Immunization with HIV AIDSVAX gp120 vaccines in the phase III VAX003 and VAX004 trials did not confer protection. To understand the shortcomings in antibody (Ab) responses induced by these vaccines, ...we evaluated the kinetics of Ab responses to the V1V2 and V3 regions of gp120 and the induction of Ab-mediated antiviral functions during the course of 7 vaccinations over a 30.5-month period. Plasma samples from VAX003 and VAX004 vaccinees and placebo recipients were measured for ELISA-binding Abs and for virus neutralization, Ab-dependent cellular phagocytosis (ADCP), and Ab-dependent cellular cytotoxicity (ADCC). Ab responses to V1V2 and V3 peaked after 3 to 4 immunizations and declined after 5 to 7 immunizations. The deteriorating responses were most evident against epitopes in the underside of the V1V2 β-barrel and in the V3 crown. Correspondingly, vaccinees demonstrated higher neutralization against SF162 pseudovirus sensitive to anti-V1V2 and anti-V3 Abs after 3 or 4 immunizations than after 7 immunizations. Higher levels of ADCP and ADCC were also observed at early or mid-time points as compared with the final time point. Hence, VAX003 and VAX004 vaccinees generated V1V2- and V3-binding Abs and functional Abs after 3 to 4 immunizations, but subsequent boosts did not maintain these responses.
Understanding the mechanisms underlying autoantibody development will accelerate therapeutic target identification in autoimmune diseases such as systemic lupus erythematosus (SLE)
. Follicular ...helper T cells (T
cells) have long been implicated in SLE pathogenesis. Yet a fraction of autoantibodies in individuals with SLE are unmutated, supporting that autoreactive B cells also differentiate outside germinal centers
. Here, we describe a CXCR5
CXCR3
programmed death 1 (PD1)
CD4
helper T cell population distinct from T
cells and expanded in both SLE blood and the tubulointerstitial areas of individuals with proliferative lupus nephritis. These cells produce interleukin-10 (IL-10) and accumulate mitochondrial reactive oxygen species as the result of reverse electron transport fueled by succinate. Furthermore, they provide B cell help, independently of IL-21, through IL-10 and succinate. Similar cells are generated in vitro upon priming naive CD4
T cells with plasmacytoid dendritic cells activated with oxidized mitochondrial DNA, a distinct class of interferogenic toll-like receptor 9 ligand
. Targeting this pathway might blunt the initiation and/or perpetuation of extrafollicular humoral responses in SLE.
Emerging evidence supports that mitochondrial dysfunction contributes to systemic lupus erythematosus (SLE) pathogenesis. Here we show that programmed mitochondrial removal, a hallmark of mammalian ...erythropoiesis, is defective in SLE. Specifically, we demonstrate that during human erythroid cell maturation, a hypoxia-inducible factor (HIF)-mediated metabolic switch is responsible for the activation of the ubiquitin-proteasome system (UPS), which precedes and is necessary for the autophagic removal of mitochondria. A defect in this pathway leads to accumulation of red blood cells (RBCs) carrying mitochondria (Mito+ RBCs) in SLE patients and in correlation with disease activity. Antibody-mediated internalization of Mito+ RBCs induces type I interferon (IFN) production through activation of cGAS in macrophages. Accordingly, SLE patients carrying both Mito+ RBCs and opsonizing antibodies display the highest levels of blood IFN-stimulated gene (ISG) signatures, a distinctive feature of SLE.
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•HIF2α degradation promotes UPS activation and mitophagy during human erythropoiesis•Defective HIF2α degradation leads to accumulation of Mito+ RBCs in SLE patients•Uptake of opsonized Mito+ RBCs by macrophages induces IFN production via cGAS/STING•Highest ISG scores define SLE patients with Mito+ RBCs and opsonizing antibodies
A subgroup of SLE patients fail to engage HIF-regulated metabolic and proteasomal pathways causing the accumulation of mitochondria-containing red blood cells. These cells, when engulfed by macrophages, activate cGAS/STING-dependent inflammation.
