Hepatitis B is a potentially deadly liver disease transmitted by the virus of hepatitis B (HBV). Globally, 2 billion people have contracted hepatitis B, according to WHO estimates, and approximately 1.5 million people get hepatitis B each year; the combined figure equals 800,000 deaths annually, largely from liver cirrhosis and hepatocellular carcinoma that occurs later in hepatitis B. The WHO estimates that some 4.5 million premature deaths could be prevented with the vaccine against hepatitis B by 2030. Preventive hepatitis B vaccines do much to avert HBV infection. Therapy vaccines are the best immunotherapies for hepatitis B. The effective hepatitis B vaccines have been created so far in recombinant protein vaccines, DNA vaccines, lipopeptide vaccines and adenovirus-vector vaccines. But no therapeutic hepatitis B vaccine exists for clinical use because the vast majority of recombinant protein and lipopeptide vaccines in use today target only HBsAg as a single target, and cannot induce multi-antigen immunity, so activating hyporeactive specific T cells left in the body. DNA vaccines have a short action time in the body and require the help of effective delivery vehicles to function. It is difficult for a single DNA vaccine to inhibit HBV DNA replication and induce a specific immune response. Although adenovirus-vector vaccines can produce potent levels of CD8⁺ T cells, it’s unclear how bad this vaccine is for the human body. The vaccine may become less immune-effective if the body produces specific antibodies to adenovirus upon vaccination. Therefore, the search for alternative technical pathways for therapeutic hepatitis B vaccines remains urgent.

While current evidence-based applications of preventive hepatitis B vaccine in HBsAg-cleared populations has demonstrated its safety and efficacy, larger, multi-center trials are far from being completed. When it comes to HBV, scientists have developed companion in vivo and in vitro models of HBV that meet the needs of their respective research objectives. Although flawed in many ways, these models have helped individuals to gain a better understanding of HBV and assisted in the creation and testing of novel medications to prevent and treat HBV.

HBV Animal Models

Non-human primate model

Non-human primates, including chimpanzees, gibbons and baboons, are susceptible to HBV infection. These vulnerable animals can be used to investigate the viability, natural infection and pathogenesis of HBV and the body’s immune clearance of the virus. However, there are limiting factors such as mild degree of virus infection, animal ethics and species endangerment. In order to establish animal models similar to HBV natural infection in humans, researchers have never stopped exploratory research on the macaques. These exploratory animal models play an important role in studying the mechanism of HBV infection and the host’s immune response during infection, but the scale is extremely limited and there is a lack of more confirmatory experiments on HBV infection.

Tree shrew model

Tree shrews are an animal model discovered after the above-mentioned primates that can be easily infected by HBV. After infection with HBV, they will experience temporary viremia, virus clearance process, and HBsAg seroconversion. The infection process is similar to that of humans, with a short infection cycle (about 40 days). In this type of model, tree shrews are mostly captured in the wild. The infection situation is related to the animal’s age, breed, HBV genotype, etc., and there is currently a lack of specific detection of hepatitis B antibodies for it, so the reliability, controllability, repeatability of this model and the test results are still controversial.

Mice model

Currently, the most commonly used hepatitis B animal model is mice, but HBV cannot directly infect mice. It’s easy to classify mature mouse models of HBV infection into two main types: the chimeric mouse model, and the most common genetically deficient mouse model. These mouse models are susceptible to HBV infection and are mostly used to study the immune response, virus replication, virus pathogenesis, etc. HBV infection) and to screen and test anti-HBV medications. However, they have shortcomings such as low reproduction rate and high mortality rate, slow process of HBV infection and certain immune tolerance to the virus. The other type is a transgenic mouse model, which includes two types: HBV expressing and NTCP (Na⁺-Taurocholate cotransporting polypeptide) deficiency transgenic mouse, with the former being the most used. This type of model plays an important role in studying HBV replication and evaluating anti-HBV drugs and vaccines. However, there are defects that HBV is not naturally infected and viral RNA comes from the integrated HBV genome. Therefore, it cannot be used to study early viral infection and viral infection. Mechanism, and is not suitable for studying mechanisms that affect cccDNA regulation and transcription activity and evaluating the pharmacodynamic effects of drugs on cccDNA production.

Woodchuck hepatitis virus (WHV) infection model

HBV is part of the Hepadnavirus family, and is subdivided into the orthohepadnaviruses and avihepadnaviruses. Beyond human HBV (receptive to human and non-human primates), the most identifiable, and widely distributed, member of the orthohevivirus genus is woodchuck hepatitis B virus (WHV). The adult woodchuck model established using WHV is mainly used to study the process of clearing infected cells by the body during acute virus infection, the mechanism of chronic infection and cancer, and to evaluate anti-HBV drugs and vaccines. However, due to differences in genetic background, the conclusions drawn from this model cannot fully reflect the status of HBV in the human body. In addition, insufficient purification of the WHV strain, incomplete antibody sets to study immune responses, and high animal prices remain bottlenecks that restrict the widespread use of this model.

Duck hepatitis B virus (DHBV) infection model

Duck hepatitis B virus (DHBV) is more commonly used in the avian hepatitis virus genus. At present, DHBV is mainly used to infect 1-3-day-old ducks for modeling research. This model can be used to evaluate new anti-HBV drugs and study cell surface virus receptors and virus clearance mechanisms. Compared with other types of models, the DHBV animal model has the following advantages: (1) The model is easy to obtain: compared with mammals, ducks have a wider source, simpler breeding conditions, shorter breeding cycles, and DHBV will not infect humans and have safer characteristics; (2) High infection success rate: Although there are differences in the natural infection rates among different duck species, different duck species have no obvious effect on the artificial infection rate. Intravenous injection has a higher infection rate than intraperitoneal injection; (3) The virus replication mechanism is similar: the replication mechanism of DHBV is highly consistent with HBV. Unlike transgenic animal models, the virus in DHBV-infected ducks can self-replicate and can spread vertically; (4) Liver pathological damage can occur: DHBV animal models can continue to express DHBV DNA highly within a certain period of time, and can cause pathological damage to the liver, which is the main difference from HBV full-genome transgenic mice. The flaw with this model is that ducks and humans differ in species, HBV and DHBV differ in structure, and there are many variables which impact the stability of the model, such as duck species, age, source of virus, batch, route of infection etc.

Testing Vaccine Effectiveness in Animal Models

Evaluation of HBV vaccine efficacy in animal models is a complicated process, including animal model selection, immunization routes and doses, and immunogenicity and protection of the vaccine.

Choice of the animal model: First, an animal model should be chosen considering the vaccine target pathogen and the anticipated immune response.

Work out the immunization route and dose: Experiment on the best immunization route and dose. For instance, antibody titters to HBV are analyzed using ELISA to determine the most effective route and dose of immunization.

Immunogeneity measurement: Immunogeneity measurement is done by counting the number of antibodies made in the animal. For instance, a vaccine is judged to be immunogenic by measuring serum antibody concentrations.

Test the vaccine’s safety: The safety of the vaccine is measured using challenge tests. For example, the protective effect of the vaccine is determined by comparing the differences in clinical symptoms, pathological changes, relative daily weight gain, pathogen nucleic acid load, etc. between the vaccinated group and the control group after challenge.

The application of animal models is also reflected in the optimization of vaccine research and development strategies. Through experiments on different animal models, researchers can explore different vaccine formulations, vaccination routes and adjuvants to improve the effectiveness and safety of vaccines. The ideal HBV animal model should be an economical, practical and easy-to-standardize animal model that has close kinship with humans, has a high infection rate for human HBV, has a long maintenance time, and can cause corresponding pathological changes after infection. It is best to have a normal immune system at the same time to facilitate evaluation of immune responses. Although great progress has been made in the research and application of HBV experimental animal models and made outstanding contributions to HBV-related research, so far there is no HBV animal model that meets all the above conditions at the same time. In addition, because the source of human liver tissue samples is very limited and it is difficult to meet relevant experimental research, HBV animal models are still the only effective means to explore and understand the pathogenesis and treatment methods of chronic hepatitis B. At present, researchers can select relatively suitable animal models of HBV infection or replication among the above models for experimental research based on the specific research content.

