Epstein-Barr virus serology

CLINICAL

Epstein--Barr Virus Serology Jean H. Bowdre University of North Carolina Hospitals, Chapel Hill, North Carolina

he association of the Epstein--Barr virus (EBV) with infectious mononucleosis (IM) was discovered fortuitously during a seroepidemiologic study of the role of EBV in African Burkitt's lymphoma.1 A laboratory technologist whose serum had been consistently used as a negative control developed IM and, on return to work, was discovered to have seroconverted to EBV. Subsequent extensive serologic and virologic study has confirmed the role of EBV in the vast majority of cases of IM. The best laboratory test for IM in adults and older children is still a screening test for heterophile antibodies; if positive in a patient with the appropriate clinical picture, no further testing is indi-

T

cated. A latex agglutination test (Monolatex, Wampole Laboratories) is used for IM screening by the Clinical Immunology Laboratory at the UNC Hospitals. The antigen for this latex test is extracted from bovine erythrocyte membranes, and the sensitivity and specificity were 98.8% and 99.6%, respectively, relative to a differential red cell slide test in a study coordinated by the manufacturer. More than 90% of patients with IM develop heterophile antibodies during their acute illness. However, it has long been recognized that the heterophile antibody response frequently is absent in young children. A recent study2 has clarified this diagnostic problem. Sumaya and Ench studied 113 children with

symptomatic IM and primary EBV infection documented by EBV-specific serology and/or isolation of EBV from oropharyngeal secretions. They noted that the proportion of children with a positive heterophile test increased with age up to 4 years and then stabilized. The Paul-Bunnell-Davidsohn (PBD) differential absorption test was more sensitive than the rapid slide test in children under 4, but not in older children. Only 19% of 44 children under 4 years old with IM developed heterophile antibodies by a rapid slide test, compared to 32% positive by the PBD test. The corresponding percentages in 55 children 4--16 years old were 80% and 84%. In young children, continued on page 82

Epstein--Barr Virus a n d L y m p h o p r o l l f e r a t i v e Disorders: B a s i c Concepts a n d Diagnostic Approach Amalio Telenti Institute for Medical Microbiology, University of Berne, Berne, Switzerland

he human immune system, particularly its cellular components, exerts a careful control on the replication of Epstein--Barr virus (EBV). Deficits in immunosurveillance caused by the T-cell immunosuppressant agents used in transplantation, or by HIV infection, have resulted in increasing numbers of

T CIMNDC 11(6)81-96,1991

Elsevier

patients with EBV-related lymphoproliferative disorders. 1,11,16,29,3o,35,37 continued on page 85 Editor's Note: "Epstein-Barr Virus Serology" by Jean H. Bowdre is an expanded version of an article that appeared in the UNC Hospital's Bulletin of Laboratory Medicine 113:1-4, 1990. 0197-1859/91/$0.00 + 2.20

82 C L I N I C A L I M M U N O L O G Y Newsletter

therefore, specific EBV serology is needed if heterophile-based tests are negative and may be the test of choice for initial evaluation. The best established use for specific EBV serology is to diagnose heterophilenegative IM and to distinguish it from similar syndromes caused by cytomegalovirus, Toxoplasma gondii, or other agents. Serologic criteria have been used to propose links between EBV and a wide variety of other diseases, some of them well-established and some tenuous. 3'4 This review wilt describe the antigens used in EBV serology, the methods currently in use at the UNC Hospitals, and the principles and pitfalls of interpretation.

LB~ICLI6ATION _

TEST

~11

YEARS

LkTER

5,NTI-VCA s1 640 o- " (IgG) TITER 'tO__.

