Measles virus persistence and its consequences

Abstract

Clearance of measles virus is complex. Infectious virus is cleared by the adaptive immune response manifested by the characteristic maculopapular rash. CD8⁺ T cells are major effectors of infectious virus clearance, a process that may fail in individuals with compromised cellular immune responses leading to progressive giant cell pneumonia and/or measles inclusion body encephalitis. In contrast to the usual rapid clearance of infectious virus, clearance of viral RNA is slow with persistence in lymphoid tissue for many months. Persistence of MeV RNA may contribute to the late development of the slowly progressive disease subacute sclerosing panencephalitis in children infected at a young age and to measles-associated immune suppression but also to maturation of the immune response and development of life long immunity.

Introduction

Measles is a systemic rash disease with several unique pathogenic features and complications of infection. Measles virus (MeV), the causative agent of measles, is a highly infectious myelotropic, lymphotropic and epitheliotropic negative strand RNA virus with important effects on the immune system. Measles remains an important disease in much of the world and continues to cause the deaths of more than 100,000 children each year despite the availability of a safe and effective attenuated live virus vaccine [1,2].

Most of the mortality caused by infection with wild type MeV is due to a measles-induced increased susceptibility to infection with bacteria or other viruses [3,4]. Nervous system complications are less frequent, but in older children and adults, measles can be complicated by development of encephalomyelitis, an autoimmune demyelinating disease that often results in permanent neurologic damage [5]. Other complications are associated with failure to clear virus so that in immunocompromised individuals MeV infection can result in progressive lung and/or nervous system infection [6,7]. In addition, in children infected at a young age the uniformly fatal nervous system disease subacute sclerosing panencephalitis (SSPE) may become manifest many years after the original infection [8].

However, despite abundant evidence of immunologic abnormalities associated with MeV infection, the immune response to MeV is highly effective and recovery from infection results in development of life-long immunity to reinfection [9,10]. Our understanding of the pathogenesis of measles, some of its complications, as well as mechanisms and time course of the immune response and virus clearance comes from studies of natural infection of humans and experimental infection of nonhuman primates, primarily rhesus and cynomolgus macaques that develop a disease very similar to that of humans [11–14]. This review will focus on virus clearance, diseases due to clearance failure and consequences of prolonged persistence of MeV RNA.

Clearance of infectious virus

MeV spreads rapidly from the respiratory tract to multiple organs through a cell-associated viremia fueled by extensive replication in lymphoid tissues. Virus dissemination occurs over several days during a clinically asymptomatic incubation period prior to the appearance of the rash. MeV replication occurs in multiple types of cells including lymphocytes, dendritic cells, macrophages, epithelial cells and endothelial cells [15–20]. Immune cells expressing the MeV receptor SLAM/CD150 in lymphoid tissue are the main sites of virus amplification with replication in memory CD4⁺ and CD8⁺ T cells and in both naïve and memory B cells [21–24]. Infection is accompanied by an acute depletion of these cells from circulation and lymphoid tissue followed by a rapid rebound in cell numbers accompanied by changes in the subtypes of immune cells in circulation [24,25].

Measles disease is typically first recognized with the appearance of the characteristic maculopapular rash 10–14 days after infection. The rash is a manifestation of the MeV-specific adaptive immune response with infiltration of T cells into sites of virus replication in the skin and other tissues and initiation of infectious virus clearance [22,26] (Figure). MeV clearance from peripheral blood mononuclear cells (PBMCs) correlates with the appearance of IFN-γ-producing CD4⁺ and CD8⁺ T cells in blood and several pieces of evidence suggest that CD8⁺ T cells are the most important effectors for elimination of infectious virus. At the time of the rash and initiation of clearance MeV-specific CD8⁺ T cells appear in circulation during natural and experimental infection [27–29]. In vitro, CD8⁺, but not CD4⁺ T cells can control virus replication in B cells [30]. Furthermore, CD8⁺ T cell depletion in macaques leads to higher levels of viremia and delayed MeV clearance [31]. It is presumed that CD8⁺ T cells eliminate infected cells through cytotoxic mechanisms but may also suppress virus production in surviving infected cells through non-cytotoxic mechanisms such as production of IFN-γ.

Figure -.

Infection of rhesus macaques with WT MeV results in viremia and rash with clearance of infectious virus and prolonged clearance of viral RNA from PBMCs with RNA persistence in lymphoid tissue (LN). Gradual clearance is associated with induction of MeV-specific T cells producing first IFN-γ, then IL-17 with continued increases in peripheral T follicular helper (pTfh) cells, antibody titer and avidity. This process is associated with failure of clearance in individuals with compromised cellular immune responses (giant cell pneumonia, MIBE) or infection at a young age (SSPE), with increased susceptibility to other infections and establishment of life long immunity to reinfection.

