Human Cytomegalovirus (CMV)

Foreword: The previous three pages at the "Evasion of the Immune Response" homepage present the fundamentals of immune evasion from cytokines, the complement system, and antigen presentation. This page is intended to illustrate specific examples of immune evasion by Cytomegalovirus and demonstrate how the immune responses to CMV shape vaccine development. A brief introduction to Cytomegalovirus is included to motivate the study of CMV and vaccine development and to provide background information.

General Information about Cytomegalovirus

What is CMV?

Cytomegalovirus is a ubiquitous beta-herpesvirus. Acute CMV infections are usually silent in immunocompetent individuals, although acute CMV infection may also cause a brief mononeucleosis-like malaise in immunocompetent adults. Approximately 70% of the population possess antibodies to CMV. Following infection, the virus resides in endothelial cells, macrophages, or granulocyte stem cells¹ and may cause reinfection if the host is rendered immunosuppressed, as by HIV or by immunosuppressive agents used during transplantation and chemotherapy. Additionally, fetuses are at risk of congenital defects, as they have undeveloped immune systems.

Transmission and Epidemiology

Despite being thought to lie completely latent, virus can be isolated from 61% of saliva samples,² 10% of uterine secretions, and 37% of urine samples,² and is also found in blood, semen, vaginal secretions, milk, and stool.^3,4,5 This suggests that, like HIV infection, the virus may occasionally cause a smoldering sub-clinical infection.⁶ CMV may therefore be transmitted by multiple means, including blood transfusion, transplantation, nursing, sexual contact, aerosol droplets, and direct person-to-person contact. Thus, it is not surprising that the presence of specific antibodies to CMV correlates with increasing age and with sexual promiscuity.⁷ CMV is more prevalent and contracted earlier in populations of developing nations and within lower socio-economic groups. One well-documented route of CMV transmission is the spread among toddlers in day-care centers, from them to their pregnant mothers, and thence to fetuses (vertical transmission).⁸

CMV in immunosuppressed individuals

When an individual is immunosuppressed by HIV or immunosuppressive drugs such as those used during transplantation, chemotherapy, or steroid therapy, unnoticed smoldering CMV infections or virus previously lying latent may cause severe illness or death. Since most individuals harbor the virus, the majority of CMV disease is thought to be caused by reactivated rather than by newly acquired virus, although infection can also be caused by transplant of organs or blood from a CMV-infected donor.⁶ Over 40% of AIDS patients and 30% of seronegative recipients organs from seropositive donors develop CMV,⁹ and mortality is as high as 80-90% in such groups.³ Common manifestations include retinitis (which may cause blindness), hepatitis, cholangitis, encephalitis, pneumonitis, gastrointestinal ulcerations, colitis, abdominal pain, fever, and weight loss. As CMV is itself immunosuppressive, it may aggravate other opportunistic infections such as Pneumocystis carinii.⁸

Neonatal CMV

CMV is the most common infectious cause of congenital defects,^9,10 sometimes resulting in mental retardation, deafness, blindness, quadriplegia, jaundice, and motor disability.⁴ The presence of antibodies in the urine of 1% of neonates³ reveals that, as in adults, the disease often passes unnoticed. However, 5-17% of CMV-infected infants eventually suffer from hearing impairment or cognitive dysfunction.^11,9 Although as many as 30% of seropositive pregnant women may shed the virus at some point during pregnancy,⁴ fetuses are far more likely to contract CMV and develop deficits if their mothers suffer from primary infection (in which case transmission rates reach 40%).¹² Indeed, 90% of clinical manifestations are caused by primary maternal infection.^11,13,14 The greater severity of neonatal CMV caused by primary infection¹⁵ might be explained in that (1) latently-infected mothers give their children both virus and protective maternal IgG, (2) viral titers are greater during primary infection such that infants receive more virions, or perhaps (3) as in HIV infection, the virus evolves within the host such that virions from primary infection are more virulent.

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Immune Evasion Strategies

A virus capable of causing lifelong persistent infection must be able to avoid the host's immune response. CMV, a large virus which codes for over 200 proteins,¹⁵ has developed a copious armamentarium of evasion strategies to deal with almost every immune effector mechanism. Additionally, it appears that CMV takes advantage of the immune system to reproduce or remain latent within leukocytes.

