Which antigen has been associated with infectious mononucleosis infections?

Herpesviruses: Epstein–Barr virus (EBV)

EBV is the aetiological agent of infectious mononucleosis. It is the virus that is most strongly related to human malignant tumours (see also Chapter 31). EBV was first detected by electron microscopy in cultured cells from Burkitt's lymphoma (BL). BL is a malignant lymphoma that occurs in children in certain tropical areas of East Africa and New Guinea. The EBV genome and EBV nuclear antigen (EBNA) have been demonstrated in fresh biopsy specimens of BL. EBNA is analogous to the T antigens induced by papova- and adenoviruses. Virus products are usually not observed directly in the biopsy, but become detectable in the lymphoblastoid cell lines established from the tumours. The only cells that permit infection in vivo are B lymphocytes and certain endothelial cells. Cells which are infected with EBV either start to produce infectious virus in connection with a lytic infection or become transformed. The virus-genome in transformed cells is covalently bound to cell-DNA or occurs in the cytoplasm in the form of an episome represented by circular DNA. In vitro, too, B cells are easily transformed by EBV to form immortalized cell lines that replicate continuously in culture. As a rule only EBNA is expressed.

Burkitt's lymphoma most likely is not caused only by an EBV infection. In addition, one or more cocarcinogenic factors such as genetic predisposition, stimulation of the reticuloendothelial system or immune suppression have to be present. Holoendemic malaria (monthly exposures to the parasite) may be one factor of importance.

EBV has been implicated also in the pathogenesis of lymphoproliferative diseases in immunosuppressed recipients of organ transplants. In patients with an X-linked genetic defect, EBV may cause a severe lymphoproliferative disease.

Nasopharyngeal carcinoma is a common tumour in African and Chinese people. The tumour cells are of epithelial origin and contain EBV-DNA and EBNA in their nuclei. Although the complete virus is not produced, early antigens can be detected frequently. The patients have high titres of IgG and IgA antibodies against EBV capsid antigen. It has been shown that patients with the highest IgA titres have the poorest prognosis. The reason for this is not clear, but it may be due to a blocking of tumour cells by IgA for cytotoxic IgG antibodies. It seems likely that an EBV infection is related to the appearance of nasopharyngeal cancer, but the pathogenesis of the disease has not yet been defined.

Abstract

Human cytomegalovirus is generally acquired as a clinically silent infection, infrequently accompanied by a mononucleosis-like syndrome. This herpesvirus remains with the host for life and is a medically significant opportunist in two settings: (1) transplacental transmission leading to intrauterine infection and transplacental transmission during pregnancy that is responsible for significant neurodevelopmental damage in newborns, recognized as congenital disease, and, (2) transmission of infection with organ or tissue transplantation, as well as reactivation of latent virus in immunocompromised individuals, such as individuals with HIV-mediated acquired immunodeficiency syndrome (AIDS), allograft hematopoietic stem cell recipients and solid organ transplant recipients, where infection results in various diseases. Patterns of infection and disease with cytomegalovirus depend on a complex pathogen-host balance such that natural levels of immunity reduce, but do not prevent reactivation from latency or reinfection such that infection with multiple distinct strains of virus are relatively common. Antiviral drugs are available for prophylaxis and treatment of human cytomegalovirus, guided by rapid diagnostic testing. Vaccines are needed to prevent congenital disease as well as diseases affecting immunocompromised hosts.

EPSTEIN-BARR VIRUS

It is now believed that the Epstein-Barr virus (EBV) is the causal agent of infectious mononucleosis and also concerned in the aetiology of Burkitt's lymphoma (Epstein, Achong and Barr, 1964; Niederman et al., 1968). Some have even suggested that this virus, which is a member of the herpesvirus group, may be associated with other malignant tumours of man (Oboshi et al., 1969; Beltran et al., 1971). Although the diagnosis of EBV infection at this stage does not fall into the category of rapid diagnosis, it would appear wrong and incomplete to ignore this subject, expecially when immunofluorescence has played so important a part in its discovery. The researches on EBV have also shown the development of two antigens in cells infected by this virus which can be demonstrated by two different techniques: the first is based on the standard techniques described elsewhere in this book and the second is the technique of cell membrane immunofluorescence. It would seem profitable, therefore, to describe this latter technique, as it has a place in the investigation of many virus infections.

EBV was first detected by Epstein, Achong and Barr in 1964 in a continuous cell culture derived from a child with Burkitt's. lymphoma. This tumour had originally been described in East Africa but has also been found in the West Indies, New Guinea and certain parts of South America. The occasional case has now been described in other countries, including the United States. The presence of virus was soon confirmed in the many cell lines which have been established from these tumours and those examined by electron microscopy all showed it to have the morphological appearance of a virus of the herpes group. However, the virions appear incomplete and are never observed with membranes whilst most of the capsids appear empty. These electron microscopic observations may account for the failure of cultivation of the virus. Patients with Burkitt's lymphoma develop antibodies which can be used for the detection of virus in these continuous tumour tissue cell cultures.