Our hepatitis virus animal models and related services:

Animal Models

HBV Full-Genome Pseudovirus-Infected Mouse Model

Duck Hepatitis B Virus (DHBV) Infection Liver Necrosis Model

Duck Hepatitis B Virus (DHBV) Infection Model

Woodchuck Hepatitis Virus (WHV) Infection Model

Services

Cell-based ELISA

CC50/IC50 Assay

Cytopathic Effect Inhibition Assay

ELISA Based Assays

Virus Yield Reduction Assay

Microneutralization Assay

Plaque Reduction Assay

Viral Replicon Assay

TCID50 Assay

References

1. Jose-Abrego, Alexis, et al. Host and HBV interactions and their potential impact on clinical outcomes. Pathogens. 12.9 (2023): 1146.
2. Menne, Stephan, and Paul J. Cote. The woodchuck as an animal model for pathogenesis and therapy of chronic hepatitis B virus infection. World journal of gastroenterology: WJG. 13.1 (2007): 104.
3. Hu, Jianming, et al. Cell and animal models for studying hepatitis B virus infection and drug development. Gastroenterology. 156.2 (2019): 338-354.
4. Guo, Wei-Na, et al. Animal models for the study of hepatitis B virus infection. Zoological research. 39.1 (2018): 25.
5. Liu, Yongzhen, Stephanie Maya, and Alexander Ploss. Animal models of hepatitis B virus infection–success, challenges, and future directions. Viruses. 13.5 (2021): 777.

The post Bridging the Gap: Evaluating HBV Vaccine Efficacy in Animal Models first appeared on Creative Diagnostics.

What is cAMP?

It was identified in 1957 and is an information molecule that is made after the first messenger attaches itself to the target cell. It’s involved in all manner of biological processes: hormone release, glycogen-processing, smooth-muscle relaxation, heart beat, learning, memory, etc. The standard cAMP signalling system is extracellular agonists bind to G protein-coupled receptors (GPCRs) and alter their conformation. Gs protein activates ACs and activates AC to oxidise adenine triphosphate (ATP) to produce cAMP. cAMP’s intracellular rise will also activate protein kinase A (PKA) and exchange protein (Epac) directly induced by cAMP, signaling to direct downstream reactions. GPCRs, ACs and PDE actively manage cAMP signaling. Intracellular cAMP level is modulated by PDE, which turns cAMP into 5′-adenine nucleotides (5’-AMP) and stops its signal transduction. Further, AKAP keeps cellular cAMP compartmentalised through the creation of a complex, which regulates certain cellular effects. Already in preliminary work, cAMP once raised also activated PKA and Epac, which in turn induced effectors on various channels, resulting in relaxation of airway smooth muscle, inhibition of airway smooth muscle growth, and modulation of cytokine production.

Figure 1. Overview of compartmentalization of cAMP signaling.

Proteases that Regulate cAMP

PDEs

PDEs can hydrolyse cAMP and cyclic guanosine monophosphate. We now recognise 11 isoenzyme-containing PDE subtypes (PDE1-PDE11) in mammals. PDE4 is the main cAMP hydrolyser among them. There are four subtypes of PDE4: PDE4A, PDE4B, PDE4C and PDE4D. It’s abundantly present in tissues and plays roles in physiology: the modulation of inflammation, microvascular permeability and fibrosis, contraction and expansion of smooth muscles of the bloodstream and airways. The negative correlation between PDE4 and cAMP is the primary driver of several pathological and physiological conditions including airway inflammation, COPD, asthma, psoriasis and rheumatoid arthritis. PDE4 is mostly found in lung structural cells (smooth muscle cells, airway epithelial cells, inflammatory cells) that maintain and relax airway smooth muscles and engage in a broad range of cell-life processes.

ACs

In 1989, the first mammalian AC was discovered in the bovine brain and purified and cloned. Subsequently, 9 AC subtypes were discovered. Currently, it has been determined that there are 10 subtypes of mammalian AC, 9 transmembrane AC subtypes (mAC) and one soluble AC (sAC). As a soluble enzyme, sAC is different from other AC subtypes in mammals. sAC is not regulated by G protein. The stimulation of calcium and bicarbonate is the main factor affecting it. It is mainly related to sperm motility and the growth of neuronal processes. mAb consists of a nitrogen terminus (N-terminus), two transmembrane domains, which are connected by C1a and C1b regions, and connected to the carbon terminus (C-terminus) by C2a and C2b in the cell. The N-terminus and C-terminus extend into the cytoplasm. C1 and C2 are two highly homologous cytoplasmic regions, which are the catalytic domains of AC and affect the activity of AC. The sequence and length of the N-terminus are significantly different in different ACs. Most tissues and many cell types express more than one AC subtype, and the expression of each subtype is inconsistent between different species. AC1 is highly expressed in the brain, mainly affecting neuronal function, and is a suitable neuron-specific drug target for chronic pain. AC8 is mainly expressed in the brain and is believed to play a role in learning and memory; AC3 is most abundant in the olfactory epithelium and plays a role in olfaction; AC5 and AC6 are dominant in the heart and are related to cardiac contraction; AC4, AC7, and AC9 are commonly found in mammalian tissues; AC2 is more widely distributed and is expressed in the brain, lungs, skeletal muscle, and heart, and the subtypes highly expressed in the lungs are AC2, AC6, AC8, and AC9.

cAMP and COPD

Complex reaction signalling in cells is extremely controlled by cAMP. When it detects and amplifies signals, it regulates related physiological effects through cAMP-dependent enzymes or proteins. After the cells are ‘inflamed’ by the outside world, it affects the functions of AC and PDE enzymes, modulates intracellular cAMP, which affects downstream PKA, EPAC and AKAP, then nuclear factor B (NFB) and cAMP response element binding protein. (CREB), and extracellular signal-regulated kinase (ERK) activation, which leads to airway smooth muscle relaxation and diminished inflammation and airway fibrosis. Consequently, controlling the cAMP pathway is a great candidate for both prevention and cure of long-term respiratory conditions like COPD.

cAMP and Airway Smooth Muscle Relaxation

In the respiratory system of COPD patients, we see the obvious signs of airflow inhibition: wall remodeling and mucus formation. Among them, distorted growth and hypertrophy of airway smooth muscle cells (ASMCs) is one of the most important features of COPD airway remodelling. One of the most important medications for treating airflow obstruction in COPD is 2-adrenergic receptor agonists. They act as bronchodilators because intracellular cAMP levels spike. The two main effectors of cAMP, PKA and EPAc, are then mobilised and participate in smooth muscle relaxation of the airway. EPAc reverses the phenotypic conversion of airway smooth muscle with ERK, regulates contraction and relaxation of ASMCs, and skewed Ras homologous gene family member A and Ras-associated C3 botulinum toxin substrate (Rac) to Rac, to suppress myosin. light chain Phosphorylation ultimately relaxes airway smooth muscle. Another research has shown that a boost in the levels of cAMP in the smooth muscle of the airway and the bronchial epithelium can both decrease intraepithelial acetylcholine and ameliorate bronchial symptoms. Recently, it has been discovered that mutating or demethylating ATP-binding box subfamily C member 1 decreases the release of cAMP from ASMCs that is stimulated by 2 receptors, increasing intracellular cAMP, easing tension on airway smooth muscle cells and inhibiting COPD airway smooth muscle remodeling in several different ways.

cAMP and Inflammation

COPD’s root cause is chronic inflammation, whose airway inflammation is defined by increased numbers of neutrophils, alveolar macrophages and lymphocytes. The cAMP anti-inflammatory effect is varied, it regulates several biological processes, and its regulatory effect depends on the type of cell. In the COPD inflammatory process, neutrophils generate inflammatory mediators and proteases. We have found that COPD patients’ neutrophils migrate irregularly, and that they migrate to the chemoattractant leukotriene B4 (LTB4) and the chemokine CXC ligand 1 (CXCL1) with increased velocity. We also discovered that increasing the intracellular cAMP level can prevent neutrophils from chemotaxis to CXCL1 and LTB4. Bronchial macrophages, interstitial macrophages, and alveolar macrophages are the most prominent ones in the lungs; alveolar macrophages are the major players here. On alveolar macrophages, researchers have observed that macrophages spit out cytokines and chemokines after they have been activated and stimulated by CSE or other stimulants, and that cAMP induces the release of pro-inflammatory factors like tumor necrosis factor- (TNF-) and the chemokine C-C ligand 3 (CCL3), but not the anti-inflammatory cytokines like IL-10.

