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.

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.

The post Research Progress on the Immune Mechanism of Varicella-Zoster Virus first appeared on Creative Diagnostics.

Figure 1. Schematic Representation of Acute and Latent Herpes Simplex Virus-1 (HSV-1) Infection in Humans.

HSV Type

There are 2 serotypes of HSV: herpes simplex virus ⁃1 (HSV⁃1) and HSV 2, which share 83% homology in their amino acid sequences. HSV⁃1 infection can cause inflammation of the lips in the human body. When the infection is severe, it can induce conjunctivitis and lead to blindness. It may also invade nerve cells and damage the nervous system, leading to encephalitis. HSV ⁃2 is the main causative agent of genital herpes. Once infected, patients will carry the virus for life and develop periodic genital herpetic lesions. Recent epidemiological studies have shown that genital damage caused by HSV⁃2 infection increases the risk of HSV⁃2 carriers contracting and transmitting human immunodeficiency virus (HIV). Even if viral shedding occurs without causing genital damage, will significantly increase the probability of HIV transmission. Different herpesviruses have similar viral structures and can enter host cells through similar membrane fusion mechanisms. Membrane fusion requires the interaction of four basic glycoproteins (gB, gD, gH and gL) on the virus surface. The complex composed of these four glycoproteins is called the core fusion mechanism. They are usually located on the surface of the viral envelope and can pass through the cascade The action transmits the activation signal from gD to gB, causing conformational changes in gB, forming a fusion pore on the host cell membrane to achieve membrane fusion, allowing the virus to successfully release the nucleocapsid and DNA into the host cell for a new round of replication.

The Mechanism of HSV Entry into Host Cells

HSV can infect a variety of cells including neurons, epithelial cells, and fibroblasts with the help of fusion proteins. In the current research model, specific binding of HSV to receptors on host cells can cause conformational changes in viral surface glycoproteins, thereby mediating HSV entry into cells. The mechanism of action is achieved through two similar pathways: 1. Entering cells through the direct fusion of the viral envelope and the host cell plasma membrane, and the virus uses this pathway to enter fibroblasts and lymphocytes; 2. The virus triggers the invagination of the host cell plasma membrane to form endosomes, Endocytosis into the cytoplasm is the pathway used by the virus to enter neuronal cells. However, the specific entry route may depend on the type of host cell surface receptors, such as the paired immunoglobulin-like receptor α (PILRα) of gB, and the integrin receptors αvβ3, αvβ6, and αvβ8 of gH⁃gL, which may be related to It is related to the role of virus non-essential envelope proteins (gC, gE, gG, gI, gJ, gK, gM, gN), and the number of receptors on the host cell membrane may also affect the entry method of the virus.

The post What is Herpes Simplex Virus first appeared on Creative Diagnostics.

EBV Invasion Mechanism

EBV Invasion B Lymphocytes

EBV is a virus transmitted through saliva. Oral mucosal epithelial cells are the first threshold for their invasive host cells. EBV’s primary infection is considered to be caused by viruses through the oropharyngeal epithelium. Infection of naive B cells present in Waldeyer’s ring of tonsils.  EBV shows obvious tendency to B lymphocytes, which is easy to infect B cells and convert initial B cells into proliferative lymphocytes.  Viral glycoproteins including gp350, gHgL, gB and gp42 mediate the preferential binding of EBV to B cells by interacting with the complement receptor CR2 (CD21) on the surface of B cells, and then the envelope glycoprotein gp42 and gp85/gp25 form a fusion protein triple molecule Complex.  The GP42 in the complex is combined with the HLA II molecular molecules, and caused the virus cell fusion under the participation of GP85/GP25 and GP110 glycoprotein.

EBV Invades T/NK Lymphocytes

EBV can also infect T/NK lymphocytes, but the mechanism by which EBV attaches and invades T/NK lymphocytes has not been elucidated. T/NK lymphocytes neither express CD21 nor HLA class II, but T/NK lymphocytes express some integrins, which increase when stimulated and can act as receptors for T/NK lymphocytes. Studies have shown that both primitive T lymphocytes and lymphoid progenitor cells can express CD21, and EBV can also infect primitive T cells and lymphoid progenitor cells by attaching to T lymphocytes through CD21. The double infection of T lymphocytes and NK cells in patients with chronic active EBV infection (CAEBV) has been reported in the literature, further supporting that EBV may infect the common progenitor cells of T cells and NK cells. EBV can also be transmitted from EBV-infected B cells or epithelial cells to T/NK lymphocytes by cell-to-cell infection. EBV-infected B cells can activate NK cells to acquire CD21 molecules through synaptic transfer, and the ectopic receptor leads to the binding of EBV to NK cells. Studies have shown that EBV-infected T/NK lymphocytes often express cytotoxic molecules, such as perforin, granzyme B, and T cell intracytoplasmic antigen (TIA-1). NK cells, CD8+ T cells, and γδ T cells observed in EBV-associated T/NK lymphocyte tumors are typical of EBV-infected cells and belong to the type of killer cells that attempt to kill EBV-infected B cells or epithelial cells. Cells may become infected with EBV through close contact at immune synapses.

