Identification of the critical attribute(s) of EBV gp350 antigen required for elicitation of a neutralizing antibody response in vivo

Vaccine prophylaxis with EBV glycoprotein 350 (gp350) subunit plus adjuvant has been demonstrated clinically to protect individuals against infectious mononucleosis (IM), but the specifications of the antigen required to elicit this protection has remained largely theoretical. Previous studies have shown that antibodies to gp350 comprise the principle component of EBV-neutralizing sera. Further, a murine monoclonal antibody against gp350 (clone 72A1) is able to prevent infection by the virus both in vitro and in vivo. In the present study, we identify the 72A1 epitope on recombinant gp350 antigen as the site required for binding to CD21 on human B cells. We also identify the need for conformational-dependence of the antigen to generate EBV-neutralizing antibodies in vivo. Further, we have characterized the glycosylation status and antigenicity profiles of both native and denatured CHO-produced soluble gp350 as well as non-glycosylated protein produced in Escherichia coli. Collectively our in vitro and in vivo data demonstrate the requirement for a conformationally accessible 72A1 epitope on gp350 to elicit EBV-neutralizing responses, and establish this as a critical attribute of this vaccine antigen. These data provide direction for commercial vaccine development, as the absence of this epitope on either E. coli-expressed or denatured gp350, may limit production and purification options for the antigen.     

The effects of nitroglycerin-methoxamine combination on infarct size in conscious dogs

The effect of nitroglycerin combined with methoxamine in reducing infarct weight was studied in conscious dogs. Ten minutes after permanent left anterior descending (LAD) coronary artery occlusion, 10 dogs received nitroglycerin (450 micrograms bolus IV, then 300 micrograms/min for 4 hours) and methoxamine as needed to maintain blood pressure and heart rate. No dogs in heart failure. Ten control dogs received saline solution. Dogs were sacrificed 3 days later. The region at risk of infarction was delineated by simultaneously perfusing the aortic root with Evans blue and the distal LAD artery with saline solution under equal pressures. Slices of stained hearts were incubated with tetrazolium to identify infarct. Total weight of left ventricle (LV), risk region, and infarct was measured. Nitroglycerin-treated dogs showed no difference from control dogs in infarct weight (26.2 +/- 5.9 gm +/- SE vs 27.7 +/- 5.6 gm), percent risk region/LV (36.0 +/- 1.4% vs 37.9 +/- 3.1%), or present infarct/LV (23.5 +/- 5.2% vs 24.8 +/- 4.9%). In a subgroup with risk region/LV less than or equal to 35%, nitroglycerin reduced infarct weight by 45% (8.8 +/- 8.5% vs 15.9 +/- 7.9%), percent infarct/LV by 49% (7.1 +/- 6.8% vs 13.8 +/- 6.6%), and percent infarct/risk region by 41% (23.0 +/- 22.0% vs 38.9 +/- 15.9%). Because of the small number of dogs in the study, differences were not significant. In dogs with risk region/LV greater than 35%, nitroglycerin had no effect. Thus, in dogs without overt heart failure, nitroglycerin may salvage ischemic tissue within small areas at risk of infarction, but the results are not definitive. However, our results clearly demonstrate that in the absence of failure, nitroglycerin does not reduce the size of large infarcts.     

Effects of calcium channel blocking agents on reperfusion arrhythmias

The effect of the calcium antagonists nifedipine (NF) and diltiazem (DT) on reperfusion after release of circumflex coronary artery (CX) occlusion was studied in open-chest dogs. Dogs were randomized to receive a bolus of 5 μg/kg NF (seven dogs), 1 μg/kg NF (nine dogs), or vehicle (nine dogs). After bolus, high and low dose NF dogs were infused with 1 μg/kg/min NF. All dogs then underwent 30 minutes CX occlusion followed by reperfusion. Dogs that did not develop ventricular fibrillation (VF) in the first 10 minutes of reperfusion were considered survivors. NF caused a dose-related increase in CX blood flow and decrease in mean arterial pressure (MAP), significant at the higher dose. Reperfusion VF occurred in five of nine low dose NF dogs, five of seven high dose NF dogs, and five of nine controls. Another 21 dogs were randomized to receive a bolus of 0.2 mg/kg DT (11 dogs) or vehicle (10 dogs). Infusion rates (and an additional bolus injection, if necessary) were chosen to produce a 10 to 20 mm Hg drop in MAP. CX occlusion and reperfusion were performed as above. Reperfusion VF occurred in 9 of 11 DT dogs vs 8 of 10 controls. Thus neither nifedipine nor diltiazem enhanced survival during reperfusion of myocardium previously subjected to 30 minutes of ischemia.     

T lymphocytes targeting native receptors

The adoptive transfer of T cells specific for native tumor antigens (TAs) is an increasingly popular cancer treatment option because of the ability of these cells to discriminate between normal and tumor tissues and the corresponding lack of short or long-term toxicities. Infusions of antigen-specific CD4⁺ and CD8⁺ T cells targeting viral antigens derived from Epstein–Barr virus (EBV) induce sustained complete tumor remissions in patients with highly immunogenic tumors such as post-transplant lymphoproliferative disease, although resistance occurred when the infused T-cell population had restricted antigen specificity. T cells specific for EBV antigens have also produced complete remissions of EBV-positive nasopharyngeal carcinomas and lymphomas developing in immunocompetent individuals, even though in these patients tumor survival is dependent on their ability to evade T-cell immunity. Adapting this strategy to non-viral tumors is more challenging, as the target antigens expressed are less immunogenic and the tumors lack the potent danger signals that are characteristic of viruses. The goals of current studies are to define conditions that promote expansion of antigen-specific T cells ex vivo and to ensure their in vivo persistence and survival by combining with maneuvers such as lymphodepletion, checkpoint inhibition, cytokine infusions, or genetic manipulations. More pragmatic goals are to streamline manufacturing to facilitate the transition of these therapies to late phase trials and to evaluate closely histocompatibility antigen (HLA)-matched banked antigen-specific T cells so that T-cell therapies can be made more broadly available.     

Prognostic significance of CD20 expression and Epstein‐Barr virus (EBV) association in classical Hodgkin lymphoma in Japan: A clinicopathologic study

To investigate the clinicopathological significance of CD20 expression and Epstein‐Barr virus (EBV) association in Hodgkin and Reed–Sterberg cells of classical Hodgkin lymphoma (CHL), CD20 expression and EBV positivity (by EBER in situ hybridization) were investigated in 389 CHL patients in Japan. They included 74 CD20‐positive cases (19%) and 315 CD20‐negative cases (81%). CD20‐positive cases showed significantly older age at onset (P = 0.018) and higher association with EBV (P = 0.002). Multivariate analysis identified EBV‐positivity (but not CD20‐positivity), presence of B symptoms, thrombocytopenia, elevated serum lactate dehydrogenase and performance status >1 as poor prognostic factors for overall survival (OS). We constructed a new prognostic model with these five factors classifying patients into three groups: low risk, 0–1 adverse factor; intermediate risk, 2–3 factors; high risk, 4–5 factors. This prognostic model could stratify the prognosis of CHL patients (P < 0.0001). For 144 patients (58%) classified into the low‐risk group, the 5‐year OS was 91%. For 92 patients (37%) in the intermediate group, the 5‐year OS was 66%; for 11 patients (5%) in the high‐risk group, the 5‐year OS was 36%. In conclusion, EBV is identified as an independent poor prognostic factor for CHL patients. Therefore, examination of EBV association in CHL is recommended as routine pathologic practice especially in countries where EBV infection prevails.