Henle and Henle (1966) described the positive cells as exhibiting apple- green fluorescence, which had a granular appearance. The cells often contained larger fluorescent aggregates and many showed irregular outlines. The above authors failed to show any relationship of EBV to herpesvirus hominis, varicella, cytomegalovirus or reovirus types 1, 2 and 3, when they tested tissue cultures containing these viruses against antibody from patients with Burkitt's lymphoma. In established cultures only a small proportion of cells can be shown by immunofluorescence to contain virus; the proportion of cells staining specifically by immunofluorescence can be increased by using an arginine-deficient medium (Niederman et al., 1968).

A combined study by workers in Sweden, the United States and Kenya (Klein et al., 1968a) showed by immunofluorescence the existence of two types of antigen in Burkitt tumour cells. Two tests were carried out. In one, the cell membrane immunofluorescence test, cells from fresh biopsies or from continuous cultures established from Burkitt's lymphoma tumours were stained vitally by the indirect fluorescent antibody technique; the sera of patients with this tumour were used for the first stage of the fluorescent antibody test. Approximately 5 × 105 lymphoma cells were centrifuged and a known positive antiserum added to the sediment. This mixture was incubated for 30 minutes at 37 °C. After incubation, the cells were washed three times with a balanced salt solution and incubated for 20 minutes at 37 °C with a suitably diluted antihuman goat globulin conjugated with FITC. One drop of 50 per cent glycerol was added which appeared to preserve the staining and morphological appearance of the cells for several weeks. A drop of the suspension was placed on the slide under a coverslip for inspection by fluorescent microscopy. This technique was fully described by Klein et al. (1967). Klein et al. (1968a) referred to their second test as the anti-EBV test, which is basically the ordinary indirect immunofluorescent technique, using Burkitt's lymphoma cells which had been dried in air and fixed in acetone. Their findings showed that cell membrane immunofluorescence in established cell lines appeared to depend not only on the presence of EBV but also to a considerable degree on the extent of the persistence of virus infection in cultures. Biopsy cells in general and young continuous cultures exhibited strong membrane immunofluorescence activity but few, if any, EBV positive cells by the anti-EBV test. On occasions, in a few established cell cultures, the reverse was seen, with relatively large numbers of cells containing EBV antigen but little significant membrane immunofluorescent reaction. They concluded that different antigens were involved in the two tests.

As might be expected, the serological investigation of all children with Burkitt's lymphoma showed that they had antibodies to EBV of a high order and also, not unexpectedly, 50 per cent of matched controls in the same geographical location showed antibodies, but at a lower level. Although Burkitt's lymphoma is an extraordinarily uncommon condition in the United States, it was surprising when Henle and Henle (1967) showed that antibodies to EBV were acquired by normal American children at a similar age to that when antibodies to other common virus infections develop. The age of acquisition of these antibodies depended to a large extent on the socio-economic group, but nevertheless the great majority of persons acquired the antibody by the time they were 30 years of age. Essentially similar findings were described by Pereira, Blake and Macrae (1969) in London. Prospective studies on freshmen at Yale University showed the coincidence of the development of antibodies to EBV and the clinical picture of infectious mononucleosis; it appeared that this virus is the probable causal agent of the illness in the majority of patients with this syndrome (Evans, Niederman and McCollum, 1968; Niederman et al., 1968). Diehl et al. (1968) showed that leucocytes of patients in the acute stage of infectious mononucleosis contained EBV and that these infected cells could be grown easily in continuous culture. The presence of EBV in leucocytes facilitates the establishment of a continuous cell line; it is difficult to accomplish even the maintenance of normal white cells for any length of time. Klein et al. (1968b) showed not only that continuous cell lines could be developed from leucocytes of patients with infectious mononucleosis and that they contained EBV but that at least 1 per cent of such cells contained antigen in their membranes. Similarly, sera from patients with infectious mononucleosis when tested against continuous cell lines derived from Burkitt's lymphoma reacted with the cell membrane antigen; sera from patients prior to the onset of their illness did not react in this way. It became clear that the antibodies involved in membrane immunofluorescence were distinct from those reacting with EBV by the anti-EBV test and also from the ordinary heterophil antibodies which occur in infectious mononucleosis. Maximal membrane immunofluorescence activity is obtained long after the antibody to EBV and the heterophil antibody reach their peaks but both the EBV antibody and the membrane antibody persist for many years, in contrast to the heterophil reaction which disappears rapidly. Figure 74 illustrates a culture of EB3, a standard tissue cell culture of Burkitt's lymphoma, stained with a human serum and antihuman globulin conjugate.