EBV-IgM TITER ANTI-EA

640

r,,@

-

16o40-

I0-

G40

.,.'~ ~"~

-

t6o-:

o 640

~NTI-EBNA iso TITER

40

,¢

~N-N.\%-N'r,.N--~

-

ig_

EUKOCYTE JooCULTURES s o % +

Description of EBV-Specific Antibodies Routine EBV serologics measure antibody responses to three main viral antigens. These are the viral capsid antigen (VCA), early antigen (EA), and EBV nuclear antigen (EBNA). These antigens have been described by immunofluorescence reactions using infected lymphocytes, and each is actually a complex consisting of more than one component. The time course for development of the various antibodies in primary EBV infection is shown in Figure 1. The sequence is the same whether the patient has IM or is asymptomatic, as are most children with primary EBV infection. IgM antibodies to VCA appear early in the course of infection and subsequently disappear. VCA-specific antibodies of the IgG class appear soon after IgM and persist for life at a stable or slowly decreasing level. By the time symptoms of IM develop, IgG anti-VCA titers are already high; therefore, testing paired sera for a rise in antibody titer usually is not helpful. Antibodies to EBNA develop late in the convalescent phase of illness (about four months after infection) and remain for life. Antibodies to EA appear during primary infection in about 70% of pa-

Vol. 1 !, No. 6, 1991

.,.,.,:~ ~ - ~

/

/

EBV IN

Ioo-

/

THROAT

50 -

,'

~

~--'~

~

Figure 1. Time course of development of EBV antibodies and of viral shedding in IM. Adapted from Henle, et al., Hum Pathol 5:551-656, 1974.

tients and traditionally have been considered a transient indicator of active infection. However, Horwitz et al. -s documented that persistence of anti-EA is common after recovery from IM, sometimes for months or years. Thus, the interpretation of anti-EA as an indicator of acute or reactivated infection is open to question.

Antigens A variety of EBV transformed human lymphoblastoid cell lines has been derived from patients infected with EBV. All of these cells carry latent EBV DNA in their nuclei. Some, called producer cell lines, undergo lytic infection and produce infectious EBV virions. Others, the non-producer lines, do not undergo the viral replicative cycle, and the EBV genome remains latent. Viral capsid antigens are expressed in producer lymphoid cell lines, such as

P3HRI cells. Only about 5-10% of the cells in such cultures (those cells undergoing lytic infection at the time) express the capsid antigens that are used to detect antibody by the indirect immunofluorescence test. EBV early antigens (EA) are non-structural viral proteins which can be induced in non-producer lymphoblastoid cell lines (eg, Raji) by superinfection with virus concentrates from producer cell lines or by induction with phorbol esters or sodium butyrate. 4 These cells (normally expressing only EBNA) then undergo an abortive infection in which the early antigens, but not VCA, are expressed in some of the cells. Two components of EA are seen by immunofluorescence; they are termed diffuse (D) and restricted (R) for their intracellular distribution. Most laboratories do not attempt to distinguish between antibodies to EA-D and EA-R, but it is useful to know that both may be

CLINICAL IMMUNOLOGY NEWSLETTER (ISSN 0197-1859) is issued monthly in one indexed volume per year by Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010. Subscription price per year: $125.00. For surface airlift add $37.00. Second-class postage paid at New York, NY, and at additional mailing offices. PcamWter: Send address changes to Clinical Immunology Newsletter, Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010. NOTE: No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use of operation of any methods, products, instructions for ideas contained in the material herein. No suggested test or procedure should be carried out unless, in the reader's judgment, its risk is justified. Because of rapid advances in the medical sciences, we recommend that the independent verification of diagnoses and drug dosages should be made. Discussions, views and recommendations as to medical procedures, choice of drugs and drug dosages are the responsibility of the authors. 0197-1859/91/$0.00 + 2.20

© 1991 Elsevier s c i e n c e Publishing C o . , Inc.