EBV Invades Squamous Cells

EBV initially enters the body through the oropharyngeal mucosa and infects B cells through the binding of the viral envelope protein gp350 to CD21 on the surface of B cells. Epithelial cells do not express CR2, and the mechanism of how EBV invades and releases from epithelial cells is not yet clear. Studies have shown that EBV can enter tongue and pharyngeal epithelial cells through 3 pathways independent of CD21 (CR2): 1) direct cell-cell contact with EBV-infected lymphocytes through the apical cell membrane; 2) through β1 or α5β1 integrin The interaction with the EBV BMRF-2 protein mediates the entry of EBV free virus particles into the basement membrane; 3) After primary infection, EBV spreads directly across the lateral membrane to adjacent epithelial cells.  The anti-EBV antigen polymer IgA can mediate the invasion of EBV into pharyngeal epithelial cells through endocytosis, and EBV bound to IgA can invade pharyngeal epithelial cells through endocytosis mediated by secretory corpuscle (SC). Elevated levels of anti-EBV-specific antigen IgA were found in mucosal secretions of NPC patients, and this EBV-IgA-SC-mediated endocytosis may represent a physiological pathway for EBV to invade nasopharyngeal epithelial cells in vivo.

EBV Invades Glandular Epithelial Cells

EBV infection of glandular epithelial cells can cause gastric cancer and bile duct cancer, and the mechanism is unclear. At least 3 models are currently speculated as the mechanism by which EBV attaches to glandular epithelial cells, which may overlap with the mechanism by which EBV invades squamous epithelial cells: 1) EBV virions with IgA specific for gp350/220 have been shown to effectively Binds to polymeric IgA receptors. Polymerized IgA is normally present in human saliva and binds to transmembrane proteins expressed on the basolateral surface of polarized epithelial cells. Internalization of the EBV-IgA-SC complex into glandular epithelial cells via an endocytic pathway is associated with an infection mechanism through the basolateral surface of the epithelial cell, possibly similar to the physiological infection of the virus in vivo. 2) It has been demonstrated that in the absence of CD21 (CR2), the gH and gL complexes can act as epithelial ligands, and EBV derived from B cells can bind to CD21 (CR2) negative epithelial cells with high affinity, but lack gH The EBV/gL complex loses its ability to bind, suggesting that the gH/gL complex present on the surface of EBV can directly bind to epithelial cell-specific receptors (such as integrins αVβ6 and αVβ8) to trigger fusion of EBV with the epithelial plasma membrane. 3) The interaction of the EBV-encoded membrane protein BMRF2 with integrins on polarized epithelial cells is another model for EBV attachment to the cell surface. The tripeptide Arg-Gly-Asp (RGD) motif in the BMRF2 molecule is presented as a ligand for rβ1, α5, α3 and αV integrins. However, BMRF2 is not a membrane protein necessary for cell-cell fusion, and very few BMRF2 molecules are present in virions. It is unclear whether the interaction of BMRF2 with integrins is primarily responsible for attachment and/or post-attachment events.

The post Mechanism of EBV to Invade Host Cells first appeared on Creative Diagnostics.

Clinical studies have found that SARS-CoV-2 can affect multiple organs such as the lungs and kidneys, and can also cause neurological symptoms, including temporary loss of smell and taste. Therefore, the symptom spectrum of COVID-19 is quite complex. In 2003, a related coronavirus-SARS-CoV caused a much smaller outbreak. This may be because the virus infection was limited to the lower respiratory system, making the virus less spread. On the contrary, SARS-CoV-2 will also infect the upper respiratory system, including the nasal mucosa, so it will spread rapidly through active virus shedding (such as when sneezing).