As you read the following, keep in mind that CMV is strictly an opportunistic pathogen: in immunocompetent individuals it is easily controlled. Thus, for each of the following mechanisms of immune evasion, the immune system possesses counter-mechanisms which, when summed, result in a delicate balance between CMV and the immune system whereby CMV is easily controlled yet never eliminated. Only when this balance is upset by immune suppression can CMV pose a serious threat to its host.

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Evasion of Antigen Presentation

CMV avoids detection by CD8+ CTLs via several mechanisms

(1) Viral product US3 binds newly synthesized MHC I heavy chain and retains it in the endoplasmic reticulum, preventing it from being expressed on the cell surface.¹⁶ (2) US11 and US 2 cooperate with US3 by transporting retained MHC I heavy chain alone and heavy chain-b -2-microglobulin dimers from the endoplasmic reticulum to the cytosol, where they are degraded by proteosomes.^{5, 17, 18, 19}(3) US6 binds to the luminal face of TAP and inhibits translocation of antigenic peptides.²⁰ (4) Before the above viral products can be produced, an early transcription factor (early intermediate protein, IE) is abundantly expressed. A viral envelope glycoprotein (UL83-encoded pp65) has been shown to phosphorylate IE and thus render it nonsusceptible to presentation in MHC I, perhaps by preventing IE degradation in proteosomes.^15,21

Evasion of MHC I isn't perfect

Depletion experiments with the closely related animal model murine CMV (MCMV) have established the CD8+ T-cells to be the single most important cell population in immunity against CMV, absence of which causes lethal infection.²² Therefore, CMV's block of MHC I expression must be less than 100% effective. This seeming paradox is explained by the production of IFNg (well-known to be important in CMV infections) by CD8+ T-cells.^23,24 IFNg can induce the synthesis of large quantities of MHC I and proteosomes that overwhelm viral inhibitory proteins and "rescue" the CTL response.^{20, 25, 26, 27} Additionally, it appears that professional antigen presenting cells such as macrophages and dendritic cells, due to their highly efficient antigen processing and presentation, may be resistant to MHC I blockade.²⁸

CMV evasion of NK cells with decoy MHC I

Since CMV potently inhibits MHC I expression, it was expected that it should be targeted by NK cells. However, the failure of NK cells to eliminate CMV was explained by the discovery of a viral protein designated UL18 which has homology to MHC I heavy chain.²⁹ Experiments have demonstrated that UL18 associates with endogenous b -2-microglobulin and is expressed on the cell surface, where it serves as a "decoy MHC" molecule, capable of presenting endogenous peptides and inhibiting NK lysis by binding to killing-inhibitory receptors (KIR) present on the NK cell.^29,30,31 Indeed, UL18 has been used to identify a novel killing inhibitory receptor, Lymphocyte Immunoglobulin-Receptor I.³² In the closely related animal model, murine Cytomegalovirus, disruption of UL18 severely restricts viral pathogenesis and reduces viral titers more than 400-fold, but virulence can be restored by the depletion of NK cells.³³

CMV also interferes with MHC II presentation

Under normal circumstances, interferon gamma (IFN g ) is a potent inducer of MHC II expression in antigen presenting cells and other cell types such as endothelial and epidermal cells. However, in CMV-infected cells, IFNg is unable to induce MHC II expression or even the production of MHC II mRNA, suggesting an interference with the Jak/Stat signal transduction pathway.⁶ There is also considerable evidence that CMV may disrupt constitutive MHC II expression in infected monocytes.⁶

Down-regulation of integrins may decrease adhesion with NK cells and CTLs

CMV infection results in a preferential decrease in integrin a1 and b1 synthesis and plasma membrane expression.³⁴ The decreased adhesion and impaired function of NK and CTLs in individuals with leukocyte-adhesion deficiency (which is due to deficient b integrin)³⁵ suggests that this down-regulation of integrins may protect against NK- and CTL- adhesion and lysis.

