A booster regime of liposome-delivered live-attenuated CHIKV vaccine RNA genome protects against chikungunya virus disease in mice.

Mosquito-transmitted chikungunya virus (CHIKV) is the causal pathogen of CHIKV disease and is responsible for global epidemics of arthritic disease. CHIKV infection can lead to severe chronic and debilitating arthralgia, significantly impacting patient mobility and quality of life. Our previous studies have shown a live-attenuated CHIKV vaccine candidate, CHIKV-NoLS, to be effective in protecting against CHIKV disease in mice vaccinated with one dose. Further studies have demonstrated the value of a liposome RNA delivery system to deliver the RNA genome of CHIKV-NoLS directly in vivo, promoting de novo production of live-attenuated vaccine particles in vaccinated hosts. This system, designed to bypass live-attenuated vaccine production bottlenecks, uses CAF01 liposomes. However, one dose of CHIKV-NoLS CAF01 failed to provide systemic protection against CHIKV challenge in mice, with low levels of CHIKV-specific antibodies. Here we describe CHIKV-NoLS CAF01 booster vaccination regimes designed to increase vaccine efficacy. C57BL/6 mice were vaccinated with three doses of CHIKV-NoLS CAF01 either intramuscularly or subcutaneously. CHIKV-NoLS CAF01 vaccinated mice developed a systemic immune response against CHIKV that shared similarity to vaccination with CHIKV-NoLS, including high levels of CHIKV-specific neutralising antibodies in subcutaneously inoculated mice. CHIKV-NoLS CAF01 vaccinated mice were protected against disease signs and musculoskeletal inflammation when challenged with CHIKV. Mice given one dose of live-attenuated CHIKV-NoLS developed a long lasting protective immune response for up to 71 days. A clinically relevant CHIKV-NoLS CAF01 booster regime can overcome the challenges faced by our previous one dose strategy and provide systemic protection against CHIKV disease.

Altered Spatial and Temporal Gait Parameters in Mice Infected with Ross River Virus.

Infection with mosquito-borne arthritogenic alphaviruses, such as Ross River virus (RRV) and Barmah Forest virus (BFV), can lead to long-lasting rheumatic disease. Existing mouse models that recapitulate the disease signs and immunopathogenesis of acute RRV and BFV infection have consistently shown relevance to human disease. However, these mouse models, which chiefly model hindlimb dysfunction, may be prone to subjective interpretation when scoring disease. Assessment is therefore time-consuming and requires experienced users. The DigiGait system provides video-based measurements of movement, behavior, and gait dynamics in mice and small animals. Previous studies have shown DigiGait to be a reliable system to objectively quantify changes in gait in other models of pain and inflammation. Here, for the first time, we determine measurable differences in the gait of mice with infectious arthritis using the DigiGait system. Statistically significant differences in paw area and paw angle were detected during peak disease in RRV-infected mice. Significant differences in temporal gait parameters were also identified during the period of peak disease in RRV-infected mice. These trends were less obvious or absent in BFV-infected mice, which typically present with milder disease signs than RRV-infected mice. The DigiGait system therefore provides an objective model of variations in gait dynamics in mice acutely infected with RRV. DigiGait is likely to have further utility for murine models that develop severe forms of infectious arthritis resulting in hindlimb dysfunction like RRV. IMPORTANCE Mouse models that accurately replicate the immunopathogenesis and clinical disease of alphavirus infection are vital to the preclinical development of therapeutic strategies that target alphavirus infection and disease. Current models rely on subjective scoring made through experienced observation of infected mice. Here, we demonstrate how the DigiGait system, and interventions on mice to use this system, can make an efficient objective assessment of acute disease progression and changes in gait in alphavirus-infected mice. Our study highlights the importance of measuring gait parameters in the assessment of models of infectious arthritis.

Liposomal Delivery of the RNA Genome of a Live-Attenuated Chikungunya Virus Vaccine Candidate Provides Local, but Not Systemic Protection After One Dose.