C L I N I C A L I M M U N O L O G Y Newsletter 83

Vol. 11, No. 6, 1991

seen by immunofluorescence, and that if anti-D is present it will obscure anti-R. Antibody to the D component is usually seen in acute IM; anti-R may appear during late convalescence. Anti-R alone may be present in young infants and was the persisting antibody in most of the post-IM cases studied by Horwitz et al. 5 EA-D is stable to methanol fixation, which destroys EA-R; both are stable to acetone. This differential fixation is the basis for distinction between the two components; unfortunately, no method has been discovered for selectively destroying EA-D. Since the diffuse component, if present, obscures the restricted in the immunofluorescence pattern, antibody to EA-R will be detected only if its titer exceeds anti-EA-D. EBV nuclear antigens (EBNA) are found in all EBV infected lymphoblastoid cell lines, but are masked when the cells are also expressing VCA or EA. Therefore, the non-producer cell lines (eg, Raji) are used to detect antibodies to EBNA. The anti-complement immunofluorescence test (ACIF) is the method of choice for detecting antibodies to EBNA. The test utilizes an amplification step in which the patient's antibody binds to EBNA and fixes complement, which is detected by a fluorescein-labeled antibody to complement. EBNA is normally expressed by nearly all cells in the culture. Six proteins have been identified to date as part of the EBNA complex. Nearly all persons infected with EBV produce antibodies to EBNA-1; the response to the other EBNA proteins is variable. Failure to produce antibodies to EBNA-1 has been noted in cases of prolonged or fatal IM and of EBV-associated B-cell malignancy. 6

Methods Currently the UNC Hospitals Special Microbiology Laboratory determines IgG antibodies to VCA by indirect immunofluorescence (IFA) in P3HR1 cells using a fourfold dilution series of the patient's serum from 1:20-1:5120. IgM anti-VCA is determined using commercially available substrate slides and a screening dilution of 1:10. A high-titer anti-human IgG reagent (Gullsorb, Gull Laboratories, Salt Lake City, Utah) is used to precipitate the

IgG from sera before testing for IgM antiVCA to avoid non-specific reactions and interference. Anti-EA is determined by IFA using commercially prepared slides and screening dilutions of 1:20 and 1:320. Anti-EBNA is determined by ACIF at a screening dilution of 1:5; control lymphocytes not infected by EBV are used to assure the specificity of the reaction. Methods have been developed to detect EBV antibodies by ELISA using synthetic peptides or affinity-purified antigens. 7 The correlation of these assays with standard immunofluorescence tests appears promising, but they are not yet in general use. With careful evaluation, ELISA procedures may permit more standardized interpretation of EBV serologic patterns. It is important to select the antigens with care because existing clinical correlations are based on empirically-defined fluorescence patterns. These patterns, as noted above, consist of multiple virus-specified proteins which may vary in both sequence and host response. Fortunately, the genome of EBV was the first of the herpesviruses to be completely sequenced. 8 As knowledge of the relevant antigens becomes more refined at the molecular level, diagnostic tests will become more precise.

Interpretation in the Normal Host The standard antibody patterns observed by immunofluorescence can be readily interpreted in the immunologically normal host (Table 1). Primary infection of seronegative persons, with or without symptoms of infectious mononucleosis, results in the early development of IgG and IgM antibodies to VCA. Anti-EA also is seen in 70% of such persons. According to Chang et al.,4 3.3% of IM patients tested during the first two weeks after onset of symptoms were negative for both IgG and IgM anti-VCA. A repeat serology would

be helpful to confirm the diagnosis in this small group. Antibodies to EBNA are typically absent during the acute phase of illness and begin to appear during convalescence, and approximately half of IM patients demonstrate anti-EBNA by three months after onset of symptoms. 4 Thus the presence of anti-EBNA is usually interpreted as consistent with EBV infection at some time in the past. Chang 4 reported that anti-EBNA was detected in ~<9% of acute phase IM patients (based on a compilation of published data). Rarely, the suspected IM patient demonstrates only low-titered (eg, 1:80) IgG anti-VCA, with no other antibodies detected. If the clinical picture warrants, a second serum should be tested two weeks later for a rising titer and/or appearance of the two "acute-phase" antibodies. We have seen patients who subsequently developed the typical "acute-phase" pattern and others in whom the "IgG only" pattern remained stable, presumably as a result of declining titers from a previous EBV infection.