The Leader into Cell

In order to infect humans, SARS-CoV-2 must first attach to the surface of human respiratory or intestinal epithelial cells. Once attached, the virus invades the cell and then replicates multiple copies of itself. These replicated viruses are released, leading to the spread of SARS-CoV-2. The process of SARS-CoV-2 attaching and invading human cells is accomplished by a viral protein called “spike” protein, and it has tissue chemotaxis. This tissue chemotaxis depends on whether there are docking points on the cell surface, the so-called receptors. These receptors allow the virus to dock and invade the cell. Interestingly, the researchers found that SARS-CoV, which uses ACE2 as the receptor like SARS-CoV-2, produces disease symptoms that are quite different from SARS-CoV-2.

Figure 1. SARS-CoV-2 supposed life cycle.

In order to understand the differences in the tropism of these tissues, these researchers observed the “spike protein” of SARS-CoV-2. The SARS-CoV-2 spike protein differs from its older relatives in that it inserts a furin cleavage site. Similar sequences have been found in the spike proteins of many other highly pathogenic human viruses. When a protein is cleaved by furin, a specific amino acid sequence is exposed at its cleaved end. Such a protein substrate that can be cleaved by furin has a characteristic pattern sequence known to bind to neuropilin on the cell surface.

Experiments using laboratory-cultured cells, artificial viruses that mimic SARS-CoV-2, and naturally-occurring viruses have shown that NRP1 can promote viral infections in the presence of ACE2. By specifically blocking NRP1 with antibodies, this viral infection can be suppressed. The relationship between the two is like the entrance and the guide. Among them, ACE2 is a door into the cell, and NRP1 may be an important factor that guides this virus to find and enter this door. Because the expression level of ACE2 in most cells is very low, it is not easy for the virus to find the door into the cell. Other factors such as NRP1 may be necessary to help this virus enter the cell.

Potential Access to the Nervous System
Given that loss of smell is one of the symptoms of COVID-19, and NRP1 is mainly found in the cell layer of the nasal cavity, these researchers examined tissue samples from dead patients. Other experiments in mice have shown that NRP1 can transport virus-sized nanoparticles from the nasal mucosa to the central nervous system. These nanoparticles are chemically engineered to bind to NRP1. In contrast to control particles that have no affinity for NRP1, when these NRP1-binding nanoparticles are applied to the noses of these mice, they reach the neurons and capillaries in the brain within a few hours.

The post Neuropilin-1 Promotes SARS-CoV-2 Entry into Host Cells first appeared on Creative Diagnostics.

In a new study, researchers from the University of California, San Diego School of Medicine found that cholesterol-25-hydroxylase (CH25H) can prevent the entry of  the coronavirus SARS-CoV-2 by removing cholesterol from the cell membrane.

According to existing research, it is known that the coronavirus will induce interferon (IFN) after infection. Interferon is an active protein (mainly glycoprotein) produced by monocytes and lymphocytes with multiple functions. They have broad-spectrum antiviral activities on the same cells, affecting cell growth, differentiation, and regulating immune function. After detecting the molecular pattern associated with viral infection, the IFN pathway is activated, prompting the further activation of hundreds of interferon-stimulating genes (ISG), thereby interfering with the virus life cycle process. Human type I interferons including IFN-α and IFN-β work through the ubiquitously expressed type I interferon receptor (IFNAR), while type III interferon IFN-λ and epithelial restricted type III interferon receptor Combine and function. In vitro and clinical studies have shown that SARS-CoV-2 is sensitive to type I IFN, and type I IFN treatment may be a promising treatment strategy for COVID-19.

Cholesterol 25-hydroxylase (CH25H) is a gene that encodes the enzyme that synthesizes hydroxysterol 25-hydroxycholesterol (25HC) from cholesterol. CH25H is an interferon-inducible gene that is strongly upregulated in SARS-CoV-2 infected lung epithelial cell lines and COVID-19 infected patients. Clinical studies have found that 25HC, the product of interferon-induced enzyme CH25H, has broad-spectrum antiviral activity against human coronaviruses. Previous studies have shown that 25HC can inhibit a variety of viruses from entering cells, including VSV, HIV, NiV, EBOV and ZIKV. However, the mechanism by which 25HC regulates virus entry is unclear. Existing studies have shown that the endoplasmic reticulum localization enzyme ACAT uses fatty acyl-coenzyme a and cholesterol as substrates to produce cholesterol esters, which are stored in cytoplasmic lipid droplets and induce the consumption of accessible cholesterol of plasma membrane through an unknown mechanism. When exploring the mechanism of 25HC function, the researchers found that 25HC triggers the consumption of accessible cholesterol in the plasma membrane by activating acyl coa:cholesterol acyltransferase (ACAT), thereby inhibiting the entry of viruses. Cholesterol has multiple effects on the lipid bilayer. The increase or decrease of cholesterol may be accompanied by changes in membrane fluidity, polarity, thickness, and inherent curvature. In addition, the change of cholesterol can affect the function of the entire membrane protein (including viral receptors or co-receptors) by changing its conformation or distribution on the plasma membrane. These changes will directly or indirectly affect the fusion of the virus and the cell membrane, and cell membrane fusion is critical to the release of the viral genome. Due to the importance of membrane cholesterol in virus-cell fusion, the mechanism of 25HC may extend to the cell membrane fusion process of other viruses.

