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The immune system is a complex system of cells and tissues, its main goal: to provide protection against the mirade of pathogens that we breathe in, swallow and colonize our skin. It consists of millions of cells and primary and secondary lymphoid tissues and weighs in total approximately 1 kilogram. Yet pathogens have evolved a number of ways to evade the intricate defense mechanisms of the immune system to invade the body and cause infection.
the immune system. It allows the pathogen to sustain in infection in a host or to re-infect a host after a previous infection with the same pathogen if it has been cleared by the immune system. Antigenic variation is defined as the periodic expression of homologous, but antigenically distinct, surface molecules within a clonal population and can be divided into two forms: antigen shift, which is largely associated with the Influenza virus, and antigenic switching which is closely associated with parasites such as plasmodium.
An example of a pathogen which uses antigenic variation is the malaria parasite Plasmodium Falciparum. Although the species has strains that differ in several polymorphic proteins, antigenic variation within a single strain also occurs. PfEMP1 antigens, expressed on the surface of the infected red cell are switched as often as 10 2 times per generation. By changing this protein that is expressed, the parasite evades the immune system, which would otherwise destroy the infected red cell. The Influenza virus is one of the best known viruses which display antigenic variation in two forms antigenic shift and antigenic drift. Antigenic drift may be described as a minor change taking place over a long period of time and usually involves a minor change such as an amino acid substitution resulting in antigenic change. Antigenic shift, in contrast is the formation of a new virus subtype with mixed HA and NA from different subtypes. These two forms of antigenic variation result in new antigens being presented to the immune system, thus even though a person may have previously been infected with Influenza, antigenic shift and antigenic drift means that it is unlikely that the person will be able to mount a secondary immune response.
The complement system is a crucial part of the defense systems of the body against infection. The complement pathway may be activated through three different routes, the classical, lectin or alternative pathway. Each of these leads to the activation of C3 that results in the deposition of opsonins C3b and iC3b on cell surfaces. These opsonins recruit Phagocytes to the site of infection. They all result in the formation of the Membrane Attack Complex (MAC), a channel formed of complement proteins which allows the influx of water, causing the pathogen or infected cell to lyse. It also promotes an inflammatory response. Microorganisms have a variety of ways to evade the complement system. One of these strategies is to not activate the cascade at all. This may be done by acquiring sialic acid from the host itself. Sialic Acid reduces binding of C3b fragments which are the first step in activating the alternative pathway. Another popular way of avoiding complement activation is by resistance to MAC induced lysis. Gram positive bacteria use this method of evasion as their capsid does not allow MAC to enter the cytoplasmic membrane.
Viruses are also use complement evasion. Herpes Simplex 1 encodes a protein known as Glycoprotein C (gC). This protein causes the dissociation of the C3 convertase, C3bBb into its component parts.
Many pathogens use the multiple steps involved in antigen processing and presentation to evade the host's immune response. They use immunomodulatory proteins to usurp the control of the pathways by interrupting molecular interactions, redirecting cellular products and destroying certain cellular protein functions. An example of this would be the interference by the Intermediate Early Protein ICP47, produced by the Herpes Simplex Viruses 1 & 2This protein inhibits the transporter associated with antigen processing (TAP)-dependant peptide translocation. This results in the MHC class I molecules being retained in the endoplasmic reticulum. Therefore no recognition of HSV infected cells takes place, resulting in no immune response by Cytotoxic T lymphocytes (CTL's). Adenovirus also affects TAP. A viral protein encoded by Adenovirus results in down regulation of transcription of TAP 1 and TAP 2.
Adenoviruses use a virally encoded protein E19 to bind MHC class I molecules. This results in the MHC class I being retained inside the cell in the endoplasmic reticulum. As a result of this, cells which are virally infected cannot be recognized or killed by CD8+ CTLs. Tumor necrosis factor (TNF) usually stimulates MHC genes, thereby enhancing T-cell recognition; in E19-expressing cells, however, TNF stimulates the production of viral proteins that neutralize the lytic activity of CTLs. This strategy assures more efficient viral reproduction and survival in the early phase of the immune response when TNF is produced.
Pathogenic interference with host immune signaling molecules is one of the most effective means of evading the immune system. Large DNA viruses have evolved a strategy which involves the encoding of homologues of cytokines, chemokines and their receptors. This process, referred to as host cytokine mimicry may result in the stimulation or down-regulation of certain cytokines.