Interpretation in the Immunologically Compromised Host Because EBV is a herpesvirus, it remains latent in the host after initial infection and may subsequently reactivate (undergo replicative cycles which may stimulate changes in antibody levels) with or without clinical symptoms. Suppression of cell-mediated immunity commonly permits reactivation of EBV. Such reactivation often results in fluctuating titers of IgG anti-VCA; anti-EA and even IgM anti-VCA may reappear. 3'6 In some patients with impaired T-cell function, the antibody response to EBNA is weak or absent. 6'9'1° Thus, compromised hosts frequently have unusual or fluctuating patterns of EBV antibodies. Numerous publications have discussed

TABLE 1. PATTERNS OF ANTIBODIES TO EBV ANTIGENS Anti-VCA IgG

IgM

Anti-EA

Anti-EBNA

Interpretation

+ +

+ -

_ _

+

Primary infection (-+IM) Past EBV infection

© 1991 Elsevier Science Publishing Co., Inc.

0197-1859/91/$0.00

+ 2.20

84 C L I N I C A L I M M U N O L O G Y Newsletter

the complex relationship between host immune function, EBV infection or reactivation, and overt disease. 6"9'1° The major cell type infected by EBV is the B lymphocyte, in which the virus usually enters a latent state and induces proliferation. Interferon, natural killer cells, and EBV-specific cytotoxic T cells control this proliferation in the immunologically normal host. The majority of the atypical lymphocytes seen in the peripheral blood of IM patients are CD8 positive T cells, which may impair normal T-cell responses. Immunodeficient persons may develop uncontrolled lymphoproliferative disease associated with EBV infection. Some of these patients, especially children with primary immunodeficiency diseases, fail to produce antibodies to EBNA; titers to VCA and EA may be unusually high or low. It is thought that a cytotoxic T-cell response is necessary for release of EBNA from the nuclear membrane of infected B cells before an antibody response can occur. Impaired T-cell function would also permit productive cycles of viral replication in B cells with production of VCA and EA. Patients with nasopharyngeal carcinoma usually have IgA antibodies to VCA and EA, and the fluctuations in titer may have prognostic significance. The chronic fatigue syndrome has received considerable attention in the United States within the last five years. Current consensus defines it as a clinical syndrome which sometimes, but not always, begins with a particularly severe or protracted case of IM. While statistical trends in EBV serologic panels have been reported for groups of chronic fatigue patients, there is no pattern that is diagnostic of this syndrome. 6 A word about antibody titers is appropriate here. While repeated testing can document changing titers in problem cases, there are no "threshold" levels which have proved useful for making a diagnosis. While the patterns of antibodies in the EBV panel are reproducible among sera, the actual titers are dependent on the reagents used and vary widely among experienced laboratories. Improved methods of fixation ~ and antigen induction, 4 as well as the commercial availability of a variety of substrate 0197-1859/91/$0.00 + 2.20

slides, have improved the ability to detect antibodies to EBV. We have recently expanded our dilution scheme because the vast majority of sera showing previous infection patterns had anti-VCA titers ~>320. We also screen for anti-EA at low and high dilutions and have found that among patients positive for lgG antiVCA and anti-EBNA only 11% lacked detectable anti-EA, 38% had titers ~>160, and 51% were positive at 1:20 but negative at 1:160. Based on this information, we have increased our high screening dilution for anti-EA to 1:320.

Other Diagnostic Approaches In diseases other than IM, it is difficult to define the role of EBV solely by serologic means. The potential for reactivation of herpesviruses under stress, including unrelated disease, complicates the interpretation of an increase in EBV antibody titers in the presence of a disease process such as hepatitis, ocular lesions, or malignancy. Does such a response indicate a causal role for EBV, or an antibody response to replicating EBV reactivated by an unrelated disease process? This problem is being approached by more direct means, including the detection of EBNA or EBV DNA in affected tissues. Ongoing work is increasingly documenting the molecular biology of the EBV genome in its latent and replicative states and its interactions with the immune system of a normal or compromised host. It is now possible to distinguish whether the EBV DNA in tissue is latent or replicating, based on probed Southern blots of restriction endonuclease fragments. Latent EBV DNA exists in lymphoid cells as a circular episome; in complete virions it is linear. Because EBV genomes contain varying numbers of terminal repeats, opening to the linear form may involve separating at any point between them. Then cleavage with restriction endonucleases generates small fragments of varying length which appear as a "ladder" on the gel if the DNA is linear; the latent circular episome yields only one much larger genomic fragment. Katz, Raab-Traub, and Miller 12 recently applied this technique to 35 tissue specimens from 30 patients with a variety of EBV-associated lymphopro© 1991 Elsevier Science Publishing Co.. Inc.