Although essential for protective immunity, direct roles in clearance for CD4⁺ T cells and antiviral antibody are less clear. IFN-γ-producing Th1 cells are generated in large numbers during the rash when plasma levels of IFN-γ are elevated [27,32]. Depletion of CD20⁺ B cells from macaques delays the appearance of MeV-specific antibody, but does not prolong the viremia [33]. However, monkeys depleted of both CD8⁺ T cells and CD20⁺ B cells develop a desquamating skin rash, in addition to prolonged viremia, suggesting a role for CD4⁺ T cells in pathology [33]. After resolution of the rash and fever, infectious virus can no longer be recovered from blood or respiratory secretions and there is a rapid decline in the numbers of IFN-γ-producing T cells in circulation [27,32] (figure).

Persistence of MeV

Although infectious virus is generally cleared during the rash, this may not occur in individuals with compromised cellular immunity. For instance, individuals with congenital immunodeficiencies, HIV infection or other types of acquired immune suppression may not develop a rash in response to MeV infection (indicating a failure of the adaptive immune response) and are at risk for developing progressive CNS infection (measles inclusion body encephalomyelitis/MIBE) and/or measles giant cell pneumonitis [6,7]. These often fatal complications of MeV infection usually present within a few weeks of the original infection and then steadily progress.

In addition, approximately one in 1000 children infected at a young age will develop the slowly progressive uniformly fatal CNS disease SSPE several years after the original infection [8,34–36]. At the time of the appearance of clinical signs of neurologic disease (e.g. behavioral changes, decreased school performance, seizures, etc) MeV is widely distributed in neurons of the CNS, inflammation is present and antibody levels are high [35,37]. Therefore, an immune response is generated to infection, but is not effective in eliminating virus or controlling replication in the CNS.

For both MIBE and SSPE, entry of MeV into the CNS may have occurred through replication in brain endothelial cells [16] or trafficking of activated infected mononuclear cells from the blood into the CNS. Neurons lack SLAM/CD150 and nectin4, the known receptors for wild type MeV, so the mechanism by which neuronal infection is established is unclear. When neurologic disease is recognized, cell-free virus typically cannot be recovered from CNS tissue. For MIBE it is likely that infection of the CNS is established at the time of the initial systemic infection with selection for viral fusion (F) protein variants that allow cell-to-cell spread through the nervous system [38]. For SSPE, it is not known whether initiation of infection occurs during the acute phase of MeV infection or later during RNA persistence and whether selection for viruses with characteristic mutations in the F and matrix (M) protein genes occurs before or after CNS infection [39].

In general, the amino acid changes found in MIBE and SSPE MeV proteins serve to facilitate cell-to-cell transmission within the CNS and inhibit detection of infected cells by the immune system [39]. Both the cytoplasmic and extracellular domains of the F protein are frequently altered. Ectodomain changes decrease stability of the F trimer to increase fusogenicity and facilitate syncytia formation by neural cells [40]. Cytoplasmic domain changes alter the length of the protein tail and the interactions with M required for virion assembly. These changes work in concert with those associated with biased hypermutation in the M protein gene to impair virus assembly, reduce virus particle formation and increase surface expression of F to enhance cell fusion [41,42].

In SSPE a vigorous antiviral immune response is induced and unusually high levels of antibody in serum and cerebrospinal fluid often suggest the diagnosis. However, the genetic mutations acquired eliminate cell surface expression of MeV proteins for antibody recognition and limited neuronal MHC expression inhibits presentation of viral peptides to T cells. Therefore, the response is not effective in clearing or controlling CNS virus replication and disease is progressive.

Persistence of MeV RNA

Although infectious virus cannot be recovered from blood or respiratory secretions within a few days after the rash has cleared, MeV RNA can be detected in PBMCs, urine and the respiratory secretions in naturally infected children for several months [43,44]. In experimentally infected monkeys, MeV RNA is also found in PBMCs and respiratory secretions. In longitudinally studied rhesus macaques PBMC MeV RNA is cleared in three phases: (1) rapid decline coincident with clearance of infectious virus; (2) up to a 10-fold rebound followed by (3) a gradual decrease in RNA levels over 2 to 3 months (Figure). When no longer detectable in PBMCs, MeV RNA remains detectable in lymph nodes for at least 5 to 6 months, but is rarely found in the bone marrow likely reflecting limited initial replication in that tissue [27,32]. PBMC RNA is cleared more rapidly in macaques with a primed T cell response indicating a role for T cells in clearance of cells harboring RNA, as well as infectious virus [45]. Modeling the effects of immune parameters on levels of MeV RNA in PBMCs also indicates an important role for antibody in clearance [32].