The other cAMP signalling mechanism in the immune system is to turn on the downstream nuclear transcription factor CREB, bind PKA to the NF-B inhibitory protein (IB) molecules to create a complex, and the signal that breaks down IB turns on the catalytic subunit of the PKA, which phosphorylates the p65 molecule. Phosphorylated CREB could block the NF-B association with the gene in question and can block gene transcription via a binding to activated NF-B to prevent inflammation.

cAMP and Epithelial-Mesenchymal Transition

And from earlier research, evidence for EMT involvement in the COPD pathogenesis is mounting. – Smoke and pollution bombard the lungs with excess oxidants (especially reactive oxygen species) which increases oxidative stress and EMT leading to a radical degeneration of COPD. Moreover, mesenchymal transition of airway, lung epithelial cells, and bronchial epithelial cells in COPD is considerably elevated. They’ve discovered that cAMP sits in particular subcellular microdomains and regulates the activity of downstream effector proteins PKA and Epac; AKAP attaches to PKA in a small -helical loop to drive EMT. It’s now even known that CSE-induced release of transforming growth factor- in Be-as-2b cells is a trigger for EMT, for the activation of type I collagen (COLI) and for the inhibition of the epithelial cell marker E-cadherin. But increasing cAMP or disrupting the direct interaction between AKAP and PKA will significantly decrease COLI upregulation, impact epithelial-mesenchymal transition in COPD, and therefore reduce COPD symptoms.

Treatment of COPD with Drugs that Balance Camp Pathway

In practice, COPD prevention and treatment medications consist mostly of inhaled glucocorticoids, long-acting 2-adrenergic receptor agonists, and long-acting M receptor antagonists, all of which are very bad. There are still few drugs available to prevent and treat COPD, and no treatment that prevents the disease from spreading. The use of drugs that regulate cAMP pathway in COPD is currently limited to PDE4 inhibitors and AC agonists.

The post Research on Regulating cAMP Pathway to Chronic Obstructive Pulmonary Disease first appeared on Creative Diagnostics.

Chronic obstructive pulmonary disease (COPD) is a chronic airway inflammatory disease characterized by progressive airflow limitation. The main symptoms are cough, expectoration, and progressive worsening dyspnea. As of 2017, COPD has become the third-largest cause of death in the world and the eighth-largest cause of life loss globally in 2019. The above data suggest that COPD has attracted widespread attention as a global public health issue. Wednesday in the third week of November every year is World COPD Day. It was initiated and established by the Global Initiative for Chronic Obstructive Pulmonary Disease (GOLD) in 2002. The purpose is to increase public understanding and attention to COPD as a global health issue.

Effect of COPD on the Lung

COPD is a common chronic respiratory disease. It is accompanied by an increase in the chronic inflammatory response of the airways and lungs to harmful particles or gases, resulting in obstruction of respiratory flow, difficulty breathing, and often accompanied by coughing, expectoration and other discomfort. Most of the things people often call “chronic bronchitis” and “emphysema” belong to chronic obstructive pulmonary disease. The lungs of the human body are like an inverted tree, the trachea and bronchi at all levels are like tree trunks and branches, and the alveoli are like tree crowns. The airflow flows in the trachea, bronchi at all levels, and alveoli. The farther the branch becomes, the narrower the channel of the airflow becomes. Chronic obstructive pulmonary disease can cause bronchial obstruction and alveolar destruction, which together lead to airflow restriction. As a result, patients will experience difficulty breathing, which will gradually worsen, which is also a landmark symptom of COPD.

What Factors Can Easily Lead to COPD?

Figure 1. Risk factors and symptoms of COPD.

The incidence of COPD may be the result of long-term interaction between multiple environmental factors and the body’s own factors.

(1) Smoking: Smoking is the most important causative factor for COPD. The incidence of COPD in smokers is 2-8 times higher than that in non-smokers. And the longer you smoke and the greater the amount you smoke, the higher the risk of developing COPD. Passive smoking (i.e. second-hand smoke) can also cause varying degrees of COPD depending on the smoke concentration in the environment and contact time.

(2) Inhalation of occupational dust and chemical substances: In the absence of protection, long-term contact or repeated inhalation of dust and chemical substances with excessively high concentrations, such as organic and inorganic dust, industrial waste gas, allergens, smoke, etc., can lead to the occurrence of chronic obstructive pulmonary disease.

(3) Air pollution: People who have lived in areas with severe air pollution for a long time inhale large amounts of harmful gases such as sulfur dioxide and chlorine to continuously stimulate the respiratory tract, leading to chronic airway inflammation, which is also an important cause of chronic obstructive pulmonary disease.

(4) Long-term exposure to biofuels: In some areas, a large amount of smoke generated when people use firewood, wood, charcoal, crop poles and animal manure for cooking or heating for a long time leads to indoor air pollution and may also lead to chronic obstructive pulmonary disease.

(5) Infection: Respiratory infection is another important factor in the development of COPD. Infections such as viruses, bacteria, and mycoplasma can cause airway damage and airway inflammation. Infection is also a common cause of acute exacerbation of COPD. A history of severe respiratory infections in childhood is associated with reduced lung function and respiratory symptoms in adulthood. Creative Diagnostics’ pathogen infection animal model platform provides high-quality pharmacodynamic evaluation services for the treatment of bacterial respiratory infections.

(6) Other factors: It is now believed that the relatively clear individual susceptibility factor is the lack of the α1-antitrypsin gene. In addition, airway hyperresponsiveness is also considered to be an important risk factor for the development of COPD. The incidence of COPD is related to the patient’s socioeconomic status.

Researchers speculate that the incidence of COPD is related to genetic factors, and it is currently believed that the incidence of COPD is related to the interaction of multiple genetic mutations and environmental factors. Despite extensive research, little is known about the potential mechanisms of COPD at the molecular level, so there are currently no effective treatment drugs. Animal models are an important tool for studying the pathogenesis, prevention, and treatment options of human diseases and identifying potential treatment targets and biomarkers. They provide a valuable, ethically and economically feasible experimental platform for research and play an important role in the development of medicine.

Animal Models of Chronic Obstructive Pulmonary Disease

The lung anatomy of COPD animal models should be similar to that of humans in order to better simulate the pathophysiological characteristics of human COPD. Currently, there are many types of animals for establishing COPD models, such as rats, mice, dogs, sheep, rabbits, monkeys, miniature pigs, etc. Among them, small rodents are the most commonly used COPD and AECOPD models. Rats and mice have short life cycles, many strains, are easy to raise, grow quickly, have a gentle temperament, and have low breeding and reproduction costs; their physiological and pathological characteristics are many similar to those of humans. Its advantages include clear genetic background, good operability, low breeding and maintenance costs, saving manpower and material resources, etc. Most researchers choose SPF Wistar rats, SD rats, C57BL/6 mice, BALB/C mice, Kunming mice, etc. as animal models to simulate COPD and AECOPD.

Creative Diagnostics focuses on using respiratory pathogens to infect model animals to accelerate research to clarify the mechanisms and treatments involved. These services include animal models of the bacterial respiratory tract (including the determination of MCLD, probability of survival, lung bacterial load, and histopathological analysis), assessment of vaccine immunogenicity, challenge protection testing, in vitro antibacterial testing, serum sterilization testing and plaque testing.

Respiratory bacterial infection models

Bacterial infection is considered to be the main cause of acute exacerbation of COPD. Pseudomonas aeruginosa infection is the most common among patients with COPD, so it is often used to build COPD models. Bacterial colonization induces chronic inflammation that drives progressive airway obstruction. In addition, a weakened immune response results in permanent colonization or failure to clear the pathogen.

Respiratory virus infection models

Studies have shown that patients with chronic obstructive pulmonary disease have increased susceptibility to influenza A virus (IAV) and experience excessive immune responses after infection. In acute and chronic cigarette smoke exposure models, increased lung and systemic inflammation, reduced virus clearance, and reduced response to bronchodilators were observed after IAV infection. The excessive immune response observed after cigarette smoke exposure was associated with increased expression of Toll-like receptor 3 in the lungs of mice.

COPD can cause changes in patients’ lung function and form lung pathological changes, which seriously affects patients’ quality of life. Exploring its pathogenesis and related molecular markers is of great significance for clinical diagnosis and treatment. Establishing an animal model of human diseases, and through comparative research with human diseases, we can deeply explore the pathogenesis of the disease, find therapeutic targets and pathways, and develop therapeutic drugs, which is of great significance to disease research. However, the physiological and pathological differences between animals and humans, the unclear mechanism of induction methods, and the lack of unified standards for animal model evaluation have all had a certain impact on the study of animal models of COPD. Creative Diagnostics provides customers with good COPD animal models: they have similar anatomical, physiological, and pathological characteristics to humans, and the modeling methods have certain specifications to facilitate repeated reproduction, and researchers can select corresponding model animals and modeling methods according to different experimental purposes. At the same time, we will continue to improve animal models of chronic obstructive pulmonary disease to ultimately further understand, define and treat COPD.

