Inactivation of the Complement Lectin Pathway by Candida tropicalis Secreted Aspartyl Protease-1.

Candida tropicalisis an opportunistic fungal pathogen and is one of the most frequently isolated non-albicans species. It can cause localised as well as invasive systemic infections particularly in immunocompromised patients. Increased resistance to common anti-fungal drugs is an emerging problem. In order to establish disseminated infections, Candida has evolved several strategies to escape the host immune system. A detailed understanding of how C. tropicalis escapes the host immune attack is needed as it can help develop novel anti-fungal therapies. Secreted aspartyl proteinases (Saps) of C. albicans have been shown to be determinants of virulence and immune evasion. However, the immune evasion properties of C. tropicalis Saps have been poorly characterised. This study investigated the immune evasion properties of C. tropicalis secreted aspartic protease 1 (Sapt1).Sapt1 was recombinantly produced using a Kluyveromyces lactis yeast expression system. A range of complement proteins and immunogloublins were screened to test if Sapt1 had any proteolytic activity. Sapt1 efficiently cleaved human mannose-binding lectin (MBL) and collectin-11, which are the initiating molecules of the lectin pathway of the complement system, but not l-ficolin. In addition, Sapt1 cleaved DC-SIGN, the receptor on antigen presenting dendritic cells. Proteolysis was prominent in acidic condition (pH 5.2), a characteristic of aspartyl protease. No proteolytic activity was detected against complement proteins C1q, C3, C3b, IgG and IgA. In view of the ability of Sapt1 to cleave MBL and collectin-11, we found that Sapt1 could prevent activation of the complement lectin pathway. RT-qPCR analysis using three different C. tropicalis clinical isolates (oral, blood and peritoneal dialysis fluid) revealed relatively higher levels of mRNA expression of Sapt1 gene when compared to a reference strain; Sapt1 protein was found to be secreted by all the tested strains. Lectin pathway and its initiating components are crucial to provide front line defence against Candida infections. For the first time, we have shown that a Candida protease can proteolytically degrade the key initiating components of lectin pathway and inhibit complement activation. Findings from this study highlight the importance of exploring Sapt1 as a potential therapeutic target. We conclude that C. tropicalis secretes Sapt1 to target the complement lectin pathway, a key pattern recognition and clearance mechanism, for its survival and pathogenesis.

Generation of a Universal Human Complement Source by Large-Scale Depletion of IgG and IgM from Pooled Human Plasma.

Complement is a key component of functional immunological assays used to evaluate vaccine-mediated immunity to a range of bacterial and viral pathogens. However, standardization of these assays is complicated due to the availability of a human complement source that lacks existing antibodies acquired either through vaccination or natural circulation of the pathogen of interest. We have developed a method for depleting both IgG and IgM in 200 mL batches from pooled hirudin-derived human plasma by sequential affinity chromatography using a Protein G Sepharose column followed by POROS™ CaptureSelect™ IgM Affinity resin. The production of large IgG- and IgM-depleted batches of human plasma that retains total hemolytic and alternative pathway activities allows for improved assay standardization and comparison of immune responses in large clinical trials.

Immunological and pathological outcomes of SARS-CoV-2 challenge following formalin-inactivated vaccine in ferrets and rhesus macaques.

There is an urgent requirement for safe and effective vaccines to prevent COVID-19. A concern for the development of new viral vaccines is the potential to induce vaccine-enhanced disease (VED). This was reported in several preclinical studies with both SARS-CoV-1 and MERS vaccines but has not been reported with SARS-CoV-2 vaccines. We have used ferrets and rhesus macaques challenged with SARS-CoV-2 to assess the potential for VED in animals vaccinated with formaldehyde-inactivated SARS-CoV-2 (FIV) formulated with Alhydrogel, compared to a negative control vaccine. We showed no evidence of enhanced disease in ferrets or rhesus macaques given FIV except for mild transient enhanced disease seen 7 days after infection in ferrets. This increased lung pathology was observed at day 7 but was resolved by day 15. We also demonstrate that formaldehyde treatment of SARS-CoV-2 reduces exposure of the spike receptor binding domain providing a mechanistic explanation for suboptimal immunity.

ChAdOx1 nCoV-19 protection against SARS-CoV-2 in rhesus macaque and ferret challenge models.

Vaccines against SARS-CoV-2 are urgently required, but early development of vaccines against SARS-CoV-1 resulted in enhanced disease after vaccination. Careful assessment of this phenomena is warranted for vaccine development against SARS CoV-2. Here we report detailed immune profiling after ChAdOx1 nCoV-19 (AZD1222) and subsequent high dose challenge in two animal models of SARS-CoV-2 mediated disease. We demonstrate in rhesus macaques the lung pathology caused by SARS-CoV-2 mediated pneumonia is reduced by prior vaccination with ChAdOx1 nCoV-19 which induced neutralising antibody responses after a single intramuscular administration. In a second animal model, ferrets, ChAdOx1 nCoV-19 reduced both virus shedding and lung pathology. Antibody titre were boosted by a second dose. Data from these challenge models on the absence of enhanced disease and the detailed immune profiling, support the continued clinical evaluation of ChAdOx1 nCoV-19.

