Continuous blood exchange in rats as a novel approach for experimental investigation.

Blood exchange therapy, specifically Whole blood exchange (WBE), is increasingly being utilized in clinical settings to effectively treat a range of diseases. Consequently, there is an urgent requirement to establish convenient and clinically applicable animal models that can facilitate the exploration of blood exchange therapy mechanisms. Our study conducted continuous WBE in rats through femoral and tail vein catheterization using dual-directional syringe pumps. To demonstrate the applicability of continuous WBE, drug-induced hemolytic anemia (DIHA) was induced through phenylhydrazine hydrochloride (PHZ) injection. Notability, the rats of DIHA + WBE group all survived and recovered within the subsequent period. After the implementation of continuous WBE therapy day (Day 1), the DIHA + WBE group exhibited a statistically significant increase in red blood cells (RBC) (P = 0.0343) and hemoglobin (HGB) levels (P = 0.0090) compared to DIHA group. The rats in the DIHA + WBE group exhibited a faster recovery rate compared to the DIHA group, indicating the successful establishment of a continuous blood exchange protocol. This experimental approach demonstrates not just promising efficacy in the treatment of DIHA and offers a valuable tool for investigating the underlying mechanisms of blood exchange. Furthermore, it has a great potential to the advancement of biomedical research such as drug delivery exploration.

Recombinant Full-Length Hepatitis C Virus E1E2 Dimer Elicits Pangenotypic Neutralizing Antibodies.

An effective prophylactic vaccine would be beneficial for controlling and eradicating hepatitis C virus (HCV) infections. However, the high diversity across HCV genotypes is a major challenge for vaccine development. Selection of the appropriate immunogen is critical to elicit broad HCV neutralizing antibodies (NAbs). To increase the antigenic coverage of heterodimer glycoproteins, we designed and produced recombinant E1E2 antigens for genotypes 1a/1b/2a/3a/6a from an IgG Fc-tagged precursor protein in FreeStyle 293-F cells. The recombinant E1 and E2 antigens were localized and associated with the endoplasmic reticulum and co-purified from membrane extracts. By examining the interactions with HCV entry co-receptors and the blockade of HCV infection, we found that these purified Fc-E1E2 proteins displayed correct folding and function. Mouse immunization results showed that each recombinant E1E2 antigen could elicit a pangenotypic antibody response to itself and other genotypes. We also found that the pentavalent formula triggered a relatively higher and more uniform NAb titer and T cell response than monovalent antigens. Taken together, our findings may provide a useful strategy for the vaccine development of HCV and other viruses with highly heterogeneous surface glycoproteins.

An ultrapotent RBD-targeted biparatopic nanobody neutralizes broad SARS-CoV-2 variants.

The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, convalescent patients derived natural antibodies are vulnerable to SARS-CoV-2 Spike mutation. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb, named Nb1-Nb2, with tight affinity and super-wide neutralization breadth against multiple SARS-CoV-2 variants of concern. Deep-mutational scanning experiments identify the potential binding epitopes of the Nbs on the RBD and demonstrate that biparatopic Nb1-Nb2 has a strong escape-resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that the Nb1-Nb2 broadly neutralizes multiple SARS-CoV-2 variants at sub-nanomolar levels, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1), and Mu (B.1.621). Furthermore, a heavy-chain antibody is constructed by fusing the human IgG1 Fc to Nb1-Nb2 (designated as Nb1-Nb2-Fc) to improve its neutralization potency, yield, stability, and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1-Nb2-Fc keeps a firm affinity (KD < 1.0 × 10-12 M) and strong neutralizing activity (IC50 = 1.46 nM for authentic Omicron virus). Together, we developed a tetravalent biparatopic human heavy-chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications.

Identification of Ascomycin against Zika virus infection through screening of natural product library.

