Recruitment of immune cells by chemokines produced by cells at the site of infection is important for control of many infectious diseases. However, the A41 and U83 gene products of vaccinia virus and human herpes virus-6 (HHV-6), respectively, subvert the normal functioning of chemokines. Discuss the mechanism of action, role in viral pathogenesis and potential therapeutic use of the A41 and U83 gene products.
The human immune system is a highly evolved, complex, multi-component system which protects the body and it's organs from the harmful effects of invading pathogens. It is orchestrated by 9 types of highly adapted leukocytes. The recruitment of leukocytes to the site of infection, and to antigen present in secondary lymphoid tissue, is an essential mechanism in co-ordinating the immune response against pathogens. This mechanism is controlled by a family of chemotactic cytokine proteins called chemokines secreted by infected host cells. Chemokine can bind to receptors on specific leukocytes to activate them, and/or interact with GAG proteins on endothelial cells and in the extracellular matrix to set up a hepatatic gradient to guide leukocyte migration to the sites of infection/to lymphoid tissue 17. There are four subtypes of chemokines, based on the arrangement of their end cysteine residues: C, CC, CXC and CX3C23.
Some pathogens have evolved to evade, mimic or hijack the host immune system. For example certain large double stranded DNA viruses, such as poxviruses and herpes viruses, are able to modify the normal functioning of chemokine to facilitate viral pathogenesis. This essay will focus on the mechanism of action of the poxvirus vaccinia virus (VV) A41 gene product and human herpes virus 6 (HHV-6) gene product U83 in CC chemokine subversion and viral dissemination.
The Vaccinia Virus
The origins of the vaccinia virus are unknown. Suggestions include that it is product of genetic recombination, an ancestral relative of the variola virus, or the remnants of a now extinct virus 27. This large double stranded DNA virus is an archetypal member of the poxvirus family, which are noted to replicate their DNA in the cytoplasm rather than in the nucleus. Its genome contains around 200 genes, only half of which are involved in viral replication. The other half are thought to be involved in enhancing viral virulence and immune evasion7, 9. The vaccinia virus was brought to fame as the live vaccine used to eradicate smallpox, caused by the variola virus. Its infection is typically asymptomatic, or sometimes associated with a mild rash and fever. However, in immunosuppressed patients it can cause serious complications9.
The A41 gene is expressed early and late during vaccinia infection and encodes for a glycoprotein which is then secreted by the infected cell. This A41 glycoprotein binds to host CC chemokines, such as CCL21, CCL25 and CCL28. Although A41 does not prevent the chemokines from binding to effecter receptors, it prevents their interaction with GAG proteins. Chemokine-GAG interaction plays an essential role in chemotaxis of leukocytes to sites of infection/to secondary lymphoid tissue by facilitating the formation of solid phase heptotactic gradients which guide cell migration12, 16, 17, 7. A41 acts to disrupt the recruitment of leukocytes to sites of infection and thus increases the vaccinia viruses chances of evading the immune system and effectively disseminating 12.
The role of the A41 chemokine binding glycoprotein has at least two potential clinical implications. Firstly it could be developed as a therapeutic agent in chemokine related diseases, such as acute and chronic inflammatory diseases, where the influx of migrating leukocytes in response to chemokine gradients cause inflammation induced tissue damage to the host. A41 could potentially bind to the excessively produced chemokines and prevent the chemotaxis of leukocytes, and thus prevent tissue damage.
The second potential clinical implication of A41 is in the vaccinia viruse role as a vaccine. Vaccinia was used to inoculate people again smallpox. While the campaign eradicated smallpox, the vaccine had a poor safety record9. The vaccine recently became of interest again, because it was feared small pox may be used as a biological weapon, and vaccination was restated again in high risk groups such as the US army 18. Because of this, safer vaccines are being developed. Research has found that vaccinia virus which have had the A41 gene deleted enhanced viral immunogenicity and produced a more efficacious vaccine in murine models 9. A41 gene deletion may therefore be the next generation of vaccines against the potential biological warfare threat of smallpox.
Human Herpes Virus - 6 (HHV-6).
There are 8 known members of the human herpes virus family, which are divided into 3 subfamilies; alpha, beta and gamma. They are all noted for their ability to remain latent in the host after the primary infection. HHV-6 shares the beta herpes virus subfamily with HHV-7. Three genes (U22, U83, U94) are specific to HHV-6 and absent from HHV-7 21
HHV-6 is a ubiquitous virus thought to infect more than 95% of the population over 2 years old 20. Primary infection most commonly occurs in infants and targets CD4+ T helper cells This can be asymptomatic but is often associated with febrile illness, and less commonly exanthem subitum (a sudden rash), seizures, and encephalitis 22. Following this initial infection, HHV-6 is thought to establish latency in a number of cell types such as monocytes/macrophages, myeloid and bone marrow progenitors4, 20, 22, 23 and remains in the host for their whole lifetime 20. It is hypothosised that latently infected progenitor cells pass on latent virus through differentiation of BMPC to blood cells of different lineage 4.The latent virus can then reactivate at a later time, usually in adulthood. This reactivation is usually asymptomatic in healthy people; however serious illness can occur when HHV-6 reactivates in patients with suppressed immune responses, such as transplant recipients who immune systems are deliberately suppressed to prevent organ rejection or AID suffers 3. In the immunosuppressed this can result in a wide range of pathologies, including bone marrow suppression, pneumonitis, encephalitis, encephalopathy, multiple sclerosis (MS) hepatitis, fever, transplanted organ complication/rejection and death 11.
