Nanoparticle-based DNA vaccine protects against SARS-CoV-2 variants in female preclinical models.

A safe and effective vaccine with long-term protection against SARS-CoV-2 variants of concern (VOCs) is a global health priority. Here, we develop lipid nanoparticles (LNPs) to provide safe and effective delivery of plasmid DNA (pDNA) and show protection against VOCs in female small animal models. Using a library of LNPs encapsulating unique barcoded DNA (b-DNA), we screen for b-DNA delivery after intramuscular administration. The top-performing LNPs are further tested for their capacity of pDNA uptake in antigen-presenting cells in vitro. The lead LNP is used to encapsulate pDNA encoding the HexaPro version of SARS-CoV-2 spike (LNP-HPS) and immunogenicity and protection is tested in vivo. LNP-HPS elicit a robust protective effect against SARS-CoV-2 Gamma (P.1), correlating with reduced lethality, decreased viral load in the lungs and reduced lung damage. LNP-HPS induce potent humoral and T cell responses against P.1, and generate high levels of neutralizing antibodies against P.1 and Omicron (B.1.1.529). Our findings indicate that the protective efficacy and immunogenicity elicited by LNP-HPS are comparable to those achieved by the approved COVID-19 vaccine from Biontech/Pfizer in animal models. Together, these findings suggest that LNP-HPS hold great promise as a vaccine candidate against VOCs.

Essential role of the CCL2-CCR2 axis in Mayaro virus-induced disease.

Mayaro virus (MAYV) is an emerging arbovirus member of the Togaviridae family and Alphavirus genus. MAYV infection causes an acute febrile illness accompanied by persistent polyarthralgia and myalgia. Understanding the mechanisms involved in arthritis caused by alphaviruses is necessary to develop specific therapies. In this work, we investigated the role of the CCL2/CCR2 axis in the pathogenesis of MAYV-induced disease. For this, wild-type (WT) C57BL/6J and CCR2-/- mice were infected with MAYV subcutaneously and evaluated for disease development. MAYV infection induced an acute inflammatory disease in WT mice. The immune response profile was characterized by an increase in the production of inflammatory mediators, such as IL-6, TNF, and CCL2. Higher levels of CCL2 at the local and systemic levels were followed by the significant recruitment of CCR2+ macrophages and a cellular response orchestrated by these cells. CCR2-/- mice showed an increase in CXCL-1 levels, followed by a replacement of the macrophage inflammatory infiltrate by neutrophils. Additionally, the absence of the CCR2 receptor protected mice from bone loss induced by MAYV. Accordingly, the silencing of CCL2 chemokine expression in vivo and the pharmacological blockade of CCR2 promoted a partial improvement in disease. Cell culture data support the mechanism underlying the bone pathology of MAYV, in which MAYV infection promotes a pro-osteoclastogenic microenvironment mediated by CCL2, IL-6, and TNF, which induces the migration and differentiation of osteoclast precursor cells. Overall, these data contribute to the understanding of the pathophysiology of MAYV infection and the identification future of specific therapeutic targets in MAYV-induced disease.IMPORTANCEThis work demonstrates the role of the CCL2/CCR2 axis in MAYV-induced disease. The infection of wild-type (WT) C57BL/6J and CCR2-/- mice was associated with high levels of CCL2, an important chemoattractant involved in the recruitment of macrophages, the main precursor of osteoclasts. In the absence of the CCR2 receptor, there is a mitigation of macrophage migration to the target organs of infection and protection of these mice against bone loss induced by MAYV infection. Much evidence has shown that host immune response factors contribute significantly to the tissue damage associated with alphavirus infections. Thus, this work highlights molecular and cellular targets involved in the pathogenesis of arthritis triggered by MAYV and identifies novel therapeutic possibilities directed to the host inflammatory response unleashed by MAYV.

siRNA lipid nanoparticles for CXCL12 silencing modulate brain immune response during Zika infection.

