Werner Henle’s research while affiliated with The Children's Hospital of Philadelphia and other places

The T cell-mediated immune response of infectious mononucleosis (IM) patients to five Epstein-Barr virus (EBV)-determined nuclear antigens, EBNAs, and to the membrane antigen associated with growth-transformed cells (latent membrane protein, LMP) was measured by the leukocyte migration inhibition (LMI) assay. Two different antigen sources were used: extracts from cells that only expressed EBNA-1, EBNA-2, or LMP after transfection with the corresponding EBV-DNA fragment, and synthetic peptides deduced from the corresponding genes. Patients in the acute phase of the disease failed to respond to EBNA-1, -5, -6, and LMP, but became responsive during convalescence. The majority of the patients responded to EBNA-2 and/or EBNA-3 in the acute phase ( and , respectively). The response to EBNA-3 disappeared more often in convalescence than the response to EBNA-2; 6 of 15 patients were negative to EBNA-2 and 12 of 15 to EBNA-3 during recovery. In addition to its value in the assessment of host sensitization to virus EBV antigens, these studies and the derived hypotheses also provide certain predictions about the predominant antigen expression in the EBV-infected host under normal and pathological conditions that can be subjected to direct experimental tests.

Twenty-seven adults with a diagnosis of the chronic fatigue syndrome were enrolled in a double-blind, placebo-controlled study of acyclovir therapy. The patients had had debilitating fatigue for an average of 6.8 years, accompanied by persisting antibodies to Epstein-Barr virus early antigens (titers greater than or equal to 1:40) or undetectable levels of antibodies to Epstein-Barr virus nuclear antigens (titers less than 1:2) or both. Each course of treatment consisted of intravenous placebo or acyclovir (500 mg per square meter of body-surface area) administered every eight hours for seven days. The same drug was then given orally for 30 days (acyclovir, 800 mg four times daily). There were six-week observation periods before, between, and after the treatments. Three patients had acyclovir-induced nephrotoxicity and were withdrawn from the study. Of the 24 patients who completed the trial, similar numbers improved with acyclovir therapy and with placebo (11 and 10, respectively). Neither acyclovir treatment nor clinical improvement correlated with alterations in laboratory findings, including titers of antibody to Epstein-Barr virus or levels of circulating immune complexes or of leukocyte 2',5'-oligoadenylate synthetase. Subjective improvement correlated with various measures of mood. We conclude that acyclovir, as used in this study, does not ameliorate the chronic fatigue syndrome. We believe that the clinical improvement observed in most patients reflected either spontaneous remission of the syndrome or a placebo effect.

The authors present data from four patients with acute heterophil-negative mononucleosis-like illnesses who were initially thought to have primary Epstein-Barr virus (EBV) infections but eventually were shown to be seroconverting to the human immunodeficiency virus (HIV). Widespread lymphadenopathy and blood smears indistinguishable from those typically encountered in the acute phase of infectious mononucleosis were present in all cases. There were also varying combinations of fever, sore throat, and malaise, as well as mild abnormalities of hepatic function and elevated cold agglutinins (anti-I). Anti-HIV was detected by both enzyme-linked immunosorbent assay and Western blot techniques in all cases, with increasing titers noted in two of three serially studied cases. In one patient, a dual infection with the hepatitis B virus was also documented. Diagnostic possibilities in patients with acute mononucleosis-like illnesses dominated by prominent lymphadenopathy should include primary seroconversions to HIV.

Ten synthetic peptides containing 18-22 residues deduced from the amino-acid sequences of the EBV-encoded latent-infection-associated membrane protein (LMP) and the 2 principal nuclear antigens, EBNA-1 and EBNA-2, were tested for their ability to induce lymphokine release from sensitized T-cells of EBV-seropositive donors, as measured by the leukocyte migration inhibition assay (LMI). Only one of the 10 free peptides induced EBV-specific LMI. After Sepharose-coupling, 4 additional peptides were regularly active. In parallel, the sera of the same and other donors were screened for synthetic peptide-binding antibodies, as measured by an ELISA assay. Antibodies to 9 of the 10 peptides were detected in 25-80% of EBV-antibody-positive, but not in EBV-antibody-negative sera. A comparison of the two responses indicates that the humoral immune system tends to react with more epitopes on a given protein than the cellular immune system. Furthermore, the antibody reactivity pattern to different epitopes is more variable from individual to individual than the T-cell response. Also, the epitopes detected by antibodies and sensitized T-cells are often not identical.

An Epstein-Barr virus (EBV)-negative human lymphoid B-cell line, DG75, was stably transfected with recombinant selection vectors that carry a subfragment of the BamHI WYH region (nucleotides 44664 to 50628), the BamHI K fragment, or a subfragment of the EcoRI D region (nucleotides 166614 to 170149) of B95-8 EBV DNA. These fragments contain the coding exons for the EBV-determined nuclear antigens EBNA2 and EBNA1, and the membrane antigen LMP, respectively. Antigen expression of the cells was detected by immunofluorescence. EBNA2 was expressed in 80-100% of the transfected cells, in contrast to EBNA1 which was expressed in only 25%, and LMP in only about 5% of the cells. Humoral antibody responses were measured by immunofluorescence and compared to cellular immunity as determined by the leukocyte migration inhibition (LMI) technique. Extracts from transfected cell lines expressing EBNA1, EBNA2 or LMP elicited an LMI response with cells from healthy EBV-seropositive individuals whereas the extract from the parental DG75 cell line did not. The results demonstrate the value of stably transfected cell lines expressing a defined EBV antigen for the monospecific analysis of host responses to the EBV-encoded antigen complex in growth-transformed cells.

The sera of 12 patients with presumed chronic active Epstein-Barr virus (EBV) infection lacked antibody to a component of the Epstein-Barr nuclear antigen (EBNA) complex encoded by the BamHI K fragment of viral DNA. This anomaly, detected in ∼18% of sera obtained from patients with a diagnosis of “chronic mononucleosis,” was more often found in patients with severe disease (∼32%) who had objective clinical findings and markedly elevated antibody titers to EBV replicative antigens than in those patients with the “fatigue syndrome” (10%). The lack of antibody to the K nuclear antigen is specific because most of those who did not have antibody to the K antigen made antibody to other latent nuclear (EBNA 2) antigens or nuclear early antigens. Such patients are thus able to lyse immortalized cells, release nuclear products, and present them to the immune system. Three hypotheses are suggested to explain the lack of antibody to the K antigen: a viral mutation, a failure of immune recognition, or lack of in vivo expression of the antigen due to extensive viral replication. Lack of antibody to one component of EBNA may serve as an objective serological marker for certain patients with chronic EBV infection.

We investigated Epstein-Barr virus (EBV)-specific T cell responses in homosexual men with, and at risk for, AIDS. Westudied healthy laboratory workers, healthy homosexual men, and patients with AIDS-related complex or AIDS. The cytotoxic activity, absolute number of T4lymphocytes, and interleukin-2 (IL-2) production decreased, whereas the relative number of Ia+ lymphocytes increased with the extent of infection with the human immunodeficiency virus (HIV). Cytotoxic activity correlated positively with the number of T4lymphocytes (r = .56, P <.001) and the amount of IL-2 produced (r = .47, P < .01)but not with interferon production. Recombinant IL-2, but not γ interferon, could restore cytotoxic T cell activity to control levels in patients with early HIV infection. EBVspecific serological studies paralleled the T lymphocyte investigations. The increased EBV activity observed in progressive HIV infection may be related to a diminution in the autoreactivepopulation of the T4lymphocyte subset and may be amenable to IL-2 reconstitution.