Neil Steven

Primary infection with virus can stimulate a vigorous cytotoxic T cell response. The magnitude of the antigen-specific component versus the bystander component of a primary T cell response remains controversial. In this study, we have used tetrameric major histocompatibility complex-peptide complexes to directly visualize antigen-specific cluster of differentration (CD)8+ T cells during the primary immune response to Epstein-Barr virus (EBV) infection in humans. We show that massive expansion of activated, antigen-specific T cells occurs during the primary response to this virus. In one individual, T cells specific for a single EBV epitope comprised 44% of the total CD8+ T cells within peripheral blood. The majority of the antigen-specific cells had an activated/memory phenotype, with expression of human histocompatibility leukocyte antigen (HLA) DR, CD38, and CD45RO, downregulation of CD62 leukocyte (CD62L), and low levels of expression of CD45RA. After recovery from AIM, the frequency of antigen-specific T cells fell in most donors studied, although populations of antigen-specific cells continued to be easily detectable for at least 3 yr.

PURPOSE: To conduct a phase I clinical trial with a second-generation oncolytic herpes simplex virus (HSV) expressing granulocyte macrophage colony-stimulating factor (Onco VEXGM-CSF) to determine the safety profile of the virus, look for evidence of biological activity, and identify a dosing schedule for later studies. EXPERIMENTAL DESIGN: The virus was administered by intratumoral injection in patients with cutaneous or s.c. deposits of breast, head and neck and gastrointestinal cancers, and malignant melanoma who had failed prior therapy. Thirteen patients were in a single-dose group, where doses of 10(6), 10(7), and 10(8) plaque-forming units (pfu)/mL were tested, and 17 patients were in a multidose group testing a number of dose regimens. RESULTS: The virus was generally well tolerated with local inflammation, erythema, and febrile responses being the main side effects. The local reaction to injection was dose limiting in HSV-seronegative patients at 10(7) pfu/mL. The multidosing phase thus tested seroconverting HSV-seronegative patients with 10(6) pfu/mL followed by multiple higher doses (up to 10(8) pfu/mL), which was well tolerated by all patients. Biological activity (virus replication, local reactions, granulocyte macrophage colony-stimulating factor expression, and HSV antigen-associated tumor necrosis), was observed. The duration of local reactions and virus replication suggested that dosing every 2 to 3 weeks was appropriate. Nineteen of 26 patient posttreatment biopsies contained residual tumor of which 14 showed tumor necrosis, which in some cases was extensive, or apoptosis. In all cases, areas of necrosis also strongly stained for HSV. The overall responses to treatment were that three patients had stable disease, six patients had tumors flattened (injected and/or uninjected lesions), and four patients showed inflammation of uninjected as well as the injected tumor, which, in nearly all cases, became inflamed. CONCLUSIONS: Onco VEXGM-CSF is well tolerated and can be safely administered using the multidosing protocol described. Evidence of an antitumor effect was seen.

Epstein-Barr virus (EBV) can display different forms of latent infection in B-cell lines in vitro; however, the types of infection normally established by the virus in vivo remain largely unexplored. Here we have approached this question by analyzing the types of viral RNAs present in mononuclear cells freshly isolated from the blood of 14 infectious mononucleosis patients undergoing primary EBV infection and 6 long-term virus carriers. Reverse transcription-PCR amplifications were carried out with a panel of oligonucleotide primers and probes which specifically detect (i) the EBER1 RNA common to all forms of latency, (ii) transcripts either from the Cp and Wp promoters generating all six nuclear antigen (EBNA1, -2, -3A, -3B, -3C, -LP) mRNAs or from the Fp promoter generating a uniquely spliced EBNA1 mRNA, (iii) the latent membrane protein (LMP1 and 2A) mRNAs, and (iv) the BZLF1 mRNA, an immediate-early marker of lytic cycle. Viral transcription in infectious mononucleosis mononuclear cells (and in the B-cell-enriched fraction) regularly included the full spectrum of latent RNAs seen during EBV-induced B-cell growth transformation in vitro, i.e., EBER1, Cp/Wp-initiated EBNA mRNAs, and LMP1/LMP2 mRNAs, in the absence of lytic BZLF1 transcripts. In addition, transcripts with the splice pattern of Fp-initiated EBNA1 mRNA, hitherto seen only in vivo in certain EBV-positive tumors, were frequently detected. In long-term virus carriers, the mononuclear cells were again positive for latent (EBER1) and negative for lytic (BZLF1) markers; Cp/Wp-initiated RNAs were not detected in these samples, but in several individuals it was possible to amplify both Fp-initiated EBNA1 mRNA and LMP2A mRNA signals. We suggest (i) that primary infection is associated with a transient virus-driven expansion of the infected B-cell pool through a program of virus gene expression like that seen in in vitro-transformed cells and (ii) that long-term virus carriage is associated with a switch from Cp/Wp to Fp usage and thus to a more restricted form of latent protein expression that may render the infected cells less susceptible to recognition by the virus-specific cytotoxic T-cell response.

BACKGROUND: The role of prophylactic antibacterial agents after chemotherapy remains controversial. METHODS: We conducted a randomized, double-blind, placebo-controlled trial in patients who were receiving cyclic chemotherapy for solid tumors or lymphoma and who were at risk for temporary, severe neutropenia (fewer than 500 neutrophils per cubic millimeter). Patients were randomly assigned to receive either 500 mg of levofloxacin once daily or matching placebo for seven days during the expected neutropenic period. The primary outcome was the incidence of clinically documented febrile episodes (temperature of more than 38 degrees C) attributed to infection. Secondary outcomes included the incidence of all probable infections, severe infections, and hospitalization but did not include a systematic evaluation of antibacterial resistance. RESULTS: A total of 1565 patients underwent randomization (784 to placebo and 781 to levofloxacin). The tumors included breast cancer (35.4 percent), lung cancer (22.5 percent), testicular cancer (14.4 percent), and lymphoma (12.8 percent). During the first cycle of chemotherapy, 3.5 percent of patients in the levofloxacin group had at least one febrile episode, as compared with 7.9 percent in the placebo group (P<0.001). During the entire chemotherapy course, 10.8 percent of patients in the levofloxacin group had at least one febrile episode, as compared with 15.2 percent of patients in the placebo group (P=0.01); the respective rates of probable infection were 34.2 percent and 41.5 percent (P=0.004). Hospitalization was required for the treatment of infection in 15.7 percent of patients in the levofloxacin group and 21.6 percent of patients in the placebo group (P=0.004). The respective rate of severe infection was 1.0 percent and 2.0 percent (P=0.15), with four infection-related deaths in each group. An organism was isolated in 9.2 percent of probable infections. CONCLUSIONS: Among patients receiving chemotherapy for solid tumors or lymphoma, the prophylactic use of levofloxacin reduces the incidence of fever, probable infection, and hospitalization.