The post Cholesterol-25-Hydroxylase Inhibits Sars-Cov-2 And Other Viral Infection Mechanisms first appeared on Creative Diagnostics.

But is Vallance right? Once we have antibodies, are we protected?

Strictly speaking, not everyone who has not been infected with COVID-19 is immune to this disease. Our body does have the ability to protect itself. In addition, our immune system can learn and remove viruses during infection. This is basically the main method of treatment at present. The hospital supports COVID-19 patients while their own bodies are fighting the virus. Unfortunately, for too many people, the virus won this battle, and they left.

The immune system has several layers. The first and top layers consist of mechanical barriers, such as hair in the nose and mucus on the airways, which prevent pathogens (such as SARS-CoV-2) from entering the lung cells. Further down, these lung cells are filled with intrinsic defenses to prevent infection. But most viruses have evolved to bypass these defense systems and quickly defeat these defenses. This attack triggered the next wave of “innate” immunity. This includes a fast, broad-spectrum defense system, including direct antiviral killing mechanisms or promoting inflammation, thereby kicking the virus out of the body.

For most people, this natural response slows and controls the infection, making the last immune layer, adaptive immune system, work. Adaptive immunity consists of antibodies produced by B cells and antiviral killer cells, T cells. Both B and T cells can learn how to respond to specific threats during infection. This reaction usually takes a while to work, but it can remain for many years, forming a memory of past infections, such as MMR injections to prevent measles, mumps and rubella.

It is clear from the research conducted on SARS-CoV-2 that those infected with the SARS-CoV-2 virus will develop the above-mentioned multilayer immune response. In most cases, in the laboratory, antibodies produced during SARS-CoV-2 infection will bind, recognize, and prevent infection. However, there is not much information about the activity of T cells.

As more and more people infected this virus and survived by producing antibodies and T cells to fight against SARS-CoV-2, we may eventually reach a threshold of “herd immunity”. This does not mean that everyone has immunity, but because most people have immunity, those susceptible people who do not have immunity are less likely to contract the disease.

The problem of achieving herd immunity through natural infection is that a large number of vulnerable people, such as those with weakened immune systems, pregnant women or the elderly, are likely to get sick and die. This is why obtaining high levels of vaccine-mediated immunity is essential to protect them. Ideally, we need a safe and effective vaccine to help us get immunized.

That is to say, as the pandemic develops, herd immunity may start to play a role later and help control infection in the short term. But this should not be the only target controlled by SARS-CoV-2. On the contrary, as outlined by the World Health Organization, active detection and isolation measures are the best way to slow the spread of this pandemic.

Since some detection methods have been developed so far, identifying people with SARS-CoV-2 antibodies (infected and recovered) will undoubtedly help determine the spread of a pandemic. But there are still many problems unsolved, for example, we don’t know how long the immunity will last, and we don’t even know how many antibodies are needed to be classified as protected. A safe and effective vaccine will greatly eliminate this doubt, and should continue to maintain the key goal of combating COVID-19.

The post Can We Defend Against SARS-CoV-2 Once Antibodies Are Produced? first appeared on Creative Diagnostics.

Glycosylation is a widespread protein post-translational modification that is complex and variable in structure and plays an important role in cells and organisms. More than 50% of the proteins in the organism are glycosylated, including nucleoporin, chromatin protein, RNA polymerase II, protein translation regulators, transcription factors, etc. Their changes are involved in various life activities, such as cell recognition, cell differentiation, cell development, signal transduction and immune response. They are also related to occurrence and development of various diseases, such as tumors, neurodegenerative diseases, cardiovascular diseases, metabolic diseases, immune diseases and infectious diseases. Similarly, virus replication and invasion of the host are closely related to the glycosylation modification of its own structural proteins.