A combined study between Finnish and American workers (Klemola et al., 1970) investigated the relationship of EBV and cytomegalovirus in illnesses with a clinical picture of infectious mononucleosis but which had a negative heterophil agglutination test. Forty-four such cases were investigated and 19 were found to be caused by cytomegalovirus. High anti-EBV titres were found in 8 patients, indicating a current or very recent infection, and 12 had lower titres, but still sufficiently high to be compatible with either current or past infection. The conclusion drawn by these workers was that in heterophil antibody negative cases of infectious mononucleosis, the principal aetiological agents were EBV and cytomegalovirus, although a proportion may still be caused by other as yet unidentified agents.

Immunofluorescence has thus an important role to play in the diagnosis of Burkitt's lymphoma but a much more important one in diagnosing infectious mononucleosis. In the latter illness, it is rare to be able to obtain paired sera taken at the beginning and in the convalescent stage of the illness and to demonstrate in them a rising titre of antibodies to EBV, but the high level of antibodies which occurs in this illness may assist in making a diagnosis. Schmitz and Scherer (1972) demonstrated by immunofluorescence specific IgM antibodies to EBV. These antibodies were found only over a period of 2-3 months after onset of symptoms; the demonstration of these antibodies indicates recent infection and may therefore be useful in making a diagnosis. However, it is only in those cases where the heterophil reaction is negative that the examination of the patient's sera for antibodies to EBV by immunofluorescence can lead to a comparatively rapid diagnosis of the illness. The subject is discussed further in Chapter 18.

The presence of rheumatoid factor can give a false positive reaction in tests by immunofluorescence for virus-specific IgM when unabsorbed sera are used (see Chapter 18). Unabsorbed sera giving positive results by immunofluorescence in tests for EBV-specific IgM should be tested by the latex test for rheumatoid factor. The Rose-Waaler test for rheumatoid factor is unsuitable when EBV infection is suspected, because sera of patients with clinical infectious mononucleosis will give a false positive result (Bell, 1979).

Even more recently Nikoskelainen, Neel and Stevens (1979) have found that EBV IgA antibody directed against the capsid antigen appears early and disappears within 10 weeks. This might be a potentially useful diagnostic tool in rapid virus diagnosis of EBV infections.

Epstein–Barr Virus (Human Herpesvirus 4)

Epstein–Barr virus (EBV) disease in the immunosuppressed individual ranges from systemic presentations such as infectious mononucleosis to more localized disease including hepatitis, pneumonitis, gastrointestinal, hematological manifestations, and neoplasia. The hematological disorders can be serious, causing hemophagocytic lymphohistiocytosis or macrophage activation syndrome, which results in the uncontrolled activation of immune cells.

An important property of EBV is its oncogenic capacity caused by the integration of viral DNA into the host cells׳ genome. During immunosuppression, the lack of cytotoxic T cell responses allows EBV to transform lymphocytes, potentially resulting in their uncontrolled proliferation. In particular, post-transplantation lymphoproliferative disorders (PTLDs) pose a great challenge. EBV-related PTLD usually arises from B cells but T and NK cell-derived PTLD is also seen. The latter may be caused by increased viral replication in the immunosuppressed, allowing for infection of cells normally not targeted by EBV. About 50% of PTLDs are EBV related and mostly occur within the first year after transplantation. This disease entity is decreasing in incidence, while late presenting EBV-negative PTLD is seen more frequently. A change in induction regimens partly explains this. The application of polyclonal anti-lymphocyte antibodies, which were administered in higher doses in the past, is a known risk factor for PTLD development. An elevated risk for PTLD is seen in EBV seronegative recipients who may contract a primary infection, making the pediatric population particularly vulnerable. This is why the serostatus before transplantation should be determined and most centers measure viral capsid antigen (VCA) IgG and Epstein–Barr nuclear antigen-1 (EBNA-1) IgG.

The type of organ transplant is another determinant of PTLD risk and the highest incidence is observed after hematopoietic stem cell transplantation, particularly transplantation of haploidentical cells. Good prediction models are, however, lacking, and the individual risk is difficult to ascertain.

Clinical presentation of PTLD can range from asymptomatic to fulminant organ failure and tumor lysis. Early signs include fever, lymph node or tonsil enlargement and changes in the hematogram. Due to organ damage such as hepatitis, colitis, pneumonia and cerebritis, symptoms can be very diverse and mimic an infectious disease process or organ rejection. PTLD frequently involves extranodal sites such as the gut, solid allograft and the central nervous system.

Diagnosis is established by histology from lesion biopsy and disease can be categorized into six subclasses defined by the World Health Organization. The detection of EBV nucleic acid or proteins in the tissue establishes a link to the EBV-associated pathogenesis. Monitoring for EBV-related disease is usually performed by measuring plasma EBV DNA by PCR. Generally, high-level EBV DNAemia or a rapid increase in blood EBV DNA load is correlated with an elevated risk of developing PTLD. However, there is a lack of standardization of thresholds, frequency and time points of viral load measurement.