Chemokines are a large family of 6-14kDa cytokines showing structural homology and are involved in the attraction and activation of leukocytes from the circulatory system in to tissues by high affinity interaction with a family of 7 trans-membrane G-coupled chemokine receptors (CKRs) which recognize only a small number of chemokines restricted to a specific class. Chemokines also interact with low affinity to cell endothelial or matrix glycosaminoglycans (GAGs) which drive the formation of haptotactic or immobilized gradients of chemokines at the site of inflammation. This directs leukocyte recruitment to the site of inflammation by the process of rolling, adhesion and extravastation. Chemokines have also been shown to be involved in haematopoiesis, and angiogenesis. Based on the presence and positioning of the N-terminal cysteine residue(s) chemokines are subdivided into 4 classes:
The CXC chemokine family have a single amino acid separating their cysteine residues (Î±)
CC chemokines have 2 cysteine residues which are adjacent to one another(Î²)
The CX3C family of chemokines have 2 cysteine residues with 3 amino acids separating them(Î³)
C chemokines have a single cysteine residue(Î´).
Based on the expression of different chemokines combined with the differential expression of the chemokine receptors on leukocytes, determine which cells are recruited to the site of inflammation.
Fig. 2. The basic structure of the chemokine family.
Murine gamma herpesvirus-68(MHV-68) is a viral pathogen which uses chemokine signaling evasion as one of its strategies to evade the immune system. MHV-68 is a naturally occurring pathogen which infects and establishes latency in small rodents causing lifelong infection. It closely related genetically to herpesvirus saimiri (HVS), both of which are members of the gamma-2-herpes virus subfamily. It is also closely related to the human infection Ebstein-Barr virus (EBV) and Kaposi's sarcoma associated virus human herpesvirus-8(HHV-8), although these are members of the gamma-1-herpesvirus family. It shares the biological characteristics of these viruses including the ability to establish latent infections with the host system, and their close association with the development of lymphoproliferative disorders.MHV-68 consists of a 118 kbp genome of unique DNA, having a G+C content of 46%, flanked by variable numbers of 1.23 kbp terminal repeats.
The virus replicates in the nucleus of the cell and causes an infection that leads to lytic replication and production of infectious viral progeny or latency, a dormant state that enable persistence of the viral genome. The genetic structure of the virus consists of co-linear genes interrupted by open reading frames (ORF) which encode for proteins. The MHV-68 contains 80 ORFs. Within these 80, 16 ORFs named M1-M14 (M10 consists of 3 ORFs named M10abc) confer a number of attributes to the virus.  MHV-68 also contains a number of genes that are unique to the virus. They are located on the left side of the genome and are the genes M1 to M4 and also eight vtRNAs.
These genes code for several homologues of the host's cellular genes including genes such as bcl-2(M11), and a complement -regulating protein encoded for by ORF 4. Of the proteins encoded for by these open reading frames, M3 is one of the most studied. The M3 gene encodes a 44-kD secreted protein consisting of 406 amino acids which is transcribed from unspliced mRNA during acute infection and the early stages of latency in the spleen and is secreted as a soluble protein, independent of the surface of the cell in which the MHV-68 has infected. Known as vCKMB-3, the protein has no structural homology to any other known CKBP's, or to any proteins, however it does share some sequence homology with the M1 gene product of MHV-68 (25%).
In Vitro, the role of the vCKBP-3 is to block the interaction of chemokines with their cellular receptors and thus to prevent the induction of intracellular signaling pathways. This was proven by a series of experiments which involved cross-linking radiolabelled CC and CXC chemokines in the presence of different concentrations of unlabelled chemokine competitors. This experiment showed that vCKBP-3 is a broad-spectrum binding protein with the ability to bind all four classes of chemokine in vivo. What this effectively means is that the vCKMP-3 has the ability to stop a number of different cell types being recruited to the site of infection, meaning that it is extremely difficult for the immune system to mount an effective response.