Because HHV-6 is lymphotrophic - particularly for CD4+ T cells which are important in activation of humoural and cell mediated antigen specific immune responses 2, 3, 4, 21 - there are implications that HHV-6 may cause immune suppression in a similar manner to the AIDs causing HIV virus, though further research is needed. 4. HHV-6 is also highly neurotrophic and linked to several neuroinflammatory diseases 21.
HHV-6 has evolved to encode for the production of 2 chemokine receptors (U12 and U51), which bind endogenous host chemokines to activate cellular replication to promote viral dissemination, and one viral chemokine, U83, which interacts with host receptors 2. Two strains of HHV-6 virus have been identified: HHV-A and B. U83 is the only HHV-6 hypervariable gene identified, and so is thought to be highly significant in the different between their pathologies 3. HHV-6B is significantly more prevalent variant in the USA, Europe and Japan, though in Africa the two variants are thought to be equally common 3. Due to variant B prevalence it is thought to cause the commonly seen febrile illness, where as variant A is proposed to be linked to more severe pathology 6, 26. This may be due to the ability of HHV-6A to not only infect CD4+ T helper cells, but also other cytotoxic effector cells such as CD8+ cytotoxic T cells, NK and gamma delta T cells which involved in antiviral defence. This is thought to aid HHV-6A's evasion of the immune system 4.
HHV-6 enter cells via an interaction with the human glycoprotein CD46 receptor present on all nucleated cells - though there is some evidence that certain strains may enter through a different receptor e.g the HST HHV-6B strain and may further explain the differences in cellular tropism and pathology shown between different strains. This HHV-6-CD46 interaction is also associated with a number of immunomodulator effects, including the deregulation of the complement system - possibly causing complement induced lymphoid tissue damage 4, 21.
While both variants secrete a CC chemokine encoded by U83 gene, small differences in the gene between the variants result in structural and functional differences between the produced glycoprotein chemokines. The chemokine produced by HHV-6A's gene U83A is called pU83A and by HHV-6B's gene U83B is called pU83B. 20
HHV-6B and U38B
U83B chemokine has been found to be an effective agonist of the CCR2 receptor, despite having a relatively low affinity interaction 3, 20. CCR2 are commonly found on monocytes 6, and U83B has been found chemoattract such CCR2 bearing cells as efficaciously as the endogenous CCR2 ligand chemokine CCL2 20. Expression of the U83B gene is thought to occur late in infection, after viral DNA replication, and is thus thought to play an important role in dissemination of the virus. Infected cells, primarily CD4+ T helper cells, expressing the viral U83B secrete the encoded chemokine product, which mimics the effects of CCL2: it binds to CCR2 to induce cellular calcium immobilisation and initiate chemotaxis of the cell to the site of viral infection, enabling viral dissemination to migrating monocytes 1. It is thought that as well as being a mechanism to spread the disease, HHV-6B then established latent infection in CCR2 bearing monocytes 1, 20.
It has been suggest that latent infection of CCR2 bearing cells by HHV-6 may play a role in the pathogenesis of the neuroinflammatory disease multiple sclerosis (MS) during viral reactivation. During MS it is hypothesised that microglial cells of HHV-6B infected monocyte linearage secrete U83B upon viral reactivation, and cause the migration of CCR2 bearing leukocytes across the bloodbrain barrier and cause the inflammatory neural damage, such as demylination, associated with MS 20. Further research is needed, but this would imply that U83B is a potential therapeutic target for the treatment of MS 23.
Another potential therapeutic application of U83B is administration at the sites of tumours. The attraction of tumour necrosis factor secreting monocytes to the cancerous area could aid the apoptosis of tumour cell and reduce the need for surgical removal 23.
HHV-6 and U83A
Two subtypes of U83A like CC chemokine exist, a spliced variation with a N terminal truncation (U83A-N) and the full length form (U83A). U83A-N is expressed in the immediate-early phase of HHV-6A infection; it inhibits the chemotaxis of leukocytes such as monocytes, dendritic and T cells, through antagonism of certain CC receptors, such as CCR1, CCR4, CCR6, CCR8 and particularly CCR5 6 .This mechanism is thought to significantly reduce the host immune response to endogenously produced chemokines by competitively binding to their target receptors before the virus has even complete replication, and therefore increase the chances of viral proliferation and dissemination 3.