CXCL12 is a key chemokine implicated in neuroinflammation, particularly during Zika virus (ZIKV) infection. Specifically, CXCL12 is upregulated in circulating cells of ZIKV infected patients. Here, we developed a lipid nanoparticle (LNP) to deliver siRNA in vivo to assess the impact of CXCL12 silencing in the context of ZIKV infection. The biodistribution of the LNP was assessed in vivo after intravenous injection using fluorescently tagged siRNA. Next, we investigated the ability of the developed LNP to silence CXCL12 in vivo and assessed the resulting effects in a murine model of ZIKV infection. The LNP encapsulating siRNA significantly inhibited CXCL12 levels in the spleen and induced microglial activation in the brain during ZIKV infection. This activation was evidenced by the enhanced expression of iNOS, TNF-α, and CD206 within microglial cells. Moreover, T cell subsets exhibited reduced secretion of IFN-É£ and IL-17 following LNP treatment. Despite no observable alteration in viral load, CXCL12 silencing led to a significant reduction in type-I interferon production compared to both ZIKV-infected and uninfected groups. Furthermore, we found grip strength deficits in the group treated with siRNA-LNP compared to the other groups. Our data suggest a correlation between the upregulated pro-inflammatory cytokines and the observed decrease in strength. Collectively, our results provide evidence that CXCL12 silencing exerts a regulatory influence on the immune response in the brain during ZIKV infection. In addition, the modulation of T-cell activation following CXCL12 silencing provides valuable insights into potential protective mechanisms against ZIKV, offering novel perspectives for combating this infection.

Delivery of Plasmid DNA by Ionizable Lipid Nanoparticles to Induce CAR Expression in T Cells.

Introduction: Chimeric antigen receptor (CAR) cell therapy represents a hallmark in cancer immunotherapy, with significant clinical results in the treatment of hematological tumors. However, current approved methods to engineer T cells to express CAR use viral vectors, which are integrative and have been associated with severe adverse effects due to constitutive expression of CAR. In this context, non-viral vectors such as ionizable lipid nanoparticles (LNPs) arise as an alternative to engineer CAR T cells with transient expression of CAR. Methods: Here, we formulated a mini-library of LNPs to deliver pDNA to T cells by varying the molar ratios of excipient lipids in each formulation. LNPs were characterized and screened in vitro using a T cell line (Jurkat). The optimized formulation was used ex vivo to engineer T cells derived from human peripheral blood mononuclear cells (PBMCs) for the expression of an anti-CD19 CAR (CAR-CD19BBz). The effectiveness of these CAR T cells was assessed in vitro against Raji (CD19+) cells. Results: LNPs formulated with different molar ratios of excipient lipids efficiently delivered pDNA to Jurkat cells with low cytotoxicity compared to conventional transfection methods, such as electroporation and lipofectamine. We show that CAR-CD19BBz expression in T cells was transient after transfection with LNPs. Jurkat cells transfected with our top-performing LNPs underwent activation when exposed to CD19+ target cells. Using our top-performing LNP-9-CAR, we were able to engineer human primary T cells to express CAR-CD19BBz, which elicited significant specific killing of CD19+ target cells in vitro. Conclusion: Collectively, our results show that LNP-mediated delivery of pDNA is a suitable method to engineer human T cells to express CAR, which holds promise for improving the production methods and broader application of this therapy in the future.

A suitable model to investigate acute neurological consequences of coronavirus infection.

OBJECTIVE AND DESIGN: The present study aimed to investigate the neurochemical and behavioral effects of the acute consequences after coronavirus infection through a murine model. MATERIAL: Wild-type C57BL/6 mice were infected intranasally (i.n) with the murine coronavirus 3 (MHV-3). METHODS: Mice underwent behavioral tests. Euthanasia was performed on the fifth day after infection (5 dpi), and the brain tissue was subjected to plaque assays for viral titration, ELISA, histopathological, immunohistochemical and synaptosome analysis. RESULTS: Increased viral titers and mild histological changes, including signs of neuronal degeneration, were observed in the cerebral cortex of infected mice. Importantly, MHV-3 infection induced an increase in cortical levels of glutamate and calcium, which is indicative of excitotoxicity, as well as increased levels of pro-inflammatory cytokines (IL-6, IFN-γ) and reduced levels of neuroprotective mediators (BDNF and CX3CL1) in the mice brain. Finally, behavioral analysis showed impaired motor, anhedonia-like and anxiety-like behaviors in animals infected with MHV-3. CONCLUSIONS: In conclusion, the data presented emulate many aspects of the acute neurological outcomes seen in patients with COVID-19. Therefore, this model may provide a preclinical platform to study acute neurological sequelae induced by coronavirus infection and test possible therapies.