References

1.Czarnecka-Chrebelska, Karolina H., et al. Biological and genetic mechanisms of COPD, its diagnosis, treatment, and relationship with lung cancer. Biomedicines. 11.2 (2023): 448.

2.Berenson, Charles S., et al. Impaired alveolar macrophage response to Haemophilus antigens in chronic obstructive lung disease. American journal of respiratory and critical care medicine. 174.1 (2006): 31-40.

3.Chen-Yu Hsu, Alan, et al. Targeting PI3K-p110α suppresses influenza virus infection in chronic obstructive pulmonary disease. American journal of respiratory and critical care medicine. 191.9 (2015): 1012-1023.

4.Kimura, Genki, et al. Toll-like receptor 3 stimulation causes corticosteroid-refractory airway neutrophilia and hyperresponsiveness in mice. Chest. 144.1 (2013): 99-105.

The post How Does COPD Affect the Lungs?-Animal Models Reveal the Truth first appeared on Creative Diagnostics.

Figure 1. Treatment of febrile autoimmune hemolytic anemia.

Primary AIHA

Genetic Level

Copper/zinc superoxide dismutase genes (SOD1 genes): Superoxide dismutase (SOD) is an active substance derived from living organisms that can eliminate harmful substances produced by organisms during metabolism. Continuous supplementation of SOD in the human body has a special anti-aging effect. Studies have shown that SOD is associated with a variety of autoimmune diseases, such as autoimmune cerebrospinal meningitis and rheumatoid arthritis. SOD1 is a subtype of SOD dimer containing Cu and Zn atoms. To date, most studies on the SOD1 gene have focused on changes in the relevance of amyotrophic lateral sclerosis. However, recent studies have found that oxidative stress deficiency of SOD1 can also lead to anemia and autoimmune reactions. NZB mice are mice with autoimmune diseases that will spontaneously develop AIHA after 4 to 6 months. The study found that as the course of the disease in NZB mice prolonged, the levels of methemoglobin and lipid peroxidation products in the body continued to increase, and consistent with this, the level of reactive oxygen species (ROS) in erythrocytes also continued to increase, and was positively correlated with the degree of anemia and the incidence of AIHA. However, the level of ROS in erythrocytes in hSOD1-Tg/NZB mice carrying the human SOD1 gene decreased significantly. The SOD1 gene reduces the incidence of AIHA by inhibiting the level of ROS in erythrocytes.

Protein Level

Human erythrocyte anion exchanger 1 (AE1): Human erythrocyte anion exchanger 1 is located in the third band in erythrocyte membrane protein electrophoresis, also known as band 3 protein. It is encoded by the SLC4A1 gene and is one of the most important proteins on the erythrocyte membrane. Band 3 protein is not only the main attachment point of the membrane skeleton, but also participates in the transmembrane transmission and functional regulation of various substances and information. Any changes in its structure or content may affect the integrity of the erythrocyte membrane structure and the response deformation ability of the entire cell. Membrane protein band 3 regulates the interaction with other cytoskeleton proteins and key enzymes of metabolic pathways, and mediates the phosphorylation of Lyn kinase and Src family kinase, thereby jointly maintaining the overall balance of erythrocytes. For example, streptolysin can inhibit the activity of band 3 protein, allowing chloride ions to quickly enter erythrocytes, destroying the stability of the cell membrane, and causing hemolysis. In addition, a series of serological and immunohistochemical tests were performed on 120 AIHA patients, and it was found that warm anti-self IgG antibodies can specifically act on band 3 protein, and it was also proved that it is an important target of warm antibodies in AIHA patients. Therefore, band 3 protein plays a vital role in the destruction of red blood cells and antibodies. Regulating the function of band 3 protein may reduce the hemolysis of AIHA and thus alleviate the disease process.

Stromal interaction molecule (STIM): store-operated calcium ion channel (SOCE) is one of the important channels that mediate the entry of extracellular calcium ions into cells. Its core protein is composed of the stromal interaction molecule (STIM) located on the endoplasmic reticulum and the calcium release-activated calcium channel modulator (ORAI) located on the cell membrane. At present, studies have found that STIM protein is divided into two subtypes, STIM1 and STIM2, and their main functions are slightly different. In the process of the body fighting against pathogens, STIM protein can change due to the increase and decrease of calcium ions in the cell, accumulate and translocate to the vicinity of the plasma membrane. The accumulated STIM protein will then activate the Orai1 protein, prompting the calcium release activated calcium channel (CRAC) to open. The influx of calcium ions leads to cell activation, thereby activating immune cells. STIM actually has the dual functions of regulating the opening and selectivity of CRAC channels. If the STIM1 and STIM2 proteins of T lymphocytes are deficient, autoimmune diseases of the exocrine glands, such as Sjögren’s syndrome, will occur. Researchers have found that STIM2 is closely related to cell migration and cytokine changes downstream of G protein-coupled receptors and TLR4 activation in macrophages. Like STIM1, it can cause the release of stored calcium and phagocytosis. In addition, in the AIHA model, STIM1 also activates C5aR-mediated FCγR, thereby causing the occurrence and development of the disease. Therefore, STIM1 and STIM2 act jointly and independently on macrophages in the fatal hemolysis process. After blocking the STIM protein-related functions, the mortality rate of AIHA model mice and LPS-induced septic mice was significantly reduced, further indicating that the inhibition of STIM protein may help alleviate inflammatory and autoimmune diseases.

Secondary AIHA

Gene Level

Immunoglobulin variable heavy chain region (IGVH) gene: The immunoglobulin variable heavy chain region is currently an important molecular indicator for predicting the condition and prognosis of chronic lymphocytic leukemia. It is generally believed that patients with somatic hypermutation of the IGHV gene sequence have a better prognosis and a longer overall survival than those without mutations. In a retrospective study of 585 Chronic lymphocytic leukemia (CLL) patients, it was found that the non-mutation state of IGHV was an independent influencing factor for the secondary occurrence of AIHA in CLL patients, and the median survival of AIHA disease development in non-mutated patients was significantly shortened. It is speculated that it may play an important role in specific B cell receptor subsets, but the specific molecular mechanism of action is still unclear.

MicroRNA: MicroRNA is a type of non-coding single-stranded RNA molecule with a length of about 22 nucleotides encoded by endogenous genes, which participates in post-transcriptional gene expression regulation in animals and plants. It has been found that microRNA is differentially expressed in many immune diseases. Therefore, drugs that act on abnormal microRNAs may be a new research direction to achieve therapeutic effects, such as systemic lupus erythematosus, chronic idiopathic urticaria, etc. In the process of studying the physiological and pathological mechanisms of AIHA secondary to CLL, it was found that a total of 9 miRNAs (miR-19a, miR-20a, miR-29c, miR-146b-5p, mir-186, miR-223, mir-324-3p, mir-484miR-660) were downregulated in expression. Two of the miRNAs (miR-20a and miR-146b-5p) are involved in autoimmune phenomena, especially miR-146b-5p, which is involved in both autoimmune diseases and chronic lymphocytic leukemia. Further research on this microRNA found that miR-146b-5p is closely related to regulating CD80, which is a molecule closely related to the synapse between B lymphocytes and T lymphocytes and the restoration of cell antigen presentation ability. Therefore, research on microRNA may become a new hotspot for the treatment of related immune diseases in the future.

