Infectious Disease Models available at Aragen Bioscience
- Clostridium difficile-associated diarrhoea model in hamsters and mice
- Mouse cytomegalovirus (mCMV)
- Respiratory syncytial virus (RSV) in mice and cotton rats
- Immunogenicity studies to evaluate vaccine candidates
- Mouse coronavirus MV-A59
- Citrobacter-induced colitis in mice
As per CDC, human respiratory syncytial virus (hRSV) is a common respiratory virus that usually causes mild, cold-like symptoms. RSV and its chimeric strains affect infants, adults, elderly worldwide and cause significant morbidity. Aragen Bioscience continues to serve pharmaceutical, biotechnology and SMEs in their preclinical efficacy and safety studies. Appropriate in vivo rodent models enable development of anti-RSV antibodies, small molecules, and vaccines for the treatment of respiratory disease.
Presently, there is no targeted treatment available for human respiratory syncytial virus (hRSV) infection other than the supportive care to mitigate the signs of and symptoms. Antiviral drug development and RSV vaccine is expected to play a critical role in overall reduction of RSV infections globally. Continued effort in RSV research published in high profile scientific journals has invigorated efforts by larger and smaller pharmaceutical and biotechnology companies to develop antivirals and RSV vaccines. Preclinical development of vaccines is hindered by the lack of clinically relevant rodent models. More importantly, the safety of new vaccines, preclinical pharmacological, safety and toxicology studies should be conducted prior to initiating the clinical studies. Aragen Bioscience will enable successful IND-enabling preclinical RSV therapeutic development studies by providing appropriate study designs, right animal models, and confirming effective immune response by performing in vivo and in vitro assays. Our scientists have and will continue to establish the identity, purity, safety, and potency of the vaccine or antivirals. In this white paper, we present several case studies performed in clinically relevant mouse and rat models to assess the critical characteristics of potential RSV therapeutics. In summary, this article includes in-house as well as client-sponsored study data developed over several years using rodent models. These studies generated clinically pertinent information for proper screening of new vaccines and antivirals. Aragen Bioscience will continue to support your RSV therapeutic development effort by providing reliable and effective preclinical services in this important infectious disease area.
Model for anti-RSV therapeutic development.
We offer preclinical efficacy and safety studies using appropriate in vivo rodent models to enable development of anti-RSV antibodies, small molecules, and vaccines.
What is Cotton Rat model for RSV?
The RSV mouse model is the preferred choice for most preclinical immunological studies, ranging from simple vaccine testing to the intricate assessment of fundamental immunopathogenic responses. Therefore, Cotton Rat model is one of the most clinically relevant models for anti RSV development.
How does our model work?
- Cotton Rat received from Aragen Bioscience-approved vendor, housed in a dedicated room
- Clinical parameters measured
- Weekly body weights measured
- Daily observations for morbidity and ambulatory discomfort
- QCed by operators
Female Cotton rats 6-8 weeks old will be infected with RSV-A2 Long (ATCC: VR-26), intranasally on day 2, observed daily for clinical signs. Test vaccine (day 0 and 28) and RSV challenge (day49). Bleeds on day 28, 49 and 53 for vaccine and RSV testing and tissues harvested on day 53 for ex vivo analyses.
- Lung weights, lung and nose homogenization with viral plaque assay
- qPCR, VNA, ELISPOT, Cytokine assays, BAL related assays
Advantages of Aragen Bioscience’s RSV platform and our Cotton Rat model:
- Experts with several years of expertise, delivered over 100 successful RSV studies (30-150 animals/study)
- Clinically relevant analysis- Imaging, biochemistry, and histopathology by our highly skilled staff
- Flexible and customizable assays in RSV models. We have tested small molecules, large molecule, and vaccines for past and existing clients/ partners
- Willing and able to develop models as per client specification with customized studies
- Useful for both RSV-A and RSV-B strains
- Clinically relevant model used for Synagis® approval
- Model for FI-RSV induced enhanced disease
- USDA regulated
Cotton rat is considered a more relevant animal model for preclinical studies on RSV infection than BALB/c mice. Consequently, cotton rats are used to study RSV pathogenesis, anti-RSV drugs, and RSV vaccine efficacy and safety. For example, the cotton rat model was used for pre-clinical evaluation of unglycosylatedrecombinantE. coliproducedGprotein (REG) as a potential RSV vaccine (3).A preclinical study showed that bacterially produced REG could provide an economical, safe and effective broad protective vaccine against RSV disease (3).
BALB/C Small Molecule – Study Design
To evaluate the efficacy of XXX antibody (drug) product in comparison toSYNAGIS (reference drug),in limiting viral replication of RSV (Strain A2) in lungs of female cotton rats.SYNAGIS is a prescription medication that is used to help prevent a serious lung disease caused by RSV in children.