Oral formulation of Wnt inhibitor complex reduces inflammation and fibrosis in intraperitoneal implants in vivo.

The use of implantable biomaterials to replace physiological and anatomical functions has been widely investigated in the clinic. However, the selection of biomaterials is crucial for long-term function, and the implantation of certain biomaterials can cause inflammatory and fibrotic processes, triggering a foreign body reaction that leads to loss of function and consequent need for removal. Specifically, the Wnt signaling pathway controls the healing process of the human body, and its dysregulation can result in inflammation and fibrosis, such as in peritoneal fibrosis. Here, we assessed the effects of daily oral administration of a Wnt pathway inhibitor complex (CD:LGK974) to reduce the inflammatory, fibrotic, and angiogenic processes caused by intraperitoneal implants. CD:LGK974 significantly reduced the infiltration of immune cells and release of inflammatory cytokines in the implant region compared to the control groups. Furthermore, CD:LGK974 inhibited collagen deposition and reduced the expression of pro-fibrotic α-SMA and TGF-ß1, confirming fibrosis reduction. Finally, the CD:LGK974 complex decreased VEGF levels and both the number and area of blood vessels formed, suggesting decreased angiogenesis. This work introduces a potential new application of the Wnt inhibitor complex to reduce peritoneal fibrosis and the rejection of implants at the intraperitoneal site, possibly allowing for longer-term functionality of existing clinical biomaterials.

Preclinical development of kinetin as a safe error-prone SARS-CoV-2 antiviral able to attenuate virus-induced inflammation.

Orally available antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are necessary because of the continuous circulation of new variants that challenge immunized individuals. Because severe COVID-19 is a virus-triggered immune and inflammatory dysfunction, molecules endowed with both antiviral and anti-inflammatory activity are highly desirable. We identified here that kinetin (MB-905) inhibits the in vitro replication of SARS-CoV-2 in human hepatic and pulmonary cell lines. On infected monocytes, MB-905 reduced virus replication, IL-6 and TNFα levels. MB-905 is converted into its triphosphate nucleotide to inhibit viral RNA synthesis and induce error-prone virus replication. Coinhibition of SARS-CoV-2 exonuclease, a proofreading enzyme that corrects erroneously incorporated nucleotides during viral RNA replication, potentiated the inhibitory effect of MB-905. MB-905 shows good oral absorption, its metabolites are stable, achieving long-lasting plasma and lung concentrations, and this drug is not mutagenic nor cardiotoxic in acute and chronic treatments. SARS-CoV-2-infected hACE-mice and hamsters treated with MB-905 show decreased viral replication, lung necrosis, hemorrhage and inflammation. Because kinetin is clinically investigated for a rare genetic disease at regimens beyond the predicted concentrations of antiviral/anti-inflammatory inhibition, our investigation suggests the opportunity for the rapid clinical development of a new antiviral substance for the treatment of COVID-19.

Ionizable Lipid Nanoparticle-Mediated Delivery of Plasmid DNA in Cardiomyocytes.

Introduction: Gene therapy is a promising approach to be applied in cardiac regeneration after myocardial infarction and gene correction for inherited cardiomyopathies. However, cardiomyocytes are crucial cell types that are considered hard-to-transfect. The entrapment of nucleic acids in non-viral vectors, such as lipid nanoparticles (LNPs), is an attractive approach for safe and effective delivery. Methods: Here, a mini-library of engineered LNPs was developed for pDNA delivery in cardiomyocytes. LNPs were characterized and screened for pDNA delivery in cardiomyocytes and identified a lead LNP formulation with enhanced transfection efficiency. Results: By varying lipid molar ratios, the LNP formulation was optimized to deliver pDNA in cardiomyocytes with enhanced gene expression in vitro and in vivo, with negligible toxicity. In vitro, our lead LNP was able to reach a gene expression greater than 80%. The in vivo treatment with lead LNPs induced a twofold increase in GFP expression in heart tissue compared to control. In addition, levels of circulating myeloid cells and inflammatory cytokines remained without significant changes in the heart after LNP treatment. It was also demonstrated that cardiac cell function was not affected after LNP treatment. Conclusion: Collectively, our results highlight the potential of LNPs as an efficient delivery vector for pDNA to cardiomyocytes. This study suggests that LNPs hold promise to improve gene therapy for treatment of cardiovascular disease.