Protein Level

B cell adsorption factor 1 (BCA-1): B cell adsorption factor 1, also known as B cell chemoattractant (BLC) or CXCL13 (chemokineCXCligand13), is a member of the CXC chemokine family and is secreted by follicular dendritic cells and macrophages in secondary lymphoid organs. This chemokine selectively attracts B cells, including two subsets, B-1 and B-2, and exerts its effect by interacting with the chemokine receptor CXCR5. The CXCR5 receptor is currently the only known ligand, which is expressed in mature B cells, follicular helper T cells, Th17 cells, and regulatory T cells. Many studies have shown that abnormal expression of CXCL13 is associated with the development of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus. Since secondary AIHA is mostly secondary to autoimmune diseases, especially systemic lupus erythematosus. Through the case analysis of clinical AIHA patients, the researchers traced the relationship between it and systemic lupus erythematosus and found that CXCL13 and hemoglobin values ​​were negatively correlated, while another chemokine CCl4 (chemokine (C-Cmotif) ligand4) and reticulum erythematosus values ​​were positively correlated. They can be used as sensitive markers for the growth and decline of AIHA disease. The plasma CXCL13 level can reflect the severity of the disease, and CCl4 can be used as an indicator for determining the bone marrow hyperplasia of AIHA patients. At the same time, higher plasma soluble tumor necrosis factor receptor II levels may have a strong guiding significance for distinguishing whether it is AIHA secondary to SLE. Therefore, the use of CXCL13 antagonists may have a certain degree of delaying effect on the development of AIHA.

In short, the pathogenesis of AIHA involves the interaction of multiple factors such as genes and proteins. As the research continues to deepen, these possible influencing factors will continue to be discovered, which can provide possible drug targets for clinical treatment and provide further in-depth directions for the study of its pathogenesis.

The post Research on Autoimmune Hemolytic Anemia first appeared on Creative Diagnostics.

Animal models are indispensable tools for studying the pathogenesis of many diseases, drugs and vaccines. Primates are close relatives of humans and are highly similar to humans in terms of organizational structure, immunity, physiology and metabolism. They are extremely precious and important experimental animals, and their application value far exceeds that of other species of animals. Since the discovery of AIDS, researchers have tried to establish suitable animal models using a variety of primates. Although chimpanzees can be infected with human immunodeficiency virus (HIV), this animal model has limited its practical application due to its limited source, high cost, difficulty in management, and slow progression of disease. Many primates in Africa are naturally infected with the simian immunodeficiency virus (SIV), and SIV can continue to replicate at high levels in the body. However, these natural hosts do not develop AIDS and are therefore not suitable for studying the pathogenesis and evaluating antiviral drugs and vaccines. Infecting Asian monkeys with virulent strains of SIV and constructed simian/human immunodeficiency virus (SHIV) will soon lead Asian monkeys to the AIDS-like illness. The disease development process and multiple detection indicators are very similar to human AIDS. Therefore, this model is widely used in the study of AIDS pathogenesis, anti-HIV/AIDS drugs and vaccines.

The Necessity of Using Animal Models for Anti-HIV Drug Research

HIV remains a major global public health issue, continuing to spread in all countries around the world, killing 40.4 million people so far; new infections are reported in some countries on an increase, compared with a previous decline. As of the end of 2022, there are an estimated 39 million people living with HIV. There is no cure for HIV infection. However, as people gain effective HIV prevention, diagnosis, treatment and care measures, including measures for opportunistic infections, HIV infection has become a manageable chronic health condition and people living with HIV are able to live a long and healthy life.

Figure 1. HIV-1 life cycle.

Since no effective anti-HIV vaccine has been developed in the world, antiviral drugs are still the main means of preventing and treating HIV/AIDS. Most anti-HIV drugs are currently developed for the main stages or major enzymes of the virus life cycle (Figure 1), such as virus-cell binding and fusion, viral reverse transcriptase (RT), integrase, protease, etc. As of 2020, the FDA has approved 222 antiretroviral drugs for global HIV/AIDS relief. These drugs can be divided into:

• Nucleoside Reverse Transcriptase Inhibitors (NRTIs)
• Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs)
• Protease Inhibitors (PIs)
• Fusion Inhibitors
• CCR5 Antagonists
• Integrase Strand Transfer Inhibitor (INSTIs)
• Attachment Inhibitors
• Post-Attachment Inhibitors
• Capsid Inhibitors
• Pharmacokinetic Enhancers
• Combination HIV Medicines

Highly active-retroviral therapy (HAART) has been proven to be an effective anti-HIV/AIDS treatment option in clinical practice. It usually uses a combination of three or more drugs. This treatment strategy can maximize the inhibition of HIV replication. However, HAART treatment has problems such as high cost, inability to clear the virus, large toxic and side effects, easy development of drug resistance, and poor drug compliance. Therefore, there is an urgent need to develop new targets, high-efficiency, low-toxicity, and inexpensive anti-HIV/AIDS drugs.

Animal Species and Virus Strains Studied for Anti-HIV Drugs

The main animals used in anti-HIV drug research are Asian monkeys, such as rhesus monkeys, cynomolgus monkeys, pig-tailed monkeys, etc. The first SIV was discovered in 1985, and several strains of SIV virus were subsequently isolated from African and Asian monkeys. SIV has certain homology with HIV-1. SIV is divided into five major branches: SIVcpz, SIVsm, SIVmnd, SIVsyk and SIVagm. Inoculation of some SIV virus strains into Asian monkeys such as rhesus monkeys and cynomolgus monkeys will infect them and quickly produce AIDS-like symptoms. Among them, SIVmac and SIVsm are more pathogenic and are the main virus strains used in anti-HIV drug research. These strains are sensitive to many NRTIs, PIs, integrase inhibitors, CCR5-target entry inhibitors, etc., but are not sensitive to NNRTIs.

Using molecular biology technology, some important genes of HIV-1 were recombined with SIV, and a series of SHIV viruses containing HIV-1 and SIV genes were successively constructed and used to infect rhesus monkeys. Initial reports stated that although several strains of SHIV can infect rhesus monkeys, they do not cause disease. Recent studies have shown that several SHIV virus strains can not only infect rhesus monkeys, but also cause them to produce AIDS-like symptoms. SHIV basically maintains the biological characteristics of SIV and carries some HIV-1 antigens. It provides an ideal model for the study of vaccines and biotherapies based on HIV-1 antigen, so it is also widely used in AIDS research. RT-SHIV, which uses the SIV virus genome as the backbone and replaces the SIV reverse transcriptase gene with the HIV-1 reverse transcriptase gene, is sensitive to NNRTIs, making the RT-SHIV/primate model play an important role in evaluating NNRTIs.

Problems in Non-human Primate Models in Anti-HIV Drug Research

The most important problem faced by several widely used AIDS primate models is that they cannot completely simulate clinical HIV infection and that different primates are infected with different immunodeficiency viruses. There are large differences in immune responses, viral loads, clinical symptoms and morbidity. For example, rhesus monkeys infected with SIVmac251 have higher viremia, lower antiviral specific immune response, and faster disease progression. After African green monkeys infected with SIVagm, although they develop high viremia, the animals will still remain healthy. When the same primate is infected with the same immunodeficiency virus, the disease process will also show great individual differences, and the asymptomatic period can last for months to years. Therefore, how to establish an animal model system with stable infection characteristics in order to reduce differences caused by host factors in drug treatment is an urgent problem to be solved. In addition, the difference in drug sensitivity between SIV or SHIV and HIV is also a problem faced by non-human primate models in anti-HIV drug research. Although many SIVs are sensitive to most anti-HIV drugs, in vivo and in vitro studies have shown that some drugs with good anti-HIV effects do not have ideal antiviral effects on SIVs.

The non-human primate (NHP) models have become a powerful tool for HIV/AIDS research. In preclinical pharmacodynamics studies of anti-HIV drugs, this model provides the reference system closest to the actual efficacy of the drug. At the same time, the formulation and testing of optimal treatment plans and treatment strategies is sometimes difficult to carry out clinically, let alone through in vitro experiments. The use of primate models for related research has become the first choice. Creative established rhesus monkey models with SIVmac251, SIVmac239 and SHIV-infected, and carried out evaluation research on the pathogenesis of AIDS, drugs and vaccines, which was recognized by customers. We hope that by carefully designing research plans, our HIV infection rhesus monkey models and non-human primate models can provide more valuable reference and better guide future clinical trials.

Our HIV infection rhesus monkey models:

References

Van Rompay, Koen KA. Tackling HIV and AIDS: contributions by non-human primate models. Lab Animal. 46.6 (2017): 259-270.

Evans, David T., and Guido Silvestri. Nonhuman primate models in AIDS research. Current Opinion in HIV and AIDS. 8.4 (2013): 255-261.

Sever, Belgin, et al. A Review of FDA-Approved Anti-HIV-1 Drugs, Anti-Gag Compounds, and Potential Strategies for HIV-1 Eradication. International Journal of Molecular Sciences. 25.7 (2024): 3659.

The post The Role of Non-human Primate Models in HIV Antiviral Drug Research first appeared on Creative Diagnostics.