Study Design :
Thirty-five cotton rats (approximately 6-8 weeks old) separated into 7 groups (N=5/ group).
Gp1: Test Article (4mg/kg); Gp2: Test Article (2mg/kg); Gp3: Test Article (1mg/kg); Gp4: Test Article (0.5mg/kg); Gp5: Synagis® 4mg/kg; Gp6: Synagis® 2mg/kg; Gp7: PBS
On day -1, rats received a prophylactic intramuscular injection of test article at 4 mg/kg, 2 mg/kg, 1 mg/kg or 0.5 mg/kg or they received a prophylactic intramuscular injection of the control antibody, Synagis® at 4 mg/kg or 2 mg/kg. On Day 0, all animals were inoculated intranasally with 1×105PFU of RSV strain A2. On day 4, serum, nose and lungs were collected following euthanasia and the viral lung titers were determined by plaque assay.
The test products and Synagis® exhibited dose-dependent antiviral activity in preventing RSV replication in the lungs of cotton rats infected with RSV A2. And treatment with test products (0.5 mg/kg, 1 mg/kg, 2 mg/kg or 4 mg/kg) significantly decreased viral lung titers compared to treatment with PBS (p<0.001). Furthermore, the test products decreased viral lung titers on average 16-fold more than the same dose of Synagis® (4 mg/kg group:p=0.021; 2mg/kg group:p<0.001).
Timely completion of the project to the full satisfaction of the Client
Female BALB/c: 6 weeks old Intranasally injected with RSV strain: RSV-A2 Long (ATCC: VR-26)LeftPost SYNAGIS® treatment on day 3, clinical observations done daily until tissue harvest on day 5 (Right)
Female BALB/c: 6 weeks old were intranasally infected with RSV strain-RSV-A2 Long (ATCC: VR-26). The peak RSV replication was seen on Day 5 post infection. Live virus was not detected in lungs on day 10 post infection. However, viral RNA was detected at day 12 post infection.
Female BALB/c 6-8 weeks old mice were intranasally infection with RSV-A2 (expanded from RSV-A2 ATCC stock (VR-1540), , tissue harvested on day 5 and Bio-burden investigated.
Transient weight loss in female BALB/c, 6-8 weeks old mice intranasally transfected with varying doses of RSV measured and on day 5, lung and nasal tissues were harvested to measure RSV burden.
Aragen Bioscience participated in a WHO collaborative study to establish the 1st International Standard for antiserum to RSV. A positive control standard run on every plate in microneutralization assay. Options for microneutralization or plaque reduction assay was adapted to develop in- house or client’s protocols.
BAL cell counts and BALF MSD analysis using U-plex kit were performed in untreated and treated (Ribavirin) mice. Decrease in lung weights were slightly increased and RSV titter decreased in Ribavirin treated RSV infected mice compared to placebo (mock) group.
Female BALB/c: 6-8 weeks old were infected intranasally with RSV-A2, BAL harvested on day 5 and 7, and MSD analyzed in BAL fluid. Expression of proinflammatory markers after RSV infection are shown in the following figures.
Female BALB/c: 6-8 weeks old, were intranasally infected with RSV A2, BAL harvested on day 5 and 7, and MSD analysis performed in BAL fluid. Expression of cytokine and Th17 markers after RSV infection are shown in the following figures.
Cotton Rat RSV Model for Vaccine development
- Useful for both RSV-A and RSV-B strains
- Clinically relevant model used for Synagis® approval
- Model for FI-RSV induced enhanced disease
- USDA regulated
Ex vivo Assays performed:
- RSV titers from lungs and nasal turbinate
- Lung weights and other organ weights, snap frozen or fixed for histology
- qPCR analysis of RSV viral transcripts in lungs or other organs
- Virus neutralization assays, either client specified or in-house protocols
- ELISA serum analysis
- Cytokine analysis on BAL or lung homogenates
- ELISpot assays for B and T cell analysis
- BAL fluid collection with total leukocyte counts and differentials
- Serum or plasma
- Serum chemistry and whole blood differentials
Female Cotton rats: 6-8 weeks old were intranasally infected with RSV-A2 (ATCC VR-1540), observed daily, harvested lungs and noses on day 4, 5 or 6 and viral plaque assay performed in homogenized tissue. Result: Peak RSV replication was observed on 4-5 days post infection.
Female Cotton rats 6-8 weeks old were infected with RSV-A2 Long (ATCC: VR-26), intranasally on day 2, observed daily for clinical signs, tissue harvested on day 5 to measure RSV replication status and leukocyte counts. We observed thatSynagis®reduced RSV replication and leukocyte count in the lungs.