Abstract Summary Subcluster analysis is a powerful means to improve clustering and characterization of single cell RNA-Seq data. However, there are no existing tools to systematically integrate ...results from multiple subclusters, which creates hurdles for accurate data quantification, visualization, and interpretation in downstream analysis. To address this issue, we developed Ragas, an R package that integrates multi-level subclustering objects for streamlined analysis and visualization. A new data structure was implemented to seamlessly connect and assemble miscellaneous single cell analyses from different levels of subclustering, along with several new or enhanced visualization functions. Moreover, a re-projection algorithm was developed to integrate nearest-neighbor graphs from multiple subclusters in order to maximize their separability on the combined cell embeddings, which significantly improved the presentation of rare and homogeneous subpopulations. Availability and implementation The Ragas package and its documentation can be accessed through https://github.com/jig4003/Ragas and its source code is also available at https://zenodo.org/records/11244921.
Objective
This study was undertaken to identify blood markers of juvenile dermatomyositis (DM) disease activity (DA), which are needed to improve disease management.
Methods
The study comprised a ...total of 123 juvenile DM patients and 53 healthy controls. Results of laboratory tests (aldolase, creatinine kinase, lactate dehydrogenase LDH, aspartate aminotransferase) and clinical measures of DA in patients with juvenile DM, including the Manual Muscle Testing in 8 muscles (MMT‐8), Childhood Myositis Assessment Scale (CMAS), and disease activity scores (DAS) (total DAS for juvenile DM, the muscle DAS, and the skin DAS), were recorded when available. Surface phenotype of peripheral blood mononuclear cells was assessed using flow cytometry. Whole blood transcriptional profiles were studied using either RNA‐sequencing or microarrays. Differential gene expression was determined using DESeq and compared by pathway and gene ontology analyses.
Results
Conventional memory (CD27+IgD–) B cells expressing low CXCR5 levels (CXCR5low/– CM B cells) were significantly increased in frequency and absolute numbers in 2 independent cohorts of juvenile DM patients compared with healthy controls. The frequency of CD4+ Th2 memory cells (CD45RA–CXCR5–CCR6–CXCR3–) was also increased in juvenile DM, especially in patients who were within <1 year from diagnosis. The frequency of CXCR5low/– CM B cells correlated with serum aldolase levels and with a blood interferon‐stimulated gene transcriptional signature. Furthermore, both the frequency and absolute numbers of CXCR5low/– CM B cells correlated with clinical and laboratory measures of muscle DA (MMT‐8, CMAS, aldolase, and LDH).
Conclusion
These findings suggest that both CM B cells lacking the CXCR5 follicular marker and CXCR5– Th2 cells represent potential biomarkers of DA in juvenile DM and may contribute to its pathogenesis.
HIV still causes a global pandemic and the development of a prophylactic vaccine is much needed to eradicate this virus. The HIV envelope (Env), composed of gp120 and gp41 subunits, is the only ...surface protein present on the surfaces of virions and infected cells. Hence, HIV vaccines designed to elicit protective Abs need to target the HIV Env. The V1V2 and V3 loops, which form the apex of the trimeric HIV Env spike, contain conserved structural elements that can serve as targets for protective Abs against HIV. Crystallographic analyses of mAbs in complex with the crown of the V3 loop have revealed that these mAbs recognize conserved sites on the V3 crown via two distinct strategies: a cradle-binding mode (V3C) and a ladle-binding (V3L) mode. Similarly, crystal structures of mAbs in complex with the V1V2 loop have identified different types of epitopes in the V1V2 domain that include V2p (peptide), V2i (integrin), and V2q (quaternary). However, little has been done to understand if and how the different types of V1V2 and V3 Abs are induced after HIV Env vaccination in humans and animal models. Our findings showed that, whereas both V3C and V3L type Abs were prevalent in sera of chronically HIV infected individuals, only V3C-type Abs were produced by humans, macaques, and rabbits immunized with different HIV Env immunogens. Immunized mice, on the other hand, produced mainly the V3L-type Abs. Nonetheless, both the V3C-type and V3L-type Abs generated by vaccinations were able to mediate virus neutralization. To examine more systematically how the V3C-type and V3L-type Ab responses are elicited and boosted during immunization with HIV Env, longitudinal analyses were performed using plasma samples collected from human vaccinees who received 7 doses of the HIV AIDSVAX recombinant gp120 proteins in the phase III VAX003 and VAX004 trials. We observed that Ab responses to V1V2 and V3 were elicited and peaked after 3 to 4 immunizations, but declined after 5 to 7 immunizations. The deteriorating responses were also evident against V2p and V2i epitopes in the underside of the V1V2 β-barrel and against V3C and V3L epitopes in the V3 crown. Importantly, the functional activities of Abs measured in virus neutralization, Ab-dependent phagocytosis, and Ab-dependent cellular cytotoxicity assays were also lower at the final time point as compared to early or mid-time points. Altogether, this study showed that HIV Env vaccination elicits restricted repertoire of Abs with species-specific differences in humans and animal models. The Abs target cross-reactive epitopes in the variable domains on the HIV Env apex and exhibit antiviral functions in vitro, but the responses are transient. Future studies are needed to improve immunogen designs and vaccination strategies to increase the durability and broaden the Ab repertoire in order to stimulate more sustained Ab responses against multiple conserved epitopes in V1V2, V3, and also other HIV Env regions.