Avian influenza virus (AIV) is a common type A influenza virus, belonging to the genus influenza virus of the family Orthomyxoviridae, and is classified as a Class A epidemic by the World Organization for Animal Health (OIE). It is usually transmitted among poultry and birds, especially when accompanied by migratory birds, which will further increase its spread and cause a large-scale epidemic across regions and countries. There are two main protein protrusions on the envelope of influenza viruses, namely hemagglutinin (HA) and neuraminidase (NA), which can be used to divide influenza viruses into four types: type A (IAV), type B (IBV), type C (ICV) and type D (IDV). Among them, type A influenza virus uses a variety of birds including waterfowl and poultry as hosts, and can also infect other species such as pigs, dogs, cats, and horses. Type B influenza virus is mainly transmitted by infecting humans, and the host of type C infection is also humans. Type D influenza virus mainly uses livestock as hosts, such as pigs, cattle and sheep. There are also obvious differences in the epidemics caused by the above four influenza viruses.

Figure 1. Avian influenza transmission flow from the natural reservoir (aquatic birds) to poultry, humans, and other animal species.

Influenza A virus and type B influenza virus can both cause widespread seasonal epidemics. On this basis, influenza A virus can further cause zoonotic pandemics, while influenza B virus has not been found to be associated with pandemics. Seasonal influenza caused by influenza A virus and influenza B virus can reach 5 million cases and 500,000 deaths each year, of which infections caused by influenza B virus account for about 25%. On this basis, according to the different pathogenicity of different avian influenza viruses, they can be further divided into low pathogenic avian influenza virus (LPAIV) and highly pathogenic avian influenza virus (HPAIV). Usually, if the HA protein in the avian influenza virus has a multi-base cleavage site, it is a highly pathogenic avian influenza virus. Most avian influenza viruses are low pathogenic avian influenza viruses, and common highly pathogenic influenza viruses include two subtypes: H5N1 and H7N9. The two subtypes of H5N1 and H7N9 can infect mammals including humans, often causing severe lower respiratory tract diseases, while low pathogenic avian influenza viruses mainly infect the upper respiratory tract, usually with complications of conjunctivitis.

Structure of Avian Influenza Virus

The genetic material of avian influenza virus of the family Orthomyxoviridae is composed of 8 single-stranded negative-strand RNA (vRNA), namely, PB2, PB1, PA, HA, NA, M and NS. It is also composed of a variety of essential and non-essential proteins. The essential proteins include hemagglutinin, neuraminidase, polymerase protein (PB1, PB2, PA), nucleoprotein (NP), non-structural protein (NS) and matrix protein (M). The diameter of the avian influenza virus particles composed of the above three is 80-120nm in spherical state. The avian influenza virus isolated from clinical samples will expand from filaments to spheres after chicken embryo culture. The filamentous virus particles are 200-300nm high and 20nm in diameter at the bottom.

Pathogenicity of H5N1 Avian Influenza Virus to Animals

Avian influenza viruses are generally classified as either low pathogenic or highly pathogenic according to their pathogenicity to poultry. H5N1 avian influenza virus belongs to the latter, which is the result of the accumulation of multiple basic amino acids in the HA cleavage site. Unlike low pathogenic avian influenza, highly pathogenic avian influenza develops rapidly and has a high mortality rate. After the outbreak of H5N1 avian influenza in 1996, H5N1 avian influenza occurred in various countries around the world to varying degrees, characterized by large-scale sudden deaths of poultry, waterfowl and wild birds, which confirmed the high pathogenicity of H5N1 to poultry. H5N1 avian influenza virus can also infect mammals, but this is determined by many factors. PA-X is a fusion protein of influenza A virus (IAV). Studies have shown that the absence of PA-X protein enhances the pathogenicity and replication of highly pathogenic H5N1 avian influenza virus to mice. At the same time, analysis of viruses isolated from Vietnam in 2012 and 2013 found that two amino acid changes in the PA protein in the polymerase complex affected the activity and virulence of the viral polymerase complex in mice. In 2009, the co-circulation of H5N1 and H1N1pdm09 raised concerns that recombination events could lead to a highly pathogenic influenza pandemic. Studies have shown that obtaining the NS fragment from H1N1pdm09 enhances the virulence of H5N1 in mice.

Detection of Highly Pathogenic H5N1 Avian Influenza Virus

According to the regulations of the World Organization for Animal Health (OIE), chicken embryo virus isolation and culture are often used for the detection of avian influenza. In recent years, many studies have used biosensors to detect viruses, such as silver nanocluster fluorescent molecular beacons based on DNA templates. As long as the corresponding recognition sequence is embedded, the well-designed molecular beacons can be conveniently used to detect a variety of virulence genes, including the genes of H5N1 virus. Aptamer sensors can also be used for detection. Aptamers represent an alternative to antibodies as recognition agents in diagnosis and detection analysis and have advantages. In 2016, scholars proposed an analytical platform for fluorescence detection of H5N1 virus gene sequences combined with near-infrared light and 2-photon excitation. At the same time, a digital microfluidic technology immunoassay method based on surface-enhanced Raman scattering has been proposed, and a sandwich immunoassay has been designed to sensitively detect H5N1 virus.

Vaccine for Highly Pathogenic H5N1 Avian Influenza Virus

Inactivated Vaccine

Using reverse genetics technology, an inactivated vaccine strain of H5NI with extremely weak pathogenicity and well adapted to growing in chicken embryos was artificially created, and the inactivated vaccine for avian influenza was made using this strain.

Recombinant Viral Vector Vaccine

Compared with inactivated vaccines, recombinant viral vector vaccines can induce more helper T cells to produce immune responses. A protective foreign antigen is injected into the viral gene to obtain the recombinant virus, and the immune system produces the corresponding target protein, thereby inducing an immune response. The recombinant viral vector vaccines currently developed for H5N1 avian influenza mainly include fowl pox virus vector vaccine, Newcastle disease virus vector vaccine, turkey herpes virus vector vaccine, duck enteritis virus vector vaccine and infectious laryngotracheitis virus vector vaccine.

DNA Vaccine

Compared with other vaccines, DNA vaccines have more advantages. Studies have reported that optiHA was inserted into the pCAGGS vector to construct a DNA vaccine, which caused a strong immune response when injected into chickens intramuscularly. In order to improve the efficacy of DNA vaccines against H5N1 influenza virus, studies have inserted three repeated KappaB (κB) motifs upstream of the cytomegalovirus promoter and downstream of the SV40 late polyadenylation signal, which were separated by a 5 bp nucleotide spacer. Variants with κB sites in chickens stimulated stronger humoral responses against the target antigen.

Inactivated IAV H5N1 Native Antigens

The post Research Progress on Highly Pathogenic H5N1 Avian Influenza Virus first appeared on Creative Diagnostics.

Animal Models for Vaccine Development

Using animal models to test the effectiveness of vaccines is one of the key steps in vaccine development and is often a key point in the long process of vaccine development and registration. Hundreds of different animal models can be used to evaluate multiple aspects of the immune response. Although many countries are promoting scientific experiments with alternative animals as much as possible and minimizing the number of animals used in scientific research, since there are currently no other feasible methods to evaluate or test the immune response after vaccination, the use of animals in experimental research in vaccine development remains the key. Choosing the most suitable animal model for this purpose is the key to the success of the experiment and to reducing the use of animals and research funding. Most vaccines have been evaluated in some way on small rodents, mostly mice. The advantages of using mice are low cost, easy manipulation, and specific genetic background and immune functions. In addition, a large number of immune components in mice can be used to explain the specific immune response caused by the vaccine. Because other species have much less immune components, this limits the level of detail in immune response analysis. However, large animal species such as pigs, cattle and sheep also have advantages, as they are closer to humans in terms of physiological functions and immune characteristics, and they are often hosts of the same or closely related pathogens. In addition, large animal species mainly reproduce outbreeding, and the normal distribution of vaccine responders and non-responders can be fully understood, which is very important for vaccine development. Animal models may be described in a number of ways: induced (experimental), spontaneous (natural), genetically modified, negative, orphan, and surrogate. These models vary widely in scope of application, cost, and their requirements for special infrastructure. The following are the advantages and disadvantages of commonly used animal models in vaccine development (Table 1):

Table 1. The advantages and disadvantages of conventional animal models in development of vaccine.

