29Del into an induced pluripotent stem cell line from a neurotypical individual. We profiled transcriptomes from isogenic cortical organoids that were aged for 2 months and 12 months, as well as day p7 perinatal mouse isocortex, all at single cell resolution. Differential expression analysis by genotype in each cell-type cluster revealed that more than half of the differentially expressed genes identified in mouse cortex were also differentially expressed in human cortical organoids, and strong correlations were observed in mouse-human differential gene expression across most major cell-types. We systematically filtered differentially expressed genes to identify changes occurring in both model systems. Pathway analysis on this filtered gene set implicated dysregulation of mitochondrial function and energy metabolism, although the direction of the effect was dependent on developmental timepoint. Transcriptomic changes were validated at the protein level by analysis of oxidative phosphorylation protein complexes in mouse brain tissue. Assays of mitochondrial function in human heterologous cells further confirmed robust mitochondrial dysregulation in 3q29Del cells, and these effects are partially recapitulated by ablation of the 3q29Del gene PAK2 . Taken together these data indicate that metabolic disruption is associated with 3q29Del and is conserved across species. These results converge with data from other rare SCZ-associated variants as well as idiopathic schizophrenia, suggesting that mitochondrial dysfunction may be a significant but overlooked contributing factor to the development of psychotic disorders. This cross-species scRNA-seq analysis of the SCZ-associated 3q29 deletion reveals that this copy number variant may produce early and persistent changes in cellular metabolism that are relevant to human neurodevelopment.

Cross-species analysis identifies mitochondrial dysregulation as a functional consequence of the schizophrenia-associated 3q29 deletion.

The 1.6-megabase deletion at chromosome 3q29 (3q29Del) is the strongest identified genetic risk factor for schizophrenia, but the effects of this variant on neurodevelopment are not well understood. We interrogated the developing neural transcriptome in two experimental model systems with complementary advantages: isogenic human cortical organoids and isocortex from the 3q29Del mouse model. We profiled transcriptomes from isogenic cortical organoids that were aged for 2 and 12 months, as well as perinatal mouse isocortex, all at single-cell resolution. Systematic pathway analysis implicated dysregulation of mitochondrial function and energy metabolism. These molecular signatures were supported by analysis of oxidative phosphorylation protein complex expression in mouse brain and assays of mitochondrial function in engineered cell lines, which revealed a lack of metabolic flexibility and a contribution of the 3q29 gene PAK2. Together, these data indicate that metabolic disruption is associated with 3q29Del and is conserved across species.

Stabilized HIV-1 envelope immunization induces neutralizing antibodies to the CD4bs and protects macaques against mucosal infection.

A successful HIV-1 vaccine will require induction of a polyclonal neutralizing antibody (nAb) response, yet vaccine-mediated induction of such a response in primates remains a challenge. We found that a stabilized HIV-1 CH505 envelope (Env) trimer formulated with a Toll-like receptor 7/8 agonist induced potent HIV-1 polyclonal nAbs that correlated with protection from homologous simian-human immunodeficiency virus (SHIV) infection. The serum dilution that neutralized 50% of virus replication (ID50 titer) required to protect 90% of macaques was 1:364 against the challenge virus grown in primary rhesus CD4+ T cells. Structural analyses of vaccine-induced nAbs demonstrated targeting of the Env CD4 binding site or the N156 glycan and the third variable loop base. Autologous nAb specificities similar to those elicited in macaques by vaccination were isolated from the human living with HIV from which the CH505 Env immunogen was derived. CH505 viral isolates were isolated that mutated the V1 to escape both the infection-induced and vaccine-induced antibodies. These results define the specificities of a vaccine-induced nAb response and the protective titers of HIV-1 vaccine-induced nAbs required to protect nonhuman primates from low-dose mucosal challenge by SHIVs bearing a primary transmitted/founder Env.

The functions of SARS-CoV-2 neutralizing and infection-enhancing antibodies in vitro and in mice and nonhuman primates.

SARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV-1 infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro , while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Nonetheless, three of 31 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo , increased lung inflammation can occur in SARS-CoV-2 antibody-infused macaques.