Use of "immune privileged" sites to avoid antigen presentation

CMV takes advantage of immune privileged sites to avoid elimination despite continued replication and shedding. For example, CMV infects the epithelial cells of the salivary glands (which express only low levels of MHC I)²²and the epithelial cells of the glomeruli of the kidney. T-cell trafficking to both sites is restricted by a strong basement membrane and microvascular endothelium.³⁶

A novel mechanism for avoidance of antigen presentation allows for latency

CMV can achieve productive yet nonlytic infection of monocytes and granulocyte/monocyte progenitor cells, continually shedding virions.^37,38,39,40 Clearly, avoidance antigen presentation is an absolute requirement for the latently infected cell to survive on a long-term basis. Immunofluorescence studies have revealed that in latently-infected macrophages CMV is sequestered from the cytosol in vacuoles such that CMV proteins are not suceptible to proteosomal degradation, suggesting yet another mechanism for evasion of antigen presentation.⁴¹

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Evasion via Interaction with Cytokines

The interaction of CMV with cytokines is especially complicated since it is beneficial to CMV both to limit CMV-specific immune responses and to cause immune proliferation of CMV-susceptible cells (e.g., activated monocytes, neutrophils, and bone marrow progenitor cells ^{37, 38, 39, 40}). Additionally, to avoid elimination of virus-shedding or virus-harboring hosts, CMV must also limit its cytopathology.

Interactions with interleukin-1 may suppress the TH1 response during times of lytic infection

Some of the principle functions of interlukin 1 (IL1) include triggering Th cells, the enhancing the inflammatory response, activating NK cells, and inducing chemotaxis. CMV infection down-regulates endogenous IL-1 production in fibroblasts by up to 99%.⁴² These findings have been confirmed in CMV-infected bone-marrow recipients.⁴³ However, the interaction between CMV and soluble IL-1 receptor (sIL1-R) is more complicated. The two CMV transactivators (IE1 and IE2) exert antagonist effects upon the secretion of soluble IL-1 receptor: IE1 down-regulates sIL-1R while IE2 up-regulates sIL-1R.⁴⁴ It has been speculated that, similar to bacteriophage l , the ratio of IE1:IE2 levels may regulate whether CMV causes lytic infection or remains dormant.⁴⁴ During lytic infection it would be advantageous to block a TH1 response with sIL-1R, while during dormancy inhibition of a TH1 response could allow for the establishment of competing viruses or for decreased host (and thus virus) survival rates.

Viral activation of TGFb suppresses the immune system and stimulates CMV replication.

Transforming Growth Factor (TGFb ) suppresses the immune system through several mechanisms, most notably the suppression of CTL and NK cells and counteraction of IL2 and TNF. CMV intermediate-early protein (IE) transactivates the increased production of TGFb in infected cells.⁴⁵ In addition to suppressing the immune response, TGFb activates the replication of CMV, possibly through a transciptional mechanism.⁴⁶

More information on TGFb ’s role in the immune evasion strategies of cancers

CMV encodes chemokine receptors which sequester extracellular chemokines.

In the animal model murine Cytomegalovirus (MCMV), the necessity for chemokines to fight infection has been definitively established.^{36, 47}The CMV genome contains 4 genes with homology to chemokine receptors, one of which (US28) has been shown to bind a wide variety of chemokines.^22,48 This protein sequesters extracellular chemokines in infected fibroblasts, possibly by a mechanism similar to that used by normal chemokine receptors (upon chemokine binding, receptors are endocytosed and the chemokine is degraded).^49,50

By recruiting leukocytes to the site of infection, chemokines play a dual role in CMV infections. Aside from the posible negative outcomes of immune activation, since many leukocytes (e.g., macrophages) are susceptible to CMV infection, chemokines could also serve to attract susceptible cells to the site of infection. That CMV produces several proteins with homology to chemokine-receptors and none with homology to chemokines suggests that overall, the accumulation of chemokines is not advantageous to CMV. It is notable, however, that murine CMV encodes a chemokine-analog (a "virokine")²², which could serve to recruit susceptible leukocytes. By producing chemokine-receptors which remove chemokines having strong anti-viral effects (e.g., TNF) while producing chemokine analogs that have mainly migratory effects, the murine CMV might manipulate host-cell chemokine responses to its advantage.

TNF may play a role in activation of latent virus.