Screening of Animal Models

During the development of vaccines, not only should appropriate animal models be selected based on the delivery route of the vaccine being developed, but also the ethical issues when using animal models should be considered. Therefore, before using animal models, various factors need to be comprehensively considered to select the most suitable animal model to solve specific problems during the experimental process. For the development of a vaccine, there may be more than one animal model suitable for it. The following table summarizes the advantages and disadvantages of common vaccine delivery routes and the corresponding animal models (Table 2).

Table 2. The vaccine delivery routes and related animal models.

All in all, animal models are crucial and indispensable for vaccine development. It is essential when determining the quality and quantity of the immune response of a vaccinator, evaluating the safety and toxicity of the vaccine formulation, determining the effectiveness of the vaccine in providing protection against attacking infections, and evaluating the likelihood that the vaccine will prevent the transmission of disease in a specific population. At the same time, the selection of the most suitable animal model should be based on the special needs of the research project, and should not be influenced by factors such as price and difficulty of handling. In addition, in the long run, selecting the right animal model when developing new vaccines can save a lot of money, time and valuable resources.

Creative Diagnostics provides a large number of infectious animal models and a large library of viral pathogens for the development of new vaccines and antiviral drugs. These models include:

In addition, we provide well-designed analytical services that allow researchers to obtain well-researched animal models of viral infection. These models provide a multi-faceted perspective on infection dynamics, covering a range of parameters such as clinical symptoms, virus proliferation rates, clinical pathological characteristics, and cellular and humoral immune responses. Working with us ensures that a single scientific study for each project is supported by carefully designed methods, thereby promoting breakthrough discoveries in the pathogenesis and treatment of the virus.

Reference

Kiros, Tadele G., et al. The importance of animal models in the development of vaccines. Innovation in Vaccinology: from design, through to delivery and testing. 2012: 251-264.

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Figure 1. Manufacturing process of CAR-T cell therapy.

CAR Structure

CAR is a specially designed synthetic protein used to enhance the ability of cells to specifically recognize and eliminate tumor cells. It consists of three main parts: the extracellular domain, the transmembrane domain, and the intracellular domain. The extracellular domain mainly includes a single-chain variable fragment (scFv) and a connecting hinge region. scFv is an engineered protein structure that helps CAR-T cells recognize and bind to specific markers on the surface of tumor cells, also known as tumor-associated antigens (TAA). This recognition mechanism is independent of the major histocompatibility complex (MHC) system, thereby helping immune cells bypass the MHC-mediated immune escape strategies that tumor cells may adopt. The connecting hinge region is a key connection site, usually derived from molecules such as IgG or CD8α/CD28. The length and structure of this region can be adjusted to regulate the CAR signal strength and immune cell function. The transmembrane domain has an important influence on the expression and stability of CAR on the cell surface, and is also a key factor in signal transmission efficiency and CAR-T cell function. Commonly used transmembrane domains are derived from proteins such as CD4, CD8α, and CD28. The intracellular domain contains a co-stimulatory domain and an activation domain. Among them, the key role of the co-stimulatory domain is to enhance the activity and persistence of CAR-modified immune cells, such as CD28 promoting T cell proliferation and initial killing function, and 4-1BB prolonging the survival time of modified cells and enhancing their memory function. The activation domain usually uses the CD3ζ chain, which contains a key signaling element, the immunoreceptor tyrosine activation motif (ITAM). When CAR binds to the target antigen, these elements can transmit activation signals to CAR-T cells, thereby prompting them to perform immune functions, such as secreting cytokines and directly killing tumor cells.

Application of T Cells in CAR

T cells are an important component of circulating lymphocytes, play a central role in the human immune system and have a variety of surface molecular characteristics. These characteristics allow T cells to be classified into several subtypes according to their function, including, but not limited to, initial T cells, cytotoxic T cells, helper T cells, regulatory T cells and memory T cells. According to the different compositions of T cell surface receptors (TCRs), T cells can be further classified into αβ T cells (expressing TCRs composed of α chains and β chains) and γδ T cells (expressing TCRs composed of γ chains and δ chains). Among these, αβ T-cells are the most widely studied T-cell subtypes, especially the CD8+ cytotoxic T-cell subtype within them, which can directly kill tumor cells and is also the most commonly used subtype of CAR-T cells in anti-tumor therapy. CAR-T therapy targeting tumor cells is the earliest and most widely used form of CAR technology. Rosenberg originally proposed the concept of using a patient’s own immune cells to attack tumor cells and demonstrated this idea in experiments using tumor-infiltrating lymphocytes (TILs) from melanoma patients.

To overcome the problem of low immunogenicity of certain tumors, Israeli scientist Professor Zelig Eshhar developed CAR technology and created the first generation of CAR-T cells that can bypass MHC restrictions. He fused scFv corresponding to specific TAA with FcεRI receptor (γ chain) or CD3 complex (ζ chain) to construct CARs, and successfully expressed these CARs on the surface of T cells using genetic engineering technology, so that T cells can directly and specifically recognise and combine with TAA to kill tumor cells. However, these cells are unable to maintain long-term viability. To overcome this limitation, Carl H. June’s team developed second-generation CAR-T cells, which significantly increased their efficacy by introducing a costimulatory domain (such as CD137). The first patient with acute lymphoblastic leukaemia to be treated with second-generation CAR-T cells has survived more than 10 years since treatment in 2012, becoming a miracle in the history of medicine. The third generation of CAR-T cells attempts to increase activity by adding a co-stimulatory domain to the second generation, but studies have shown that their anti-tumor efficacy is not significantly better than the second generation. The fourth generation of CAR-T cells introduces specific cytokines or suicide genes to improve therapeutic efficacy while controlling potential toxicity. The fifth generation aims to achieve large-scale production of universal CAR-T cells to improve the availability and cost-effectiveness of treatment, but it must overcome higher technical barriers and meet more stringent safety requirements.

Although CAR technology continues to innovate and evolve, second-generation CAR technology remains the preferred technology for cell transformation. To date, ten CAR-T cell therapy products have been approved by the FDA for worldwide marketing, but the CAR-T cell therapies currently on the market are all for blood cancers. In the treatment of solid tumours, the clinical effect of CAR-T therapy has not yet met expectations, indicating that CAR technology and CAR-T therapy still face major development challenges.

Cat. No.	Product Name	Application
CABT-L0438Y	Magic Rabbit Anti-G4S linker monoclonal antibody, clone H2	FC	Inquiry
CABT-L0438YP	Magic Rabbit Anti-G4S linker monoclonal antibody, clone H2 [PE]	FC	Inquiry
CABT-L0438YF	Magic Rabbit Anti-G4S linker monoclonal antibody, clone H2 [FITC]	FC	Inquiry
CABT-L0438YA	Magic Rabbit Anti-G4S linker monoclonal antibody, clone H2 [APC]	FC	Inquiry
CABT-L0101YU	Mouse Anti-G4S linker monoclonal Antibody, clone 746	IA	Inquiry
CABT-L0101Y	Mouse Anti-G4S linker monoclonal Antibody, clone 746 [PE]	FC	Inquiry
CABT-L0102Y	Mouse Anti-G4S linker monoclonal Antibody, clone 746 [APC]	FC	Inquiry
DMABB-JX390	Magic Rabbit Anti-Whitlow 218 linker monoclonal antibody, clone F4	WB, FC	Inquiry
DMABB-JX391	Magic Rabbit Anti-Whitlow 218 linker monoclonal antibody, clone F4 [PE]	WB, FC	Inquiry
DMABB-JX392	Magic Rabbit Anti-Whitlow 218 linker monoclonal antibody, clone F4 [APC]	WB, FC	Inquiry
DMABB-JX393	Magic Rabbit Anti-Whitlow 218 linker monoclonal antibody, clone F4 [AF488]	WB, FC	Inquiry
DMABB-JX394	Magic Rabbit Anti-Whitlow 218 linker monoclonal antibody, clone F4 [AF647]	WB, FC	Inquiry
DMABB-JX395	Magic Rabbit Anti-Whitlow 218 linker monoclonal antibody, clone F4 [AF594]	WB, FC	Inquiry
DMABB-JX396	Magic Rabbit Anti-Whitlow 218 linker monoclonal antibody, clone F4 [Biotin]	WB, FC	Inquiry

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Figure 1. M6A regulates DNA repair through the R-loop to maintain genomic stability.