Zika virus (ZIKV) infection could lead to Guillain-Barré syndrome in adults and microcephaly in the newborns from infected pregnant women. To date, there is no specific drug for the treatment of ZIKV infection. In this study, we sought to screen inhibitors against ZIKV infection from a natural product library. A ZIKV replicon was used to screen a library containing 1680 natural compounds. We explored the antiviral mechanism of the compound candidate in vitro and in vivo infection models. Ascomycin, a macrolide from Streptomyces hygroscopicus, was identified with inhibitory effect against ZIKV in Vero cells (IC50 = 0.11 µM), hepatoma cell Huh7 (IC50 = 0.38 µM), and glioblastoma cell SNB-19 (IC50 = 0.06 µM), far below the cytotoxic concentrations. Mechanistic study revealed that Ascomycin suppressed ZIKV RNA replication step during the life cycle and the regulation of calcineurin-NFAT pathway maybe involved in this inhibitory effect, independent of innate immunity activation. Moreover, we found that Ascomycin also inhibited the infection of other Flaviviridae members, such as hepatitis C virus and dengue virus. Ascomycin reduced ZIKV load in blood by up to 3500-fold in A129 mice. Meanwhile, the infection in the mice brain was undetectable by immunohistochemistry staining. Together, these findings reveal a critical role of Ascomycin in the inhibition of ZIKV and related viruses, facilitating the development of novel antiviral agents.

Zika virus degrades the ω-3 fatty acid transporter Mfsd2a in brain microvascular endothelial cells and impairs lipid homeostasis.

Zika virus (ZIKV) infection during pregnancy increases the risk of postnatal microcephaly. Neurovascular function provides a homeostatic environment for proper brain development. The major facilitator superfamily domain-containing protein 2 (Mfsd2a) is selectively expressed in human brain microvascular endothelial cells (hBMECs) and is the major transporter mediating the brain uptake of docosahexaenoic acid (DHA). We have discovered a pivotal role for Mfsd2a in the pathogenesis of ZIKV. ZIKV disrupted Mfsd2a both in cultured primary hBMECs and in the neonatal mouse brain. ZIKV envelope (E) protein specifically interacted with Mfsd2a and promoted Mfsd2a polyubiquitination for proteasome-dependent degradation. Infection with ZIKV or ectopic expression of ZIKV E impaired Mfsd2a-mediated DHA uptake. Lipidomic analysis revealed obvious differences in DHA-containing lipids after ZIKV infection. Supplementation with DHA rescued ZIKV-caused growth restriction and microcephaly. Our findings suggest endothelial Mfsd2a as an important pathogenic mediator and supplementation with DHA as a potential therapeutic option for ZIKV infection.

High-Throughput Screening Identifies Mixed-Lineage Kinase 3 as a Key Host Regulatory Factor in Zika Virus Infection.

The Zika virus (ZIKV) life cycle involves multiple steps and requires interactions with host factors. However, the inability to systematically identify host regulatory factors for ZIKV has hampered antiviral development and our understanding of pathogenicity. Here, using a bioactive compound library with 2,659 small molecules, we applied a high-throughput and imaging-based screen to identify host factors that modulate ZIKV infection. The screen yielded hundreds of hits that markedly inhibited or potentiated ZIKV infection in SNB-19 glioblastoma cells. Among the hits, URMC-099, a mixed-lineage kinase 3 (MLK3) inhibitor, significantly facilitated ZIKV replication in both SNB-19 cells and the neonatal mouse brain. Using gene silencing and overexpression, we further confirmed that MLK3 was a host restriction factor against ZIKV. Mechanistically, MLK3 negatively regulated ZIKV replication through induction of the inflammatory cytokines interleukin-6 (IL-6), IL-8, tumor necrosis factor alpha (TNF-α), and monocyte chemoattractant protein 1 (MCP-1) but did not modulate host interferon-related pathways. Importantly, ZIKV activated the MLK3/MKK7/Jun N-terminal protein kinase (JNK) pathway in both SNB-19 cells and neonatal mouse brain. Together, these findings reveal a critical role for MLK3 in regulating ZIKV infection and facilitate the development of anti-ZIKV therapeutics by providing a number of screening hits.IMPORTANCE Zika fever, an infectious disease caused by the Zika virus (ZIKV), normally results in mild symptoms. Severe infection can cause Guillain-Barré syndrome in adults and birth defects, including microcephaly, in newborns. Although ZIKV was first identified in Uganda in 1947 in rhesus monkeys, a widespread epidemic of ZIKV infection in South and Central America in 2015 and 2016 raised major concerns. To date, there is no vaccine or specific medicine for ZIKV. The significance of our research is the systematic discovery of small molecule candidates that modulate ZIKV infection, which will allow the development of antiviral therapeutics. In addition, we identified MLK3, a key mediator of host signaling pathways that can be activated during ZIKV infection and limits virus replication by inducing multiple inflammatory cytokines. These findings broaden our understanding of ZIKV pathogenesis.