Tumor Necrosis Factor (TNF) has been shown to produce viral replication in latently-infected cells by causing the activation and translocation of NFkB to the nucleus, where it activates the CMV IE promoter to produce the IE transactivator. In the absence of TNF, CMV IE gene transcription is inhibited in monocytes by ATF/CREB,¹³ producing latency. The role of TNF in causing virus reactivation may explain why CMV positively correlates with graft rejection and immunosuppressive regimens which elicit 'cytokine release syndrome.'⁵¹ Perhaps transplant recipients could benefit from anti-TNF antibodies, a strategy that has proven effective in laboratory animals and in vitro.⁶ Why it might be advantageous to reestablish infection at periods of high TNF concentration is unclear; perhaps coinfection with other pathogens could subvert immune attention from CMV. TNF's role in reactivation might also explain why polyclonal T-cell activation causes CMV reactivation:⁵² TNF might be secreted by activated T-cells.

CMV transactivators inhibit TNF-induced apoptosis.

Two CMV proteins (IE1 and IE2) have been shown to suppress the apoptotic anti-viral response induced by TNF or superinfection with an adenovirus that usually induces fibroblast apoptosis.⁵³

Manipulation of cytokines to interfere with hematopoeisis

As in AIDS, one way to avoid immune elimination is to infect and impair the immune system. However, as mentioned earlier, simply depleting leukocytes would protect CMV from immune response but also eliminate susceptible cells. CMV balances these factors by inhibiting hematopoeisis while establishing latent infection in remaining progenitor cells and their progeny.⁵⁴ Infection of bone marrow with CMV causes decreased concencrations of G-CSF and GM CSF (and thus inhibiting hematopoesis) while causing increased production of TGF b (and thus suppressing the immune response).

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Evasion of Complement and Humoral Immunity

CMV infection up-regulates complement control proteins

In contrast to the effect of CMV on production of other cellular proteins, CMV infection increases the cell surface expression of two proteins, membrane cofactor protein (a.k.a. MCP, CD46) and decay activating factor (a.k.a., DAF, CD55), two members of the regulator of complement activation (RCA) gene cluster, by up to eightfold.⁵⁵ Under normal conditions, these proteins protect host cells from complement-mediated cell lysis by inhibiting C3 convertases. The role of CD55 in protection of CMV virions against complement-mediated lysis was definitively established by stripping virions of CD55 with phosphatidylinositol-specific phospholipase C: stripped virions were more susceptible to lysis while reconstitution of CD55 (via the addition of exogenous CD55 in the absence of phospholipase) restored resistance to lysis.⁵⁶

Membrane Fc receptor protects infected cells and virions against complement and antibody-dependent cellular cytotoxicity.

CMV-infected cells have been shown to express a surface Fc-receptor distinct from endogenously produced Fc-receptors which can bind IgG with high affinity.⁵⁷ A homologous Fc-receptor produced by Herpes Simplex Virus functions to capture the Fc end of IgG bound to plasma membrane glycoproteins and thus prevent these Fc regions from mediating complement-mediated lysis or ADCC,⁵⁸ (antibody-dependent cellular cytotoxicity) suggesting a similiar role for CMV's Fc-receptor. (Figure 1,below) Such a mechanism would protect both CMV-infected cells and virions (which acquire the protein upon budding). Fc-receptors might also bind (and perhaps endocytose and degrade) free IgG, although whether this effect would appreciably reduce immunoglobulin titers is questionable. However, recent evidence reveals that in murine CMV, the gene for the Fc receptor confers an advantage both in wild-type mice and in mice incapable of producing IgG,⁵⁹ demonstrating that the role of this gene is more complicated than simply binding immunoglobulin.

Figure 1 Possible role of CMV-encoded Fc-receptor

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Vaccine Development

Given the prevalence of CMV-induced pathology in neonates, AIDS patients, and transplant recipients, a vaccine is desperately needed. Not surprisingly, different strategies must be used to protect these different populations.

Failed Attempt to vaccinate with a single T-cell epitope

Viral early protein pp89 is a good target for a CTL-oriented vaccine since it is expressed early, before complete development of the block of MHC I expression, during a time period when IFNg can rescue MHC I expression.⁶⁰ Given its immunodominance in CTL response against MCMV, the exact nine peptide T-cell epitope was identified and spliced into a vaccinia virus vector conjugated with hepatitis B core protein.^61,62 Despite the success of this vaccine in eliciting CTL responses in mice, it produces no humoral immunity and only a monoclonal T-cell response.⁶³ A similar vaccine was developed using the entire pp89 gene, but, possibly due to the above limitations, this vaccine could prevent death but not morbidity in mice.⁹ Additionally, it has since been revealed that human CTL responses are predominantly directed against matrix proteins pp65 and pp150 rather than IE genes ^64,65and other strategies of vaccine development have been persued.

