Efficient in vitro and in vivo transfection of self-amplifying mRNA with linear poly(propylenimine) and poly(ethylenimine-propylenimine) random copolymers as non-viral carriers.

Messenger RNA (mRNA) based vaccines have been introduced worldwide to combat the Covid-19 pandemic. These vaccines consist of non-amplifying mRNA formulated in lipid nanoparticles (LNPs). Consequently, LNPs are considered benchmark non-viral carriers for nucleic acid delivery. However, the formulation and manufacturing of these mRNA-LNP nanoparticles are expensive and time-consuming. Therefore, we used self-amplifying mRNA (saRNA) and synthesized novel polymers as alternative non-viral carrier platform to LNPs, which enable a simple, rapid, one-pot formulation of saRNA-polyplexes. Our novel polymer-based carrier platform consists of randomly concatenated ethylenimine and propylenimine comonomers, resulting in linear, poly(ethylenimine-ran-propylenimine) (L-PEIx-ran-PPIy) copolymers with controllable degrees of polymerization. Here we demonstrate in multiple cell lines, that our saRNA-polyplexes show comparable to higher in vitro saRNA transfection efficiencies and higher cell viabilities compared to formulations with Lipofectamine MessengerMAX™ (LFMM), a commercial, lipid-based carrier considered to be the in vitro gold standard carrier. This is especially true for our in vitro best performing saRNA-polyplexes with N/P 5, which are characterised with a size below 100 nm, a positive zeta potential, a near 100% encapsulation efficiency, a high retention capacity and the ability to protect the saRNA from degradation mediated by RNase A. Furthermore, an ex vivo hemolysis assay with pig red blood cells demonstrated that the saRNA-polyplexes exhibit negligible hemolytic activity. Finally, a bioluminescence-based in vivo study was performed over a 35-day period, and showed that the polymers result in a higher and prolonged bioluminescent signal compared to naked saRNA and L-PEI based polyplexes. Moreover, the polymers show different expression profiles compared to those of LNPs, with one of our new polymers (L-PPI250) demonstrating a higher sustained expression for at least 35 days after injection.

Lipid Nanoparticle Delivery Alters the Adjuvanticity of the TLR9 Agonist CpG by Innate Immune Activation in Lymphoid Tissue.

Pharmacological strategies to activate innate immune cells are of great relevance in the context of vaccine design and anticancer immune therapy, to mount broad immune responses able to clear infection and malignant cells. Synthetic CpG oligodeoxynucleotides (CpG-ODNs) are short single-stranded DNA molecules containing unmethylated CpG dinucleotides and a phosphorothioate backbone. Class B CpG ODNs activate robust innate immune responses through a TLR9-dependent NF-κB signaling pathway. This feature is attractive to exploit in the context of vaccine design and cancer immunotherapy. Soluble CpG-ODNs cause hepatic toxicity, which reduces its therapeutic applicability. The formulation of class B CpG ODN1826 in lipid nanoparticles (LNPs) containing an ionizable cationic lipid that complexes CpG through electrostatic interaction is reported. Upon local administration, LNP-formulated CpG drains to lymph nodes and triggers robust innate immune activation. Unformulated, soluble, CpG, by contrast, is unable to induce robust innate activation in draining lymph nodes and is distributed systemically. In a vaccination setting, LNP-formulated CpG, admixed with a protein antigen, induces higher antigen-specific antibody titers and T cell responses than antigen admixed with unformulated soluble CpG.

Lactobacillus coryniformis subsp. torquens T3 alleviates non-alcoholic fatty liver disease via reconstruction of the gut microbiota and redox system.

BACKGROUND: A high-fat diet (HFD) that induces obesity has become the most common type of diet worldwide, leading to serious global health issues. Obesity is associated with an increased risk of non-alcoholic fatty liver disease (NAFLD). Probiotic supplements have been shown to help alleviate obesity. The present study aimed to investigate the mechanism by which Lactobacillus coryniformis supsp. torquens T3 (T3L) alleviated NAFLD induced by HFD via reconstruction of the gut microbiota and redox system. RESULTS: The results showed that, compared with the HFD group, T3L inhibited obesity and relieved fat accumulation in the liver of mice with NAFLD. In addition, T3L inhibited liver inflammation and oxidative stress injury in NAFLD mice by regulating the lipopolysaccharide (LPS) inflammatory pathway in the liver. Furthermore, T3L changed the composition of the intestinal flora, reduced the abundance of harmful bacteria in the intestinal tract, enhanced the mechanical function of the intestinal barrier, and increased the short-chain fatty acid contents, thus inhibiting the secondary metabolite LPS, which directly causes liver damage through the portal vein. CONCLUSION: In summary, T3L ameliorated NAFLD induced by obesity through the liver-gut axis pathway, thus reducing oxidative stress and liver injury. © 2023 Society of Chemical Industry.

Obesity, but not high-fat diet, is associated with bone loss that is reversed via CD4+CD25+Foxp3+ Tregs-mediated gut microbiome of non-obese mice.