To measure the replication of RSV-A2 in immunocompromised mouse model, we intranasally injected placebo group with vehicle and test groups with RSV-A2 (1×105pfu), monitored body weight for over 14 days in different groups of mice before harvesting lung tissues for RSV measurement on days 6, 8, 10, 12 and 14 days. We observed that RSV-A2 replication was prolonged in SCID mice over 14 days compared to placebo (mock) group.
Female SCID mice, 6 weeks old were inoculated intranasally with RSV-A2 (1×105pfu), treated with Synagis® IP on days 2,4 and lung tissues harvested on day 14 for RSV burden analysis. We observed that RSV-A2 replication was prolonged and the administered doses of Synagis® provided partial protection against RSV infection.
Mouse Coronavirus Model (MHV-A59) development
Recent reports implicate SARS-CoV infection in causing lung fibrosis through multiple signaling pathways and TGF-β activation (5,6). Aragen Bioscience has developed a mouse corona virus model to study lung fibrosis, which most SARS-CoV-2 infected patients develop. This model can be studied with BSL-2 Containment to evaluate vaccines and antivirals for treating respiratory infection with Fibrotic effects.
We measured MHV-A59 viral load in lungs and in BAL cell counts (dpi) post infection with MHV-A59 (days 3-8) and analyzed 29 cytokines/chemokines. Viral load (TCID50) increased after 3 days post infection decreased on 6th and 8th day. Similar trend in BALF cell counts was seen after 3rd, 6th and 8th days. In this model, we observed that IL7 was significantly induced by MHV infection, which is like SARS-CoV-2-induced upregulation of critical cytokines often seen in COVID patients. Therefore, this mouse model serves as a surrogate SARS-CoV-2 model that can be studied in BSLII set up unlike BSLIII requirements for studies involving SARS CoV-2 models.
Changes in Body, Lung, Liver weights post MHA-A59 infection in mice
Cytokine profile changes seen in mice lung homogenates post MHA-159 infection
Cytokine profile changes seen in mice plasma post MHA-159 infection
Fibrosis -specific gene Expression in Lung Tissue
Fibrosis -specific gene Expression in Liver Tissue
Our rodent asthma models include induction by ova or other allergens (cedar pollen, dust mote antigen). Models might range from mild to severe disease, to evaluate a range of treatment options.
- Whole Body Plethysmograph (WBP)
- Airway hyper responsiveness
- Respiratory rate
- FlexiVent Lung Functional Analysis
- Abbott iSTAT
- Blood glass measurement to monitor hypoxia-related symptoms
- FlexiVent Lung Functional Analysis
- Detection of structures that contain high concentrations of carbohydrate macromolecules (eg. glycogen, glycoprotein, proteoglycan) typically found in mucus
- Serum and Bronchoalveolar Lavage Fluid Analysis
- Antigen-specific IgE and IgA levels
- Cytokines levels
Lipopolysaccharide/ Zymosan-induced Acute Lung Injury Model
A well characterized ALI model induced by co-administration of LPS and Zymosan for lung function analysis in bronchoalveolar lavage infiltrate, including cytokine analysis, hypoxia measurements and histopathology.
Bleomycin-induced Lung Fibrosis
Visit: Fibrosis – Aragen Bioscience
Our track record and experience in Fibrosis
Experience that counts: over 55 combined years of experience in fibrosis
Experience that delivers: over 600 successful fibrosis studies for 50+ customers
Stellar track record of success: 8 programs in clinical development, most advancement in phase II clinical trials
About Aragen Bioscience:
- Aragen Bioscience is a world leader in accelerating preclinical respiratory disease and fibrosis research.
- With over 600 fibrosis studies under our belt, our strengths lie in translational in vivo modelling of a variety of fibrotic diseases including pulmonary fibrosis.
- We have combined our strength in IPF with our strength in infectious diseases to help understand potential links between IPF and infectious respiratory diseases.
- With decades of experience in preclinical efficacy biology and discovery and development of biologics, we offer comprehensive solutions from concept to commercialization.
Animal Models of IPF
The most common preclinical animal model for IPF is the bleomycin-induced pulmonary fibrosis rodent model. Bleomycin induction causes inflammatory and fibrotic reactions within a short period of time in rodent lungs.
Advantage of the Aragen Bioscience Bleomycin-IPF Model
While the bleomycin-induced model for IPF is a useful model to study lung fibrosis, a key concern with this model is reproducibility, animal mortality, intracohort variability, and inconsistency in disease induction. With a dedicated team of fibrosis scientists, Aragen Bioscience has successfully established a highly reproducible model of IPF that has been used to evaluate many drug candidates and has helped accelerate several candidates to the clinic. We have established a robust bleomycin-induced model for IPF that shows consistent disease induction and has been used to develop both therapeutic and prophylactic candidates.