Definition and Types of RNA Methylation Modification

In nature, RNA modification is widely present in a variety of nucleotides, such as A, U, C, G, and I, of which methylation modification accounts for about 2/3 of the total modification. In eukaryotes, there are many RNA methylation modifications, such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), N7-methylguanosine (m7G), N6,2-O-dimethyladenine (m6Am) and N1-methyladenosine (m1A).

m6A

m6A refers to the methylation that occurs on the N atom at position 6 of base A and is involved in many important links in normal and abnormal biological processes such as RNA shearing, translation and degradation. It has been shown that m6A modifications are mainly distributed in the coding region of mRNA, near the splice site, the stop codon region and the 3′ non-coding region (3’UTR). At the same time, m6A is also present in the exon region, which has the conservation of the RRACH sequence (R is usually G and A, H is usually A, C and U). The most classical sequence is GGACU.

Three types of proteins are involved in the m6A modification of RNA: methyltransferases (writers), demethylases (erasers) and methylation readers. In essence, it is a dynamic and reversible process in which m6A is formed by methyltransferases, removed by demethylases, and the modified sites can be recognised by methylation readers. Previous studies have shown that m6A modification affects the stability of the mRNA itself, the efficiency of protein translation, chromatin remodelling and histone modification. Studies have shown that during RNA transcription, m6A modification can directly demethylate adjacent DNA, thereby increasing chromatin accessibility and the expression of the genes in which it is located. In addition to its function in regulating gene expression, abnormal m6A methylation levels can also cause dysregulation of downstream gene expression, leading to a variety of diseases including tumours, cardiovascular dysfunction and Alzheimer’s disease.

m5C

m5C is also a dynamic and reversible modification that is widely present in rRNA, mRNA, tRNA and many non-coding RNAs. The m5C modification in mRNA is mainly enriched in the untranslated region (3’UTR and 5’UTR), GC-rich region and near the AGO protein binding site with an AUCGANGO motif. RNA m5C methylation modification can mediate a variety of biological functions, including RNA output, ribosome assembly and translation. Similar to m6A, m5C also involves writers, erasers and readers.

m7G

m7G was first discovered in eukaryotic mRNA, tRNA and rRNA. Its most typical enzyme is METTL1, and other related enzymes are less studied. The m7G modification in mRNA is enriched at the 5’UTR and can be dynamically regulated with changes in stress. Its function is to promote translation. In rRNA, m7G modification is mediated by WBSCR22, but its role needs to be further investigated.　

DNA Damage Repair and Tumorigenesis

Causes and Repair Methods of DNA Damage

DNA is constantly attacked by exogenous and exogenous factors in cells and is damaged, resulting in genomic instability. This genomic instability is one of the important reasons for promoting the occurrence and development of tumors. Different damaging factors will cause different DNA damage. DNA damage mainly includes DNA single-strand and double-strand breaks (DSB), base mismatches, and interstrand crosslinks, among which DSB has the greatest toxic effect on cells. In order to maintain the integrity of the genome structure, cells will respond to different types of DNA damage through a variety of repair methods such as direct repair, base excision repair, nucleic acid excision repair, mismatch repair, homologous recombination repair (HRR), and non-homologous end joining (NHEJ). HRR, as the most important and precise repair method for DNA double-strand damage, occupies an important position.

Abnormal DNA Damage Repair and Tumor Treatment

Abnormal DNA damage repair in cells can lead to DNA mutations, endanger genome stability, and thus mediate the transformation of normal cells to malignancy. Studies have shown that the DNA damage repair pathway is one of the tumorigenesis pathways, and defects in DNA damage repair can promote tumorigenesis. Compared with normal tissues, cell proliferation in tumor tissues is abnormal and the degree of DNA damage increases. In this case, the apoptosis gene P53 is inactivated and the driver gene is activated, resulting in increased DNA replication pressure and an increase in the incidence of DNA damage, especially DSB. After DNA damage, on the one hand, ataxia telangiectasia-mutated (ATM) protein kinase, ATM and Rad3-related (ATR) protein kinase, etc. quickly sense the damage and start the DNA damage repair mechanism, activate the tumor suppressor gene P53, and induce cell apoptosis; on the other hand, the accumulation of DNA damage causes genomic instability, increases the incidence of tumors, and ultimately causes normal tissues to develop into malignant tumors.

Regulation of DNA Damage by RNA Methylation Modification and Chemoresistance

DSB is the most cytotoxic DNA damage. If not repaired in time, it will damage genome stability and chromosome integrity. In mammals, there are two main pathways for DSB repair: homologous recombination (HR) and NHEJ. Related studies have shown that RNA plays an important role in DNA damage response (DDR), especially dilncRNA and DDRNA have been reported to exist at DSB sites, thereby promoting DNA double-strand breaks repair (DSBR). At the same time, more evidence shows that dilncRNA can form DNA-RNA hybrid double strands at DSB sites, thereby promoting DNA repair proteins such as breast cancer susceptibility protein 1 (BRCA1), BRCA2, DNA repair protein RAD51 and meiotic recombination protein 11 (MRE11) to approach the proximal DSB site, thereby improving the efficiency of DSB repair. In addition, DSBs in the transcriptionally active regions of the genome can also induce the formation of DNA-RNA hybrid double strands, and the induction of reactive oxygen species can also play the same role.

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Figure 1. VZV life cycle and replication.

VZV Pathogenesis

Innate Immunity and Humoral Immune Response to VZV Infection

The innate immune system is the first line of defense in the host body to protect the pathogen after it enters the body. VZV infection first causes an innate immune response. Activated T lymphocytes and natural killer (NK) cells secrete interferon (IFN), which enables the host to establish a systematic antiviral system in the early stage of infection, effectively prevent and control the replication and spread of the virus, and participate in immune clearance. With the activation of VZV, the infected T cells are activated into memory T cells, which damages the body’s immune level, and eventually the virus will break through the innate immune response regulated by IFN. In the study of severe combined immune mouse models, it was found that IFN-ɑ expression was downregulated, so that VZV replication proceeded smoothly, explaining the innate immune process of VZV infection and breakthrough.

The humoral immune response can produce antiviral antibodies after the host is infected with pathogens, preventing the virus from binding to the host cell receptors, and playing a role in resisting and neutralizing the virus. VZV-specific antibodies are produced when primary infection forms chickenpox, but they cannot prevent the occurrence of HZ. The onset of HZ is related to both innate immunity and humoral immunity, especially the innate immunity.

T lymphocytes—VZV Transport and Transmission

During primary infection with VZV, the virus replicates locally in the upper respiratory tract and epithelial cells, and is transferred to T lymphocytes in the tonsils directly or through other virus-infected cells such as dendritic cells (DCs). The virus further replicates in the organs of the reticuloendothelial system such as the liver and spleen, spreads widely throughout the body, and is eventually transported to the skin and mucous membranes to produce chickenpox. At the end of the infection, VZV hides in neurons at the root of the host’s spinal cord. In the primary infection, VZV infection causes T cells to transform into memory T cells, which participate in the transport and transmission of the virus and reduce immune function. It usually takes 10 to 21 days for chickenpox to appear, which is presumably the time required for T cells to infect skin tissue. After infection, T cells increase skin homing and produce skin lesions. The SCID-hu mouse VZV infection model and the detection of VZV-infected T cells in the blood during acute infection support the role of T cells in the spread of the virus within the host.

VZV Latent Mechanism

VZV Inhibits the Expression of Molecules

The gene products encoded by VZV can downregulate the expression of MHC-I molecules in T lymphocytes, reduce the sensitivity of T lymphocytes to antigens, and reduce the immune recognition function of CD8+ T lymphocytes. Therefore, VZV has time to replicate and remain latent for a long time before being cleared by the immune system. Some researchers also believe that the nerve cells in the ganglia where VZV lurks only express a small amount or no MHC-I, thus avoiding immune recognition.

VZV Activates Cell Autophagy

Cell autophagy is one of the important defense mechanisms of the host against viruses, and is also involved in the process of the immune system clearing pathogens. However, human herpes viruses have also evolved a variety of mechanisms to avoid, destroy, and even induce the activation of cell autophagy to lurk in the human body.

VZV Reactivation Mechanism

The activation of latent VZV is the key to the onset of HZ. VZV inhibits the expression of MHC-I, MHC-II and other molecules through immune regulation, and activates T lymphocyte autophagy, making it difficult for target cells infected with VZV to be recognized by the immune system, and VZV will be reactivated. The process of VZV reactivation is closely related to the recognition of viruses by pattern recognition receptors, the interaction of T lymphocyte subsets and the release of cytokines.

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