Immunoprophylaxis and immunotherapy via external expansion of T-cells

Prophylactic or therapeutic generation of CMV-specific CTLs has also been achieved more directly. Such an approach entails isolation of lymphocytes either from a bone marrow donor or from the patient, in vitro expansion of specific CTL clones, and re-introduction of these cells to the patient. In transplant patients, the use of such a technique prophylactically has two objectives: (1) eliminating virus-harboring cells and subsequently decreasing the odds of CMV reactivation⁶⁶ and (2) reducing the severity of an infection should it occur. Both of these objectives were achieved in an experiment using the animal model MCMV, although complete eradication of the virus (as detected by PCR) was not possible.⁶⁷This approach has also been highly successful in phase I clinical trials, completely eliminating CMV infections in bone marrow recipients, although CTL activity declined after more than 3 months, possibly due to the absence of CD4+ helper T-cells.⁶⁸

Success with attenuated Towne strain vaccine

By passaging Cytomegalovirus through fibroblast cell lines, the attenuated virus strains Towne and AD169 have been developed. Unlike wild-type CMV, the Towne strain does not produce reactivation (latent infection) or shedding,⁶⁹ and is therefore not a danger to developing fetuses or transplant recipients if eliminated before the initiation of pregnancy/immunosupression.¹³ Genomic studies reveal that Towne was attenuated by several deletions, making reversion to virulent CMV unlikely.

Human trials have demonstrated that the Towne strain is safe and capable of inducing lymphoproliferative responses to CMV in 100% of subjects, CTL responses to CMV in 75% of subjects with persistence for over 6 months, and neutralizing antibodies in titers comparable to those induced by natural CMV infection.^13,70 Additionally, vaccination protected against acute mononucleosis-like symptoms in vaccinated individuals who were initially CMV-seronegative.⁹ Towne strain has also been shown to be safe and effective in prevention of disease caused by CMV infection in renal transplant recipients.⁷¹ Interestingly, vaccination doubled the probability of graft acceptance,⁷² suggesting that CMV infection can contribute to organ rejection. In the highest risk group, seronegative recipients from seropositive donors, Towne vaccination reduced the incidence of severe CMV-induced disease by 85% although infection rates were similar.⁷²

Development of a subunit vaccine

A subunit vaccine would have the advantages that (1) it is cheaper than external expansion of T-cells, (2) it is heat-stable, and (3) it may be administered to immunocompromised individuals or people in contact with immunocompromised individuals. The glycoprotein B (gB, a.k.a. UL55) has been selected for use because it is conserved in all known CMV strains and is the main target for neutralizing antibodies.¹² The adjuvant saponin (QS-21) was used due to its ability to stimulate CTLs and induce antibody class switching.¹² Together, glycoprotein B and QS-21 administration to mice have been shown to cause the production of high-affinity neutralizing antibodies, IgG subclass-switching with significant production of IgG2a (the analog of human IgG1 involved in complement fixation and ADCC), and the production of cytotoxic lymphocytes in mice.¹² Human trials have demonstrated that this vaccine induces both the production of mucosal IgA (unlike natural infection or Towne) and IgG⁷¹ and may therefore, unlike the Towne vaccine (which protects against infection but not disease), be able to prevent both infection (via mucosal immunity) and disease (via IgG and CTLs). Additionally, gB has been inserted into an attenuated yet replication-competent adenovirus and a similarly innocuous poxvirus, the latter currently being tested in phase I clinical trials.⁹

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Links

Professor Biron uses CMV to investigate the cytokine and cellular responses to viruses. (See references 36 and 47)

Immune Evasion Strategies of Cancers

The following lead to compiled links on CMV:

Congenital CMV links

CMV link 1

CMV link 2 (scroll down)

CMV link 3

CMV link 4 (scroll down, listed under betaherpesviridae)

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References

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