Variations of the Bleomycin-Induced Fibrosis Model
Aragen Bioscience has also developed bleomycin models of systemic sclerosis that develops fibrosis of both lung and skin.
Systemic/subcutaneous administration of bleomycin tends to produce more perivascular/subpleural fibrosis, while intratracheal bleomycin administration tends to cause peribronchial/bronchiolar (hilar) fibrosis.
Silica-Induced Lung Fibrosis Model
Aragen Bioscience has developed a silica-induced fibrosis mouse model. Mice were oro-pharyngeally instilled with crystalline silica or saline and longitudinally monitored with respiratory-gated-high-resolution µCT to evaluate disease onset and progress using scan-derived biomarkers. Silica-instillation increased the non-aerated lung volume, corresponding to onset and progression of inflammatory and fibrotic processes not resolving with time. Moreover, total lung volume progressively increased with silicosis. This model of non-resolving lung fibrosis provides quantitative assessment of disease progression and stabilization over weeks and months, essential towards evaluation of fibrotic disease burden and antifibrotic therapy evaluation in preclinical studies.
Preclinical Model for Acute Lung Injury: Lipopolysaccharide (LPS)-Induced Acute Lung Injury Model
The lipopolysaccharide (LPS)-induced ALI model in rodents is a very relevant in vivo model for ALI/ARDS. LPS-induction results in microvascular injury, edema, and diffuse alveolar damage with intrapulmonary characteristics that are similar to what is observed in patients with ALI/ARDS. Animals exposed to LPS display features of microvascular lung injury, including leukocyte accumulation in lung tissue, pulmonary edema, and profound lung inflammation. Similar to the bleomycin-induced model, there are multiple ex vivo analyses that can be performed in LPS-ALI model to establish the injury and determine the efficacy of test therapeutics.
Over the years, numerous agents have been shown to inhibit fibrosis in this model. It is critical to distinguish between drugs interfering with the inflammatory and early fibrogenic response from those preventing progression of fibrosis, the latter much more meaningful for clinical application. All potential antifibrotic compounds should be evaluated in the phase of established fibrosis rather than in the initial period of bleomycin-induced inflammation for assessment of its antifibrotic properties. Further care should be taken in extrapolation of drugs successfully tested in the bleomycin model due to partial reversibility of bleomycin-induced fibrosis over time in young and aged animals. The use of alternative and more robust animal models, which better reflect human IPF, are necessary.
Standard methodology: Fibrosis induced in C57BL/6 mice with infusion of clinical grade Bleomycin via oropharyngeal route or through an osmotic pump. Various routes of administration of test articles (PO, IP, IV, IM, SC, nebulization, and osmotic pumps) and option to treat animals therapeutically or prophylactically are available.
Standard readouts include body weight, survival, lung weight, leukocyte count in bronchoalveolar lavage (BAL) and lung fixation for histology (H&E and trichrome staining).
Fibrotic readouts include lung hydroxyproline, serum/BAL soluble mediators, lung FibroPanelTM Gene expression, lung fixation for histology (H&E and trichrome staining at third party CRO) and differentials from BAL cells. Lung measurements include flexiVentTM, hypoxia related parameters and whole-body plethysmography.
The graphs show the flexiVentTM analysis where bleomycin instillation increases resistance and elastance while decreasing compliance and enhanced Respiratory pause (Penh) increased by Bleomycin. Pirfenidone improved lung function measurements.
The following pictures show that we achieved highly reproducible and consistent fibrotic induction in lungs and pathology of tissues from six studies. Hence our established models allow for robust evaluation of candidate drugs.
Multiple studies were performed to achieve consistency in our methodology and the lung weight measurements as shown in the graphs below.
Bleomycin administration increased pro-inflammatory cytokines in group 2 (red) compared to no-bleomycin (control-blue) group. Pirfenidone treatment attenuated the cytokine response in group 3 (green) compared to bleomycin-administered group 2 (blue).
Bleomycin administration increased pro-inflammatory chemokines (MCP-1, MIP3a, MIP-2, IP-10) in group 2 (red) compared to no-bleomycin (control-blue) group. Pirfenidone treatment affected the chemokine response in group 3 (green) compared to groups 2 and 3.
Silica-induced Lung Fibrosis
A rodent model induced by the administration of crystalline silico dioxide (or silica) displays many pathophysiological features of chronic inflammation and pulmonary fibrosis.