The full length version of the U83A, expressed in the latent phase of HHV-6 A infection is also thought to competitively block endogenous chemokines, and more efficaciously than the truncated U83A-N form 2. However, U83A has further mediator effects. Unlike U83A-N, as well as competitively binding to CCR1, CCR4, CCR5, CCR6 and CCR8 CC chemokine receptors to block endogenous chemokine binding, it can also act as an agonist on the receptors, inducing G protein activation, receptor internalisation, calcium mobilisation and chemotaxis of leukocytes bearing these receptor types. Although al the afore mentioned CCR receptors can interact with U83A, CCR5 is of particular significance as U83A binds to this receptor with a higher affinity than any know chemokine-receptor interaction 6. Endogenous chemokines usually induce receptor internalisation via a rapid clathrin mediated pathway, leading to chemotaxis of leukocytes to the site of infection. However U83A stimulated receptors are internalised via a delayed caveolin pathway. Thus it chemoattracts leukocytes to disseminate to, but by delaying the signalling pathway, it attracts non activated CCR1, CCR4, CCR6, CCR8 and particularly CCR5 bearing cells 2. It is also likely that the preferential binding of U83A to receptors means that there are increased concentrations of endogenous chemokine, which may trigger their uptake by regulator scavenger receptors such as DARC, further reducing their ability to attract activated leukocytes to the site of infection 2.
Of the 3 chemokines reviewed, U83A currently has the widest scope for therapeutic use and adaption, which will now be discussed.
As with U83B, administration of U83A at tumour sites would encourage the influx of leukocytes and increase the antigenicity and killing of tumour cells, which tend to have low immunogenic properties. Also an endogenoud ligand of CCR6 called CCL20 has been reported to inhibit leukaemia of myeloid progenitor. As U83A is also a ligand of CCR6, there is a potential therapeutic use for U83A as a anti-cancer drug 3.
Given the wide range of receptors and immune cells U83A can activate and chemoattract, there is the potential for its administer with vaccines, as it could draw immune cells to the vaccine and enhance the immune response, thus leaving the patient with improved immunity3. This may be particularly true for use with skin mucosal-directed vaccines, as pU83A induced CCR4, CCR6, and CCR8 bearing cells have particular skin homing abilities, and pU83A also stimulates the inflammatory receptors CCR1 and 5. Thus pU83A's immunostimulatory abilities could be manipulated to improve the efficiency of vaccinations6.
Inflammatory and autoimmune diseases
Chemokine over production has been discovered to play an important pathophysiological role in several inflammatory diseases and autoimmune disease such as rheumatoid arthritis and atherosclerosis. The antagonistic properties of pU83A-N on chemokine receptors of leukocytes involved in inflammatory pathogenesis have implication as potential anti-inflammatory drugs for such diseases 23.
There is experimental evidence that U83A could have a potential role in reducing the pathogenesis of HIV-1 strains which use CCR5 as a co-receptor. Data has already highlighted CCR5 as a therapeutic target for HIV inhibition as deletion mutations seen in the population confer some resistance to HIV and in some HIV infected patients CCR5 antibodies are produced which cause down regulation of CCR5 expression and inhibition of HIV. Catusse et al 2007 found that HIV-1 infection of human PBMC was inhibited by 95% in the presence of pU83A-N due to direct competition of CCR5 binding. For reasons not stated this particular experiment was not performed with the full length pU83A, though due to the greater affinity pU83A has with CCR5 compared to pU83A-N, it could be expected that the full length chemokine would have an even greater inhibitor effect. This combined with its immunostimulating abilities mark U83A for further research as a HIV-1 treatment 6.
Evidence that suggest that HHV-6 interacts with HIV in the cooperative depletion of CD4+ T helper cells, accelerating progression to AIDs would seem to contradict the above evidence4. However given that only inhibition of HIV-1 strains which use CCR5 as a co-receptor and only by HHV-6A stains was shown, and so more detailed research into non CCR5 co-receptor strains of HIV-1 and HHV-6 co-depletion of CD4+ T helper cells is needed. Further research may lead to the development of a HHV-6 vaccine used as a combination therapy for HIV treatment to delay the progression to AIDs.
U83A is has also been linked to MS and other neuroinflammatory diseases. There are several theories of the proposed, one already mention regarding U83B inducing migration of macrophages/monocytes to neural tissue which may causes inflammation and demylination 23. A similar theory proposes that U83A induces macrophage migration to neural tissue through activation of CCR1, 5 and 8 receptors on the leukocytes, which cause neural inflammation and demylination. Supporting evidence shows that analogous HHV-7 virus, which shares many genes with HHV-6 but notably not the U83 gene, is not linked with MS. Clearly more research is needed, but experiments investigated agonistic U83A as a therapeutic target and antagonistic U83A-N as a potential therapeutic drug may yield the next generation of MS treatment 3.