Comparing with the glycosylation of the envelopes of several common viruses, there are 8 to 15 glycosylation sites for Ebola virus, 5 to 11 for IAV (influenza virus), and 4 to 11 for HCV (hepatitis C virus), and 20 to 30 for HIV (AIDS virus). It has been found that the glycosylation site of SARS-CoV-2 is at least twice that of HIV.

The figure below shows the predicted glycosylation sites on the viral protein shell with green the SARS-CoV-2 glycosylation site, and blue the SARS-CoV glycosylation site. Highly glycosylated sites phenomenon can be observed.

Fig 1. Predicted Glycan shield (spheres) of SARS-CoV-2 and SARS-CoV Spike Glycoproteins.

Source: DOI: 10.1080 / 22221751.2020.1739565

Professor Raymond Dwek, the founding director of the Oxford Glycobiology Institute at Oxford University, joined the online international discussion at the invitation of the World Association of Scientists (WLA). He said that this highly glycosylated sites phenomenon is bringing great difficulty to vaccine research and development. Scientists represented by Professor Dwek first discovered the glycosylation phenomenon in the virus protein shell. The new study found that SARS-CoV-2, as a highly glycosylated spherical particle, has at least 66 glycosylation sites and is more prone to mutation. This virus has many similarities with SARS-CoV in 2003. Studies have shown that of the 69 glycosylation sites of SARS-CoV, 54 are similar or identical to SARS-CoV-2.

“In a simple analogy, glycosylation sites are like camouflage props.” The vaccine is actually a “hollowed out” harmless virus so that the body’s immune system can recognize it and form antibodies endogenously. Professor Dwek said that the vaccine should evoke an immune response to attack the virus, but when the virus really invades the body, these highly camouflaged external sites can deceive the immune system’s detection and help the virus survive.

For such a global public health issue, researchers have been searching for solutions with great effort. Recently, many articles about S protein glycosylation sites and sugar chain types of the novel coronavirus have been published in various platforms and journals such as bioRxiv, Science, Cell, etc. These research papers have aroused widespread concern in the field of basic scientific research and the pharmaceutical industry.

The post Difficulties in Research and Development of the Novel Coronavirus Vaccine first appeared on Creative Diagnostics.

The first stage: virus invasion

The first stage of coronavirus infection is Spike(S) protein-mediated attachment to the cell surface via S protein to the ceramide acid portion (acidic carbohydrate with 9 carbon atoms) or heparan sulfate. This infection strategy is very effective because there are many types of receptor molecules on the surface of all mammalian cells, thus creating abundant conditions for plasma membrane attachment. After binding, the virus particles actively rearrange the cytoskeleton by regulating the FAK/Cofilin/Rac/Cdc42 pathway.

The second stage: transport

In the second phase, some studies revealed that actin and tubulin are complementary cytoskeletal components of intracellular transport. Researchers explored the function of F-Actin in intracellular localization. Jasplakinolide (a cell-permeable F-actin stabilizing compound) inhibits the plasma membrane binding of virus particles during virus invasion, while cytochalsin D (a F-actin depolymerization compound) does not inhibit the invasion of the virus, but disrupts the normal positioning of the virion from the surrounding nucleus to the cytoplasmic region. The C-terminal peptide (S protein) of the coronavirus spike protein binds to several β-tubulin subtypes in a coronavirus-specific manner, and the chaotic peptides show that the binding is not due to random ionic charge interactions. Therefore, the transport of virions within cells utilizes a variety of cytoskeletal structural proteins to navigate and locate specific areas within the cell.

The third stage: assembly and maturity

After the virions are transported to the perinuclear area, the coronavirus RNA leaks from the vesicles and virions, and enters the nucleus for reverse transcription and replication. The DNA replicon is then transcribed as RNA and enters the Golgi/ER/microtubule organization center from the nucleus. Initially, the nucleocapsid (N) protein binds to the RNA copy and to the vesicle membrane, and then the N and E proteins mature further, which is required for the assembly of basic virus-like particles (VLPs). If the S protein is co-expressed, it will be incorporated into the virus particles. Under the synergistic effect of a variety of cytoskeleton and membrane regulatory proteins (such as HDAC6, ubiquitin and Rab GTPases), the assembly is assisted by concentrating packaging components.

The fourth stage: release

The genetic fusion of the SARS nucleocapsid or SARS S protein with GFP makes it possible to track non-infectious virus particles by fluorescence microscopy. Scientists used this technique to monitor the release of SARS-CoV and found vesicles fused into a multi-particle mass. This transport is sensitive to Brefeldin A, indicating that the secretory pathway is being used. Other studies have found that nocodazole can effectively inhibit the transport of virions to the plasma membrane, indicating that microtubules are an important part of the virus’s export outlet. Rab11 involves KHC binding for microtubule transport and then binding to myosin to help traverse the periocular actin matrix and release from the cells.