Peptide fragments of bradykinin show unexpected biological activity not mediated by B1 or B2 receptors.

BACKGROUND AND PURPOSE: Bradykinin (BK-(1-9)) is an endogenous nonapeptide involved in multiple physiological and pathological processes. Peptide fragments of bradykinin are believed to be biologically inactive. We have now tested the two major peptide fragments of bradykinin in human and animals. EXPERIMENTAL APPROACH: BK peptides were quantified by MS in male rats. NO release was quantified from human, mouse and rat cells loaded with DAF-FM. Rat aortic rings were used to measure vascular reactivity. Changes in BP and HR were measured in conscious male rats. To evaluate pro-inflammatory effects both vascular permeability and nociception were measured in adult mice. KEY RESULTS: BK-(1-7) and BK-(1-5) are produced in vivo from BK-(1-9). Both peptides induced NO production in all cell types tested. However, unlike BK-(1-9), NO production elicited by BK-(1-7) or BK-(1-5) was not inhibited by B1 or B2 receptor antagonists. BK-(1-7) and BK-(1-5) induced concentration-dependent vasorelaxation of aortic rings, without involvement of B1 or B2 receptors. Intravenous or intra-arterial administration of BK-(1-7) or BK-(1-5) induced similar hypotensive response in vivo. Nociceptive responses of BK-(1-7) and BK-(1-5) were reduced compared to BK-(1-9), and no increase in vascular permeability was observed for BK-(1-9) fragments. CONCLUSIONS AND IMPLICATIONS: BK-(1-7) and BK-(1-5) are endogenous peptides present in plasma. BK-related peptide fragments show biological activity, not mediated by B1 or B2 receptors. These BK fragments could constitute new, active components of the kallikrein-kinin system.

Sulfonamide-Functionalized Polymeric Nanoparticles for Enhanced In Vivo Colorectal Cancer Therapy.

BACKGROUND: Colorectal cancer (CRC) is the third most common cancer in the world. 5- Fluorouracil (5-FU) is a conventional and most effective drug used in the clinic for the treatment of CRC. However, the clinical use of 5-FU is limited due to the acquired resistance and systemic toxicity, such as hepatotoxicity and gastrointestinal toxicity. OBJECTIVE: Recent advances in nanomedicine are being exploited to develop nanoparticle platforms to overcome resistance and therapeutic delivery of active molecules. Here, we developed 5-FU loaded sulfadiazine-poly(lactide-co-glycolide) nanoparticles (SUL-PLGA NPs) to be applied in the colorectal cancer model. METHODS: We assessed the in vivo efficacy of the SUL-PLGA NPs to enhance the antitumor effect of 5-FU. RESULTS: In vivo treatment with 5-FU-SUL-PLGA NPs significantly reduced tumor growth in a colon cancer xenograft model compared to free 5-FU and 5-FU loaded non-targeted NPs. Treatment with 5-FU-SUL-PLGA NPs also increased blood vessel diameters within tumors, which could act in conjunction to enhance antitumor efficacy. In addition, 5-FU-SUL-PLGA NPs significantly reduced liver mass and lung mass, which are the most common metastasis sites of CRC, and decreased liver hepatotoxicity compared to free 5-FU drug and 5-FU loaded non-targeted NPs. CONCLUSION: Our findings suggest that the use of 5-FU-SUL-PLGA NPs is a promising strategy to enhance 5-FU efficacy against CRC.

A Biosafety Level 2 Mouse Model for Studying Betacoronavirus-Induced Acute Lung Damage and Systemic Manifestations.