Vol. l l, No. 6, 1991

tiferative diseases. They found evidence of replicating EBV DNA in 40% of these tissues; the genome was in the latent episomal form in the rest of the samples studied. It is likely that such approaches will lead to molecular diagnostic techniques that will complement serologic data and shed light on the status of virushost interaction in the individual patient.

Summary Because of the ubiquitous nature of EBV, most people are infected with this virus by the time they are adults. People acquire the virus at an early age, earlier in developing countries and in socioeconomically deprived areas of the United States, where about 80% of 5-year-old children are seropositive. In economically privileged areas, only about 40-50% of children are seropositive by age 5. Infections during childhood are usually asymptomatic. In contrast, 50% of adolescents who become infected with EBV develop the fatigue, fever, pharyngitis, and atypical lymphocytosis characteristic of acute infectious mononucleosis (IM). Heterophil antibodies, which are the basis for screening tests for IM, usually appear in the serum of these patients. However, approximately 10% of patients (more commonly children) with EBV induced IM do not develop heterophil antibodies. For this reason, tests for specific antibodymediated immune responses to EBV may be necessary for diagnosis, ciN

References 1. Henle W, Henle G, Lennette ET: The EpsteinBarr virus. Scientific American 241:48-59, 1979. 2. Sumaya CV, Ench Y: Epstein-Ban virus mononucleosis in children. II. Heterophil antibodies and viral specific responses. Pediatrics 75:1011-1019, 1985. 3. Andiman WA: EBV-associated syndromes: a critical reexamination. Pediatr Infect Dis 3:198203, 1984. 4. Chang RS, Chan RCK, Cheng PNM, et al.: The diagnosis of infectious mononucleosis and Epstein-Barr virus-related diseases. In: de la Maza LM, Peterson EM (eds): Medical Virology V. Hillsdale, NJ: Lawrence Erlbaum Associates, pp. 95-139, 1986. 5. Horwitz CA, Henle W, Henle G, et al.: Longterm serological follow-up of patients for Epstein-Barr virus after recovery from infectious mononucleosis. J Infect Dis 151:1150-1153, 1986.

C L I N I C A L I M M U N O L O G Y Newsletter 85

Vol. 11, No. 6, 1991

6. Okano M, Thiele GM, Davis JR, et al.: Epstein-Barr virus and human diseases: recent advances in diagnosis. Clin Microbiol Rev 1:306-312, 1988. 7. Luka J, Chase RC, Pearson GR: A sensitive enzyme-linked immunosorbent assay (ELISA) against major EBV-associated antigens. I. Correlation between ELISA and immunofluorescence titers using purified antigens. J Immunol Methods 67:145-156, 1984.

Epsteln.l~

Disorders

continued from page 81

This article reviews some basic concepts about the relationship between EBV and lymphoproliferative disorders in the immunocompromised host, and the current status of virological diagnosis.

Pathogenesis of EBV-Related Lymphoproliferation In Vitro Experience After primary infection, EBV persists for life in the oropharyngeal epithelium and latently in B-lymphocytes. 33 If those B-lymphocytes are grown in vitro in the presence of autologous T-lymphocytes, no proliferation occurs; however, if T-cells are rendered dysfunctional by means of anti-T-cell antibodies or cyclosporine A, the B-lymphocytes will proliferate to establish immortalized clones. Long-term incubation of immortalized B-lymphocytes allows the accumulation of random chromosomal aberrations and the eventual prominence of a single clone. 15 These in vitro phenomena bear a remarkable similarity to the clinical, pathological, and molecular features of lymphoproliferative disorders that occur after transplantation or in patients with AIDS (Figure 1).