Chikungunya virus (CHIKV) is the causative pathogen of chikungunya fever, a mosquito-borne viral disease causing highly debilitating arthralgia that can persist for months and progress to chronic arthritis. Our previous studies have identified the CHIKV live-attenuated vaccine candidate CHIKV-NoLS. Like most live-attenuated vaccines, attenuated replication of CHIKV-NoLS has the potential to limit scalable production. To overcome production limits, as well as other drawbacks of live-attenuated vaccines, we developed an in vivo liposome RNA delivery system to deliver the self-replicating RNA genome of CHIKV-NoLS directly into mice, allowing the recipients' body to produce the live-attenuated vaccine particles. CAF01 liposomes were able to deliver replication-competent CHIKV-NoLS RNA in vitro. Immunodeficient AG129 mice inoculated with liposome-delivered CHIKV-NoLS RNA developed viremia and disease signs representative of this lethal model of CHIKV infection, demonstrating de novo vaccine particle production in vivo. In immunocompetent C57BL/6 mice, liposome-delivered CHIKV-NoLS RNA inoculation was associated with reduced IgM and IgG levels with low antibody CHIKV-neutralizing capacity, compared to vaccination with the original live-attenuated vaccine CHIKV-NoLS. One dose of liposome-delivered CHIKV-NoLS RNA did not provide systemic protection from CHIKV wild-type (WT) challenge but was found to promote an early onset of severe CHIKV-induced footpad swelling. Liposome-delivered CHIKV-NoLS RNA inoculation did, however, provide local protection from CHIKV-WT challenge in the ipsilateral foot after one dose. Results suggest that in the presence of low CHIKV-specific neutralizing antibody levels, local inflammatory responses, likely brought on by liposome adjuvants, have a role in the protection of CHIKV-induced footpad swelling in the ipsilateral foot of mice inoculated with liposome-delivered CHIKV-NoLS RNA. Low IgG and CHIKV-specific neutralizing antibody levels may be responsible for early onset of severe swelling in the feet of CHIKV-WT-challenged mice. These results support previous studies that suggest CHIKV is vulnerable to antibody-mediated enhancement of disease. Further studies using booster regimes aim to demonstrate the potential for liposomes to deliver the self-replicating RNA genome of live-attenuated vaccines and offer a novel immunization strategy.

Arthritogenic Alphavirus-Induced Immunopathology and Targeting Host Inflammation as A Therapeutic Strategy for Alphaviral Disease.

Arthritogenic alphaviruses are a group of medically important arboviruses that cause inflammatory musculoskeletal disease in humans with debilitating symptoms, such as arthralgia, arthritis, and myalgia. The arthritogenic, or Old World, alphaviruses are capable of causing explosive outbreaks, with some viruses of major global concern. At present, there are no specific therapeutics or commercially available vaccines available to prevent alphaviral disease. Infected patients are typically treated with analgesics and non-steroidal anti-inflammatory drugs to provide often inadequate symptomatic relief. Studies to determine the mechanisms of arthritogenic alphaviral disease have highlighted the role of the host immune system in disease pathogenesis. This review discusses the current knowledge of the innate immune response to acute alphavirus infection and alphavirus-induced immunopathology. Therapeutic strategies to treat arthritogenic alphavirus disease by targeting the host immune response are also examined.

Attenuation and Stability of CHIKV-NoLS, a Live-Attenuated Chikungunya Virus Vaccine Candidate.