The emergence of life-threatening zoonotic diseases caused by betacoronaviruses, including the ongoing coronavirus disease 19 (COVID-19) pandemic, has highlighted the need for developing preclinical models mirroring respiratory and systemic pathophysiological manifestations seen in infected humans. Here, we showed that C57BL/6J wild-type mice intranasally inoculated with the murine betacoronavirus murine hepatitis coronavirus 3 (MHV-3) develop a robust inflammatory response leading to acute lung injuries, including alveolar edema, hemorrhage, and fibrin thrombi. Although such histopathological changes seemed to resolve as the infection advanced, they efficiently impaired respiratory function, as the infected mice displayed restricted lung distention and increased respiratory frequency and ventilation. Following respiratory manifestation, the MHV-3 infection became systemic, and a high virus burden could be detected in multiple organs along with morphological changes. The systemic manifestation of MHV-3 infection was also marked by a sharp drop in the number of circulating platelets and lymphocytes, besides the augmented concentration of the proinflammatory cytokines interleukin 1 beta (IL-1ß), IL-6, IL-12, gamma interferon (IFN-γ), and tumor necrosis factor (TNF), thereby mirroring some clinical features observed in moderate and severe cases of COVID-19. Importantly, both respiratory and systemic changes triggered by MHV-3 infection were greatly prevented by blocking TNF signaling, either via genetic or pharmacologic approaches. In line with this, TNF blockage also diminished the infection-mediated release of proinflammatory cytokines and virus replication of human epithelial lung cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Collectively, results show that MHV-3 respiratory infection leads to a large range of clinical manifestations in mice and may constitute an attractive, lower-cost, biosafety level 2 (BSL2) in vivo platform for evaluating the respiratory and multiorgan involvement of betacoronavirus infections. IMPORTANCE Mouse models have long been used as valuable in vivo platforms to investigate the pathogenesis of viral infections and effective countermeasures. The natural resistance of mice to the novel betacoronavirus SARS-CoV-2, the causative agent of COVID-19, has launched a race toward the characterization of SARS-CoV-2 infection in other animals (e.g., hamsters, cats, ferrets, bats, and monkeys), as well as adaptation of the mouse model, by modifying either the host or the virus. In the present study, we utilized a natural pathogen of mice, MHV, as a prototype to model betacoronavirus-induced acute lung injure and multiorgan involvement under biosafety level 2 conditions. We showed that C57BL/6J mice intranasally inoculated with MHV-3 develops severe disease, which includes acute lung damage and respiratory distress that precede systemic inflammation and death. Accordingly, the proposed animal model may provide a useful tool for studies regarding betacoronavirus respiratory infection and related diseases.

Development of sulfadiazine-decorated PLGA nanoparticles loaded with 5-fluorouracil and cell viability.

The aim of this work was to synthesize sulfadiazine-poly(lactide-co-glycolide) (SUL-PLGA) nanoparticles (NPs) for the efficient delivery of 5-fluorouracil to cancer cells. The SUL-PLGA conjugation was assessed using FTIR, 1H-NMR, 13C-NMR, elemental analysis and TG and DTA analysis. The SUL-PLGA NPs were characterized using transmission and scanning electron microscopy and dynamic light scattering. Additionally, the zeta potential, drug content, and in vitro 5-FU release were evaluated. We found that for the SUL-PLGA NPs, Dh = 114.0 nm, ZP = -32.1 mV and the encapsulation efficiency was 49%. The 5-FU was released for up to 7 days from the NPs. Cytotoxicity evaluations of 5-FU-loaded NPs (5-FU-SUL-PLGA and 5-FU-PLGA) on two cancer cell lines (Caco-2, A431) and two normal cell lines (fibroblast, osteoblast) were compared. Higher cytotoxicity of 5-FU-SUL-PLGA NPs were found to both cancer cell lines when compared to normal cell lines, demonstrating that the presence of SUL could significantly enhance the cytotoxicity of the 5-FU-SUL-PLGA NPs when compared with 5-FU-PLGA NPs. Thus, the development of 5-FU-SUL-PLGA NPs to cancer cells is a promising strategy for the 5-FU antitumor formulation in the future.

New perspectives for leishmaniasis chemotherapy over current anti-leishmanial drugs: a patent landscape.

INTRODUCTION: Although leishmaniasis is estimated to cause the ninth largest disease burden among individual infectious diseases, it is still one of the most neglected diseases in terms of drug development. Current drugs are highly toxic, resistance is common and compliance of patients to treatment is low, as treatment is long and drug price is high. AREAS COVERED: In this review, the authors carried out a patent landscape in search for new perspectives for leishmaniasis therapy. This search encompassed patent documents having priority date between 1994 and 2014. Selected compounds were compared to current anti-leishmanial drugs regarding efficacy and toxicity, when experimental data were available. EXPERT OPINION: Most patents related to drugs for leishmaniasis have not been produced by the pharmaceutical industry but rather by public research institutes or by universities, and the majority of the inventions disclosed are still in preclinical phase. There is an urgent need to find new ways of funding research for leishmaniasis drugs, incentivizing product development partnerships and pushing forward innovation.