The role of M3 and its product in vCKBP-3 in pathogenesis are extensive. The usual route of infection for MHV-68 is through the respiratory tract and the primary site of infection is the lungs. The inflammatory response within the lungs is slow and may be due to the broad spectrum chemokine binding capabilities of vCKBP-3, which is produced by the virus during gene transcription of the M3 gene during the lytic phase infection. This was proven by a series of experiments done on the role of the vCKBP-3 in meningitis. This study showed that the gene M3 is advantageous for the induction of meningitis after inoculating the CNS of mice. Chemokine expression has been observed to rise post CNS infection, and this allows a rapid immune response. However the production of vCKBP-3 by the gene M3 slowed down the response of the immune system by stopping chemokine signalling. Although it would appear that M3 gene is essential for lytic infection, it has been shown that this is, in fact, not the case. Investigations studying the effects of removing the M3 gene have shown that it is not essential in the lytic phase and that viral replication is equal at this point with or without the presence of M3. However they do dampen immune responses as an enhanced clearance rate of the virus was noted in MHV-68 lacking the M3 gene. From the initial site of infection, the virus enters the lymph nodes and it is here that B cells and dendritic macrophages are infected. The macrophages are responsible for transporting the virus to the mediastinal lymph node and it is here that the virus infects more B cells. Rapid expansion of the infected B cells within the parafollicular region of the lymph nodes allows massive increase of the virus and allows it to be carried to the spleen and other lymphoid organs where the number of latently infected cells increases
(e.g.)1 per 107 to 1 per 104 in splenic cells. Latency is maintained in B cells, the spleen and the epithelial cells of the lung. M3 is expressed in the latently infected cells in the germinal centres of the spleen and it is here that M3 is essential in maintaining viral infection. This was shown by an experiment which compared the number of splenic follicles infected with MHV-68 expressing M3 and splenic follicles which were infected with MHV-68 but which has a M3 gene into which LacZ had been inserted, thus making it impossible to express M3. Up to day 7 post infection, there was little difference observed in infection rates between the wildtype (WT) infected cells and those infected with the mutant M3LacZ. However by day 13, there was a massive drop off in the number of cells infected with M3LacZ. In contrast those cells infected with the WT had undergone a massive clonal expansion. Thus it can be said that the clearance of MHV-68 infected cells is mediated by the immune system as the afore mentioned results clearly show. This highlights the importance of the M3 gene in pathogenesis. Although its presence is negligible during lytic infection (although it is expressed) it is during the latent phase that the presence of M3 protects the virus from clearance by the production of vCKBP-3. It is apparent that the expression of vCKBP-3 stops the recruitment of lymphocytes to the spleen by blocking the binding of chemokines produced by the infected cells to their receptors.
This ability to bind a broad specificity of chemokines has made M3 and its gene product vCKBP-3 the topic of major research into possible therapeutic uses. Chemokines in their role as cell activators and recruiters regulate and may exaserbate several diseases such as MS, diabetes or allergy induced inflammatory responses. By preventing the processes that lead to inflammation, it would be possible to prevent tissue damage associated with inflammation before it began. The fact that the M3 gene product vCKBP-3 is a broad spectrum binding protein of chamomiles means that it would be more effective at slowing or stopping the inflammatory process associated with these diseases than a specific chemokine antagonist, which would have the ability to bind only one chemokine. Due to the complexity of the chemokine network, the likelihood would be that the binding of only one chemokine would be of negligible effect.
The M3 produced vCKBP-3 may be used in the future as a way of enhancing the results of Oncolytic Virotherapy, a cancer treatment which has much promise but has been hampered by the immune system response to the virus. Currently researchers are using a recombinant VSV vector expressing equine herpes virus-1 glycoprotein G to treat hepatocellular carcinoma in rats. This glycoprotein is a broad spectrum CKBP. The results have been promising, showing enhanced oncolytic action by the virus Thus the use of M3 in an oncolytic virotherapy are a possibility as it too produces a broad spectrum CKBP.
The M3 gene and its product may also hold the key to inhibiting allograft vasculopathy following cardiac transplant. In the first six months, the main reasons for failure of the transplant are non-specific graft failure, acute rejection and infection. After the first year, the main cause of death in transplant patients is transplant vasculopathy. It is characterrised by a neointimal proliferation, which will eventually lead to ischemic allograft failure. One of the hallmarks of this process is characteristic infiltration of leukocytes in the tissue. Although the pathogenic basis of TV is not known it is believed that it may be caused by the repeated injury and repair to the graft. Injury occurs to the graft from the moment of brain death of the donor and is continuously inflicted throughout all the stages of transplant. These injuries release chemokines from the injured cells, which then recruit leukocytes to the site.
Broad spectrum chemokine binding receptors may hold the key to preventing TV. Studies done in rats show that an infusion of 300ng of M3 gene product resulted in a significantly reduced plaque area. It also resulted in a reduction in the cell invasion of the tissue This was also found in mice who had undergone femoral artery injury. The mice were transgenic, and conditionally expressed CKBP-3 in response to the injection of doxycylcine . Following femoral injury, doxyclcline was injected into a defined group of mice, to induce gene expression of M3. It was found that intimal hyperplasia was lower than in those mice in which no M3 was expressed. This result suggests that just as with the allograft transplant vasculopathy mentioned earlier, the M3 gene product vCKBP-3 bound chemokines and thus prevented the chemokines released from the damaged tissue recruiting and activation a cell mediated inflammatory response, which would be detrimental to the tissue integrity.
As we have seen, the M3 gene and its product vCKBP-3 are an ingenious method of evading the immune system and allowing the virus to infect cells. But this feature which allows the virus to infect the host may also be used to treat a variety of pathologic conditions in the future.