Our previous investigation of the nucleolar localisation sequence (NoLS) of chikungunya virus (CHIKV) capsid protein demonstrated the role of capsid in CHIKV virulence. Mutating the NoLS of capsid in CHIKV led to the development of a unique live-attenuated CHIKV vaccine candidate, termed CHIKV-NoLS. CHIKV-NoLS-immunised mice developed long-term immunity from CHIKV infection after a single dose. To further evaluate CHIKV-NoLS attenuation and suitability as a vaccine, we examined the footpad of inoculated mice for underlying CHIKV-NoLS-induced immunopathology by histological and flow cytometric analysis. In comparison to CHIKV-WT-infected mice, CHIKV-NoLS-inoculated mice exhibited minimal inflammation and tissue damage. To examine the stability of attenuation, the plaque phenotype and replication kinetics of CHIKV-NoLS were determined following extended in vitro passage. The average plaque size of CHIKV-NoLS remained notably smaller than CHIKV-WT after extended passage and attenuated replication was maintained. To examine thermostability, CHIKV-NoLS was stored at 21 °C, 4 °C, -20 °C and -80 °C and infectious CHIKV-NoLS quantified up to 84 days. The infectious titre of CHIKV-NoLS remains stable after 56 days when stored at either -20 °C or -80 °C. Interestingly, unlike CHIKV-WT, the infectious titre of CHIKV-NoLS is not sensitive to freeze thaw cycles. These data further demonstrate preclinical safety and stability of CHIKV-NoLS.

Terminalia bellirica fruit extracts: in-vitro antibacterial activity against selected multidrug-resistant bacteria, radical scavenging activity and cytotoxicity study on BHK-21 cells.

BACKGROUND: Identification of novel sources for developing new antibiotics is imperative with the emergence of antibiotic resistant bacteria. The fruits of Terminalia bellirica (Gaertn) Roxb., widely used in traditional medicine, were evaluated for antibacterial activity against multidrug-resistant (MDR) bacteria, antioxidant activity and cytotoxicity. METHODS: Twelve solvent extracts of T. bellirica fruits were prepared by direct aqueous extraction and sequential extraction with dichloromethane, methanol and water using Soxhlet, bottle-shaker and ultrasound sonicator methods. Antibacterial activity of the extracts was tested against 16 strains MDR bacteria-methicillin-resistant Staphylococcus aureus (MRSA), extended spectrum ß-lactamase (ESBL) producing Escherichia coli and MDR Acinetobacter spp., Klebsiella pneumoniae and Pseudomonas aeruginosa-and 4 control organisms, using the cut-well diffusion method. The minimum inhibitory concentration (MIC) was determined using an agar dilution method. The radical scavenging activity of six antibacterial extracts was screened against 2,2'-diphenyl-2-picrylhydrazyl (DPPH) and correlation was established between EC50 (50% effective concentration) values and the total phenolic content (TPC). Cytotoxicity was determined for the most potent antibacterial extract on baby hamster kidney (BHK-21) cells by Tryphan Blue exclusion method. Statistical analysis was carried out by one-way analysis of variance at significant level p < 0.05 using "SigmaPlot 10" and "R 3.2.0" software. RESULTS: All aqueous and methanol extracts displayed antibacterial activity (MIC 0.25-4 mg/mL) against all strains of MRSA, MDR Acinetobacter spp. and MDR P. aeruginosa. The sequential aqueous extracts (MIC, 4 mg/mL) inhibited ESBL producing-E. coli. None of the extracts exhibited activity against MDR K. pneumoniae (MIC > 5 mg/mL). The sequential methanol extract (Soxhlet) recorded high antibacterial activity and the highest DPPH radical scavenging activity (EC50, 6.99 ± 0.15 ppm) and TPC content (188.71 ± 2.12 GAE mg/g). The IC50 (50% inhibition concentration) values of the most potent antibacterial extract-the direct aqueous extract from reflux method-on BHK-21 cells were 2.62 ± 0.06 and 1.45 ± 0.08 mg/ml with 24 and 48 h exposure, respectively. CONCLUSIONS: Results indicate that T. bellirica fruit is a potential source for developing broad-spectrum antibacterial drugs against MDR bacteria, which are non-toxic to mammalian cells and impart health benefits by high antioxidant activity.