Highlights • Abs with distinct V3-binding modes (cradle and ladle) are made during HIV infection. • In contrast, HIV envelope vaccines induce either the cradle- or the ladle-type Abs. • The types of ...V3 Abs elicited differ depending on the vaccinated animal species. • V3 cradle- and V3 ladle-type Abs induced by vaccination can neutralize virus.
The V3 region of HIV-1 gp120 is important for virus-coreceptor interaction and highly immunogenic. Although most anti-V3 antibodies neutralize only the sensitive tier 1 viruses, anti-V3 antibodies ...effective against the more resistant viruses exist, and a better understanding of these antibodies and their epitopes would be beneficial for the development of novel vaccine immunogens against HIV. The HIV-1 isolate JRFL with its cryptic V3 is resistant to most V3-specific monoclonal antibodies (MAbs). However, the V3 MAb 2424 achieves 100% neutralization against JRFL. 2424 is encoded by IGHV3-53 and IGLV2-28 genes, a pairing rarely used by the other V3 MAbs. 2424 also has distinct binding and neutralization profiles. Studies of 2424-mediated neutralization of JRFL produced with a mannosidase inhibitor further revealed that its neutralizing activity is unaffected by the glycan composition of the virus envelope. To understand the distinct activity of 2424, we determined the crystal structure of 2424 Fab in complex with a JRFL V3 peptide and showed that the 2424 epitope is located at the tip of the V3 crown ((307)IHIGPGRAFYT(319)), dominated by interactions with His(P308), Pro(P313), and Arg(P315). The binding mode of 2424 is similar to that of the well-characterized MAb 447-52D, although 2424 is more side chain dependent. The 2424 epitope is focused on the very apex of V3, away from nearby glycans, facilitating antibody access. This feature distinguishes the 2424 epitope from the other V3 crown epitopes and indicates that the tip of V3 is a potential site to target and incorporate into HIV vaccine immunogens.
HIV/AIDS vaccines are crucial for controlling the HIV epidemics that continue to afflict millions of people worldwide. However, HIV vaccine development has been hampered by significant scientific challenges, one of which is the inability of HIV vaccine candidates evaluated thus far to elicit production of potent and broadly neutralizing antibodies. The V3 loop is one of the few immunogenic targets on the virus envelope glycoprotein that can induce neutralizing antibodies, but in many viruses, parts of V3 are inaccessible for antibody recognition. This study examined a V3-specific monoclonal antibody that can completely neutralize HIV-1 JRFL, a virus isolate resistant to most V3 antibodies. Our data reveal that this antibody recognizes the most distal tip of V3, which is not as occluded as other parts of V3. Hence, the epitope of 2424 is in one of the vulnerable sites on the virus that may be exploited in designing HIV vaccine immunogens.
Many neurodegenerative diseases result due to the accumulation of misfolded proteins as amyloid fibrils. Although the protein components of these fibrils from different disease states differ ...considerably, they appear to share common structure. Among these conformational disorders, Alzheimer's disease (AD) and prion diseases exhibit significant overlap in their mechanism of pathogenesis. The present report demonstrates that antibodies directed against the prion protein repeat motif, Tyr-Tyr-Arg motif, recognize recombinantly expressed human amyloid beta (A beta) aggregates in enzyme linked immunosorbent assay. In addition, these antibodies dissociate the preformed aggregates of A beta in vitro. These findings illustrate an important property of conformation dependent antibodies viz., they specifically recognize the protein deposits associated with pathology and not the protein in normal tissue. These antibodies may benefit the development of approaches towards prevention and treatment of protein misfolding diseases.
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