During persistence, viral RNA in PBMCs and lymphoid tissue is most frequently detected in B lymphocytes but occasionally in T lymphocytes and monocytes as well [46]. The nature and intracellular location of the RNA are not known and the possibility of persistence in other types of cells has not been carefully examined. It is also not known when clearance from lymphoid tissue is complete.

MeV replication in secondary lymphoid tissue likely contributes both to alterations in composition and function of cells in circulation by inducing death of some populations and expansion of others [22,24]. For months to years after apparent recovery from measles, there is an increase in susceptibility to other infectious diseases accompanied by a decrease in level and diversity of circulating antibody to other pathogens [4,47,48]. Persistent RNA in lymphoid cells may contribute to immune response dysfunction by altering the ability of cells to proliferate in response to immune signaling [49,50]. Decreases in protective antibody due to loss of long-lived plasma cells making antibody to other pathogens may be due directly to MeV infection of these cells or to eviction from bone marrow niches during the acute disease.

On the other hand, persistent MeV RNA may contribute to development of life long immunity. Development of the immune response to MeV continues for months with ongoing antibody maturation and waves of functionally distinct T cells entering the circulation [27,46,51]. Continued B cell stimulation is also reflected in the ongoing appearance of antibody-secreting cells (ASCs) in circulation with maximal numbers 6–8 weeks after infection and maturation of the avidity of MeV-specific IgG. Lymph nodes show.sustained B cell proliferation, increasing numbers of germinal centers that become hyalinized late after infection and production of ASCs for at least 5–6 months with preferential bone marrow accumulation of ASCs secreting antibody to the hemagglutinin (H) protein [46].

Although infectious virus is cleared, these observations resemble B cell responses associated with chronic infection [52–54] and suggest that persistent MeV RNA induces ongoing synthesis of MeV proteins sufficient for stimulation of immune cells and B cell selection. Although the importance of antigen persistence in development of long-lived plasma cells is not clear [55,56], ongoing viral protein production may contribute to continued development of T cells, as well as supplying antigen to follicular dendritic cells to promote B cell selection.

T cells are essential for this process and at the time of clearance of infectious virus, most MeV-specific CD4⁺ and CD8⁺ T cells produce IFN-γ while later IL-17-producing cells that are associated both with autoimmunity and B cell maturation [57,58] are increased [27] (Figure). In addition, numbers of CD4⁺ CXCR5⁺ peripheral Tfh cells in circulation steadily increase [46]. In secondary lymphoid tissue, both Th17 and Tfh cells can promote germinal center formation and production of ASCs [58,59] and appearance in circulation likely reflects increasing numbers in tissue. Changes in levels of plasma cytokines also suggest shifts in activation of functionally distinct T cell populations over time with increased levels of IFN-γ early and IL-4, IL-13 and IL-10 later [60,61]. Presumably, the evolution of T cell and B cell differentiation over time after infection reflects as yet unidentified progressive changes in the lymphoid tissue immune response environment.

These data lead to the hypothesis that persistence of MeV RNA in lymphoid tissue is associated with ongoing synthesis of viral proteins that promote continued development of long-lived plasma cells that produce high avidity neutralizing antibody important for life-long protective immunity to measles.

Conclusions

Successful immune responses to MeV infection require clearance of infectious virus to prevent progressive disease, but also development of life long immunity to prevent reinfection. A primary site of MeV replication is in lymphoid tissue and this results in depletion of some cell populations and proliferation of others. CD8⁺ T cell-mediated clearance of infectious virus is compromised in individuals with inadequate immune function due to immune deficiencies or young age with a risk for progressive disease. The immune response to MeV infection in immunologically normal individuals leads to clearance of infectious virus, but persistence of viral RNA for many months. RNA persistence may contribute to immune suppression, but is also hypothesized to promote development of life long immunity through prolonged stimulation of immune responses in secondary lymphoid tissue.

Highlights.

• Clearance of measles virus usually occurs at the time of the rash, but may be incomplete in immunocompromised patients leading to progressive lung or nervous system infection.

• Children infected at a young age may fail to clear measles virus that acquires mutations that facilitate the development of the progressive nervous system infection subacute sclerosing panencephalitis many years later.

• Clearance of measles virus RNA from lymphoid tissue is slow and associated with continuous maturation of antibody and T cell responses that lead to life long immunity to reinfection.

• Persistence of measles virus RNA in lymphoid tissue may also impair immune responses to subsequent infection leading to prolonged increased susceptibility to other infections.