Due to the lack of antiviral drugs with activity against EBV, preemptive strategies and therapy involve reducing the immunosuppressive regimen as an initial step. This conversely risks provoking an acute transplant rejection as seen in 37% of patients followed in a prospective trial. If this approach fails, administration of rituximab – a B cell depleting antibody directed towards CD20 – can be considered in CD20 positive PTLD. Rituximab was the first treatment to improve prognosis drastically. Combined with the reduction of immunosuppression, rituximab achieves a response rate of 44%–79% and complete remissions in 20% to 55%. If the PTLD is still unresponsive to treatment, the next step involves the use of chemotherapy. In rare cases of PTLD such as peripheral T cell lymphoma, Hodgkin’s lymphoma, Burkitt’s lymphoma and central nervous system lymphoma, chemotherapy is considered as first-line therapy. Further treatment modalities include the adoptive transfer of EBV-specific T cells, surgical resection and radiotherapy.

Infectious Mononucleosis

Primary infection with EBV in childhood is usually asymptomatic but when delayed until adolescence or early adulthood can manifest clinically as IM, a self-limiting lymphoproliferative disease characterized by a quartet of symptoms – sore throat, cervical lymphadenopathy, fever and fatigue. IM is associated with the massive expansion of activated CD8+ T cells that are reactive predominantly to EBV lytic cycle antigens with some limited responses against latent antigens. Immunodominant CD8+ T cell responses in IM are focussed on immediate early proteins (e.g., BZLF1 and BRLF1) and early proteins (e.g., BALF2, BMRF1, BORF2) and these reactivities are subsequently maintained in the CD8+ T cell memory pool at high levels. Activated CD4+ T cell responses specific for EBV epitopes in EBV lytic and latent antigens are also elevated in IM patients but fall rapidly as the disease resolves. IM appears to result in the global down-regulation of the alpha chain of the IL-15 receptor on T cells and NK cells, an effect which lasts for years after acute infection and may influence other disease risks. The incidence of IM is low in developing countries where asymptomatic primary infection predominantly occurs in childhood. In certain poorly defined situations IM-like symptoms can persist resulting in chronic active EBV infection (CAEBV) associated with elevated antibody titers to virus lytic antigens but low titers to the EBV-encoded nuclear antigens (EBNAs) along with elevated EBV infection in circulating T cells and NK cells. CAEBV appears to originate from an EBV-infected lymphoid progenitor and is associated with intragenic deletions in the EBV genome.

Pathogenesis

EBV infection is transmitted via the oral route and is generally asymptomatic. As such, knowledge of the primary infection has come from the study of IM. These individuals shed virus in saliva and throat washings. However, the source of this virus remains contested. It is generally assumed that because B cell-deficient individuals show no sign of EBV infection in the throat, initial infection of a naïve host is B cell-dependent. However, it is becoming more widely accepted that epithelial cells may also be sites of viral replication, with studies suggesting that virus bound to the surface of a B cell is highly efficient at infecting epithelial cells. Interestingly, recent evidence further suggests that virus released from B cells is defective for B cell infectivity but shows enhanced infection of epithelial cells, while virus released from epithelial cells has the opposite phenotype.

EBV establishes a latent infection in B cells at the site of primary infection, the tonsillar tissue. Studies suggest that latently infected B cells express a memory phenotype, and it has been proposed that infection of naïve B cells with EBV mimics the process of B cell differentiation, resulting in activation and proliferation of the infected cell population, and thus the production of an expanded pool of latently infected memory cells. It is generally accepted that the cell-mediated immune response brings the proliferating B cells under control. The memory pool of latently infected B cells, which circulates through the body, is able to disperse the latently infected cells throughout the lymphoid system. Individuals latently infected with EBV will have peripheral blood B cells that harbor virus and, following clearance of the primary infection, these individuals will continue to shed low levels of infectious virus via the oral cavity. This virus comes from the latent B cell reservoir. It is thought that memory B cells containing latent virus may undergo reactivation when they receive an activation signal, and that such cells, which localize near mucosal surfaces, would be capable of transmitting lytic virus to epithelial cells where viral replication and subsequent shedding can occur.

In a healthy individual with an intact immune system, EBV persists in this form for the life of the host with no clinical manifestations. The latent pool is constantly maintained by the virus moving forward and backward through the various forms of latency as required, and infectious virus is sporadically shed from the oral cavity with the potential of infecting other susceptible hosts. However, when immune suppression occurs, either through disease or drug intervention, this balance is destroyed and the individual is put at risk of EBV-associated disease.

Which test is most specific for infectious mononucleosis?

What is EBV antigen?

What is EBV antibody to viral capsid antigen IgG?