Quantification of SARS-CoV-2 neutralizing antibody by wild-type plaque reduction neutralization, microneutralization and pseudotyped virus neutralization assays.

Virus neutralization assays measure neutralizing antibodies in serum and plasma, and the plaque reduction neutralization test (PRNT) is considered the gold standard for measuring levels of these antibodies for many viral diseases. We have developed procedures for the standard PRNT, microneutralization assay (MNA) and pseudotyped virus neutralization assay (PNA) for severe acute respiratory syndrome coronavirus 2. The MNA offers advantages over the PRNT by reducing assay time, allowing increased throughput and reducing operator workload while remaining dependent upon the use of wild-type virus. This ensures that all severe acute respiratory syndrome coronavirus 2 antigens are present, but Biosafety Level 3 facilities are required. In addition to the advantages of MNA, PNA can be performed with lower biocontainment (Biosafety Level 2 facilities) and allows for further increases in throughput. For each new vaccine, it is critical to ensure good correlation of the neutralizing activity measured using PNA against the PRNT or MNA. These assays have been used in the development and licensure of the ChAdOx1 nCoV-19 (AstraZeneca; Oxford University) and Ad26.COV2.S (Janssen) coronavirus disease 2019 vaccines and are critical for demonstrating bioequivalence of future vaccines.

Comparison of rhesus and cynomolgus macaques as an infection model for COVID-19.

A novel coronavirus, SARS-CoV-2, has been identified as the causative agent of the current COVID-19 pandemic. Animal models, and in particular non-human primates, are essential to understand the pathogenesis of emerging diseases and to assess the safety and efficacy of novel vaccines and therapeutics. Here, we show that SARS-CoV-2 replicates in the upper and lower respiratory tract and causes pulmonary lesions in both rhesus and cynomolgus macaques. Immune responses against SARS-CoV-2 are also similar in both species and equivalent to those reported in milder infections and convalescent human patients. This finding is reiterated by our transcriptional analysis of respiratory samples revealing the global response to infection. We describe a new method for lung histopathology scoring that will provide a metric to enable clearer decision making for this key endpoint. In contrast to prior publications, in which rhesus are accepted to be the preferred study species, we provide convincing evidence that both macaque species authentically represent mild to moderate forms of COVID-19 observed in the majority of the human population and both species should be used to evaluate the safety and efficacy of interventions against SARS-CoV-2. Importantly, accessing cynomolgus macaques will greatly alleviate the pressures on current rhesus stocks.

Dose-dependent response to infection with SARS-CoV-2 in the ferret model and evidence of protective immunity.

There is a vital need for authentic COVID-19 animal models to enable the pre-clinical evaluation of candidate vaccines and therapeutics. Here we report a dose titration study of SARS-CoV-2 in the ferret model. After a high (5 × 106 pfu) and medium (5 × 104 pfu) dose of virus is delivered, intranasally, viral RNA shedding in the upper respiratory tract (URT) is observed in 6/6 animals, however, only 1/6 ferrets show similar signs after low dose (5 × 102 pfu) challenge. Following sequential culls pathological signs of mild multifocal bronchopneumonia in approximately 5-15% of the lung is seen on day 3, in high and medium dosed groups. Ferrets re-challenged, after virus shedding ceased, are fully protected from acute lung pathology. The endpoints of URT viral RNA replication & distinct lung pathology are observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease.

Amphibious fish jump better on land after acclimation to a terrestrial environment.

Air and water differ dramatically in density and viscosity, posing different biomechanical challenges for animal locomotion. We asked how terrestrial acclimation influences locomotion in amphibious fish, specifically testing the hypothesis that terrestrial tail flip performance is improved by plastic changes in the skeletal muscle. Mangrove rivulus Kryptolebias marmoratus, which remain largely inactive out of water, were exposed to water or air for 14 days and a subgroup of air-exposed fish was also recovered in water. Tail flip jumping performance on land improved dramatically in air-acclimated fish, they had lower lactate levels compared with control fish, and these effects were mostly reversible. Muscle plasticity significantly increased oxidative muscle cross-sectional area and fibre size, as well as the number of capillaries per fibre. Our results show that reversible changes to the oxidative skeletal muscle of K. marmoratus out of water enhance terrestrial locomotory performance, even in the absence of exercise training.