In general, there are many ways for coronavirus to enter the cell through the attachment and invagination of the plasma membrane. Subsequently, actin, tubulin, dynein and myosin cytoskeletal components need to be transported to the correct location for replication. After reverse transcription and transcription, the positive-strand RNA is packaged on the scaffold of the Golgi/ER/microtubule complex. Virus particles wrapped in vesicles move along the microtubules, then fuse with the plasma membrane and escape from the cells.

The post The Interaction Between Cytoskeleton and Life Cycle of Coronavirus first appeared on Creative Diagnostics.

Clinical data indicate that after the initial stage of viral infection, approximately 30% of COVID-19 patients will develop severe disease with progressive lung injury. Further research found that part of the reason was caused by excessive inflammation. Through clinical studies, it was found that pulmonary complications are caused by the destruction of the vascular barrier. Damage to the vascular barrier can lead to tissue edema, endothelitis, activation of the coagulation pathway and the development of disseminated intravascular coagulation (DIC) and uncontrolled inflammatory cell infiltration.

Under normal physiological steady-state conditions, blood vessels are usually surrounded by vascular endothelial cells to maintain the integrity of blood vessels. Vascular endothelial cells control inflammation by restricting the interaction of EC immune cells and EC platelets, inhibit coagulation by expressing coagulation inhibitors and blood clot lytic enzymes, and can produce glycogenase with anticoagulant properties. In addition, recent studies have found that a subtype of pulmonary capillary ECs has a similar profile to gene expression related to antigen presentation mediated by MHC class II. Compared with ECs of other organs, the characteristics of immunomodulation of lung ECs are more obvious. These all imply that pulmonary ECs seem to be related to coagulation and excessive inflammation in ARDS.

A recent clinical study on SARS-CoV-2 found that vascular leakage and pulmonary edema in severe COVID-19 patients are caused by multiple mechanisms. First, SARS-CoV-2 can directly infect the EC of multiple organs of patients. These infected ECs exhibit extensive endothelitis characterized by EC dysfunction, dissolution and death. Second, it is well known that SARS-CoV-2 binds to the ACE2 receptor, which can impair the activity of ACE2. It is worth noting that decreased ACE2 activity will indirectly activate the kallikrein-bradykinin pathway, thereby increasing vascular permeability. Third, activated neutrophils recruited to the lung EC produce tissue toxic mediators such as reactive oxygen species (ROS). Fourth, stimulation of immune cells, inflammatory cytokines and vasoactive molecules will increase EC contractility, which in turn leads to loosening of the connections between endothelium. This will cause the EC to separate, resulting in an endothelial space. Finally, the cytokines IL-1β and TNF activate glucuronidase and degrade glycocalyx, but also upregulate the expression of hyaluronan synthase, resulting in increased deposition of hyaluronic acid in the extracellular matrix and promoting Liquid retention. These mechanisms together lead to increased vascular permeability and vascular leakage.

In addition to vascular leakage, activation of the coagulation pathway is also an established feature of patients with severe COVID-19, and may further develop into disseminated intravascular coagulation. The study found that this is also related to EC activation and dysfunction. Because of the destruction of vascular integrity and the death of EC, the basement membrane of thrombosis is exposed, which in turn leads to the activation of the coagulation cascade. In addition, ECs activated by IL-1β and TNF initiate coagulation by expressing P-selectin, von Willebrand factor and fibrinogen binding to platelets. In turn, ECs release trophic cytokines, further increasing platelet production. In addition, platelets also release VEGF, which triggers ECs to up-regulate the expression of tissue factor, which is the main activating factor in the coagulation cascade. In response, the body will take appropriate measures to dissolve fibrin-rich blood clots, which explains why high levels of fibrin breakdown products can indicate a poor prognosis. Due to disseminated intravascular coagulation and blockage/congestion caused by inflammatory cells to small capillaries, and possible thrombosis in large blood vessels, lung tissue ischemic, triggering angiogenin and potential EC hyperplasia. The latter can aggravate ischemia, and angiogenesis can be used as a rescue mechanism to minimize ischemia. However, the newly formed blood vessels can also serve as channels for inflammatory cells and are attracted by activated ECs, thereby promoting inflammation.

The post Lethal Coronavirus-Vascular Attack first appeared on Creative Diagnostics.