Osteoporosis is characterized by decreased bone mass, microarchitectural deterioration, and increased bone fragility. High-fat diet (HFD)-induced obesity also results in bone loss, which is associated with an imbalanced gut microbiome. However, whether HFD-induced obesity or HFD itself promotes osteoclastogenesis and consequent bone loss remains unclear. In this study, we developed HFD-induced obesity (HIO) and non-obesity (NO) mouse models to evaluate the effect of HFD on bone loss. NO mice were defined as body weight within 5% of higher or lower than that of chow diet fed mice after 10 weeks HFD feeding. NO was protected from HIO-induced bone loss by the RANKL /OPG system, with associated increases in the tibia tenacity, cortical bone mean density, bone volume of cancellous bone, and trabecular number. This led to increased bone strength and improved bone microstructure via the microbiome-short-chain fatty acids (SCFAs) regulation. Additionally, endogenous gut-SCFAs produced by the NO mice activated free fatty acid receptor 2 and inhibited histone deacetylases, resulting in the promotion of Treg cell proliferation in the HFD-fed NO mice; thereby, inhibiting osteoclastogenesis, which can be transplanted by fecal microbiome. Furthermore, T cells from NO mice retain differentiation of osteoclast precursors of RAW 264.7 macrophages ex vivo. Our data reveal that HFD is not a deleterious diet; however, the induction of obesity serves as a key trigger of bone loss that can be blocked by a NO mouse-specific gut microbiome.

A 113-amino-acid truncation at the NS1 C-terminus is a determinant for viral replication of H5N6 avian influenza virus in vitro and in vivo.

Virulence of highly pathogenic avian influenza viruses (AIV) is determined by multiple genes and their encoded proteins. In particular, the nonstructural protein 1 (NS1) of viruses is a multifunctional protein that plays an important role in type I interferon (IFN) antagonism, pathogenicity, and determining viral host range. Naturally-occurring truncation or mutation of NS1 during virus evolution attenuates viral replication and pathogenicity, but the mechanisms underlying this phenomenon remain poorly understood. In the present study, we rescued an H5N6 AIV harboring a 113-amino-acid (aa) truncated NS1 at the C-terminus that had previously naturally occurred in an H3N8 equine influenza virus (designated as rHN109 NS1/112). The replication and pathogenicity of the rescued and parental viruses were then assessed in vitro in cells and in vivo in chickens and mice. Replication of rHN109 NS1/112 virus was significantly attenuated in various cells compared to its parental virus. The attenuation of rHN109 NS1/112 virus was subsequently clarified by investigating the effects on IFN and apoptosis signaling pathways via multiple experiments. The results indicated that the 113-aa truncation of NS1 impairs viral inhibition of IFN production and enhances cellular apoptosis in avian and mammalian cells. Animal studies further indicated that replication of the rHN109 NS1/112 virus is remarkably attenuated in chickens. The results of this study improve our understanding of C-terminal region function for NS1 proteins of influenza viruses.

A novel genotype VII Newcastle disease virus vaccine candidate generated by mutation in the L and F genes confers improved protection in chickens.

Administration of vaccines combined with the good management and strict biosecurity is an effective way for Newcastle disease (ND) control. However, vaccine failure is continuously reported in some countries mainly because the antigenic difference between the used vaccine and field strains even they are of one serotype. Therefore, development of antigen-matched ND vaccines is needed to improve the vaccine efficacy in birds. In this study, we introduced four site mutations, K1756A, D1881A, K1917A and E1954Q, respectively, into the large protein gene of the virulent genotype VII Newcastle disease virus (NDV) G7 strain using reverse genetics technology. Four rescued NDVs were sharply attenuated for the pathogenicity in chickens. One of these mutants, E1954Q, was further manipulated by replacing the F cleavage site sequence of typical velogenic strains with that of the LaSota vaccine, resulting in a new mutant, G7M. Biological characterization showed that G7M was safe and genetically stable after serial passages in embryos and chickens. Vaccination of chickens with G7M induced a progressive elevation of the homologous antibodies and markedly higher CD8+ T cell percentage, T cell proliferation and IFN-γ than LaSota. G7M conferred full protection against genotype VII NDV challenge, and more importantly, it effectively reduced the challenge virus replication and shedding in chickens. Together, our data suggest that G7M is a promising genotype VII vaccine candidate, and the novel attenuation approach designed in this study could be used to develop new antigen-matched NDV vaccines.

Two single mutations in the fusion protein of Newcastle disease virus confer hemagglutinin-neuraminidase independent fusion promotion and attenuate the pathogenicity in chickens.

The fusion (F) protein of Newcastle disease virus (NDV) affects viral infection and pathogenicity through mediating membrane fusion. Previously, we found NDV with increased fusogenic activity in which contained T458D or G459D mutation in the F protein. Here, we investigated the effects of these two mutations on viral infection, fusogenicity and pathogenicity. Syncytium formation assays indicated that T458D or G459D increased the F protein cleavage activity and enhanced cell fusion with or without the presence of HN protein. The T458D- or G459D-mutated NDV resulted in a decrease in virus replication or release from cells. The animal study showed that the pathogenicity of the mutated NDVs was attenuated in chickens. These results indicate that these two single mutations in F altered or diminished the requirement of HN for promoting membrane fusion. The increased fusogenic activity may disrupt the cellular machinery and consequently decrease the virus replication and pathogenicity in chickens.