8. Kieff E, Liebowitz D: Epstein-Barr virus and its replication. In: Fields BN, et al. (eds): Virology, 2rid edition, New York: Raven Press, 1990. 9. Sixbey JW, Pagano JS: New perspectives on the Epstein-Barr virus in the pathogenesis of lymphoproliferative disorders. Curr Clin Top Infect Dis 5:146-176, 1984. 10. Henle W, Henle G: Epstein-Barr virus-specific serology in immunologically compromised indi-

viduals. Cancer Res 41:4222-4225, 1981. 11. Gallo D, Walen KH, Riggs JL: Improved immunofluorescence antigens for detection of IgM antibodies to EB viral capsid antigen and antibodies to EB virus nuclear antigen. J Clin Microbiol 15:243-248, 1982. 12. Katz BZ, Raab-Traub N, Miller G: Latent and replicating forms of Epstein-Barr virus DNA in lymphomas and lymphoproliferative diseases. J Infect Dis 160:589-598, 1989.

specimens of lymphoproliferative disease nations from intermediate to high grade lymphoid neoplasms, s, 11 occurring in the setting of transplantation, 17"31 in a significant percentage assoExperimental and clinical experience ciated with AIDS lymphomas, 29 and in suggests that the spectrum of disease exsome lymph nodes from HIV-infected tends from the simple accumulation of patients with diffuse lymphadenopathy. 13 proliferating EBV-infected cells (the in Both AIDS and transplantaton patients vivo correlate of immortalization) that have also been shown to have elevated could be controlled by immune reconstinumbers of EBV-infected B-cells in petution, to full transformation into an irreripheral blood. 1,31 versible malignant process 15'16 (Figure 1). The clinical and pathological presentaTherefore, considerable research effort tion of these disorders is diverse. Some has been directed at establishing prognospatients present with febrile illnesses and tic parameters to differentiate, at the mowidespread, multi-organ invasion with a lecular level, potentially reversible from polymorphihc infiltrate; others develop irreversible lymphoproliferation. localized monomorphic nodal or extraStudies of Clonality and Oncogenes nodal tumors that have been described as immunoblastic, large cell lymphomas, Studies of clonality by immunotyping or Burkitt-like lymphomas, or other denomiby immunoglobulin gene rearrangement Figure 1: Pathogenesis of EBV-related lymphoproliferation: parallels between in vitro experimenml dam and climcM, pathological, and molecular findings. IN VITRO

IN VIVO

]

Culture of infected B-lymphocytes

Primary infection of B-lymphocytes

Proliferation controlled by T-lymphocytes

Clinical or subclinical Infectious Mononucleosis Latency

Removal of T-cells or addition of cyclosporin or anti-T monoclonals

Immunosupression (transplant, AIDS)

Outgrowth and proliferation of B-cells

Polyclonal,oligoclonal or monoclonal proliferation of B-cells

Immortalized clones

Lymphoproliferative disorder (potentially reversible)

Random chromosomal damage: or experimental activation of oncogene c - m y c

Cytogenetic abnormalities and/or activation of c - m y c or other oncogenes

neoplastic clone

Neoplastic process malignant lymphoma (irreversible)

Clinical and Pathological Features Immunosuppressive protocols used in organ transplantation, particularly those with cyclosporine A, and anti-rejection treatment with anti-T antibodies such as OKT3 or ATG, have been associated with substantial EBV-morbidity and mortality: 1-10% of transplants are complicated by lymphoproliferative disorders. 3°'35 Similarly, up to 2% of patients infected with HIV develop nonHodgkin's lymphoma. 27 Viral sequences are found almost universally in tissue

© 1991 Elsevier Science Publishing Co., Inc.

0197-1859/91/$0.00 + 2.20