Novel trimeric human cytomegalovirus glycoprotein B elicits a high-titer neutralizing antibody response.

Human cytomegalovirus (HCMV) is a major cause of disability in congenitally infected infants and in the immunosuppressed. There is currently no licensed prophylactic HCMV vaccine. The HCMV envelope glycoprotein B (gB) is considered a major vaccine target antigen based on its critical role in mediating viral-host cell fusion and thus viral entry. The natural conformation of HCMV gB within the viral envelope is a trimer, but there has been no reported success in producing a recombinant trimeric gB suitable for vaccine use. Phase II clinical trials of a monomeric recombinant gB protein demonstrated 50% efficacy in preventing HCMV infection in seronegative women of reproductive age, and in reducing viremia in solid organ transplantation recipients. We now report the production of a uniformly trimeric recombinant HCMV gB protein in Chinese ovary cells, as demonstrated by Western blot analysis under modified non-reducing conditions and size exclusion chromatography with multi-angle scattering. Immunization of mice with trimeric HCMV gB induced up to 11-fold higher serum titers of total gB-specific IgG relative to monomeric HCMV gB using Alum + CpG as adjuvants. Further, trimeric HCMV gB elicited 50-fold higher complement-independent and 20-fold higher complement-dependent HCMV neutralizing titers compared to monomeric HCMV gB using the fibroblast cell line, MRC-5, and up to 6-fold higher complement-independent and -dependent HCMV neutralizing titers using the epithelial cell line, ARPE-19. The markedly enhanced HCMV neutralizing activity in response to trimeric HCMV gB was also observed using an additional four distinct clinical HCMV isolates. These data support a role for trimeric HCMV gB as an important component for clinical testing of a prophylactic HCMV vaccine.

Trypsin and splanchnic protein turnover during feeding and fasting in human subjects.

Knowledge of the stimulatory effects of enteral and parenteral (intravenous) feeding on the synthesis and turnover of trypsin would help in the management of acute pancreatitis, because the disease is caused by the premature activation of trypsin. To investigate this, we labeled intravenous infusions with [1-(13)C]leucine and enterals with [(2)H]leucine and measured isotope enrichment of plasma, secreted trypsin, and duodenal mucosal proteins over 6 h by duodenal perfusion/aspiration and endoscopic biopsy. Thirty healthy volunteers were studied during fasting (n = 7), intravenous feeding (n = 6), or postpyloric enteral feeding [duodenal polymeric (n = 6), elemental duodenal (n = 6), and jejunal elemental (n = 5)]. All diets provided 1.5 g x kg(-1) x day(-1) protein and 40 kcal x kg(-1) x day(-1) energy. Results demonstrated that compared with fasting, enteral feeding increased the rate of appearance (71 +/- 4 vs. 91 +/- 5 min, P = 0.01) and secretion (546 +/- 80 vs. 219 +/- 37 U/h, P = 0.01) of newly labeled trypsin and expanded zymogen stores (1,660 +/- 237 vs. 749 +/- 133 units, P = 0.03). These differences persisted whether the feedings were polymeric or elemental, duodenal, or jejunal. In contrast, intravenous feeding had no effect on basal rates. Differential labeling of the plasma amino acid pool by enteral and intravenous isotope infusions suggested that 35% of absorbed amino acids were retained within the splanchnic bed during enteral feeding and that mucosal protein turnover increased from a fasting rate of 34 +/- 6 to 108 +/- 8%/day (P < 0.05) compared with no change after intravenous feeding. In conclusion, all common forms of enteral feeding stimulate the synthesis and secretion of pancreatic trypsin, and only parenteral nutrition avoids it.

Trypsin secretion and turnover in patients with acute pancreatitis.

Studies in humans have shown that pancreatic enzyme secretion is reduced during acute pancreatitis. It is not known, however, whether the reduction is due to impaired synthesis or disruption of the secretory pathway. The rate of secretion and turnover of trypsin was measured in 12 patients with acute pancreatitis of variable etiology and severity (median Ranson's score 2.5, range 0-5, 4 with severe necrotizing disease) and eight healthy volunteers by 4-h primed/continuous intravenous infusions of 1-(13)C-labeled l-leucine, and collection of pancreatic secretions by duodenal perfusion and sampling. Trypsin secretion was reduced from 476 +/- 73 to 153 +/- 60 U/h (means +/- SE, P = 0.005) in acute pancreatitis, with the greatest reductions being observed in patients with necrotizing disease (32 +/- 7 U/h, P = 0.003). The time for newly labeled trypsin to first appear in digestive juice was not, however, delayed in pancreatitis patients (87.2 +/- 11.1 vs. 94.7 +/- 4.9 min); on the contrary, there was an early appearance of newly labeled trypsin at 30 min in patients with severe necrotizing pancreatitis (P < 0.05). Calculated zymogen pool turnover was unchanged, but pool size was decreased (P = 0.01). Despite low rates of luminal secretion, trypsin continues to be synthesized in patients with acute pancreatitis. Our findings could be explained by post-Golgi leakage of enzymes from acinar cells or by loss of synthetic function in some cells with preservation in others.

Physiological effects of enteral and parenteral feeding on pancreaticobiliary secretion in humans.

In the nutritional management of digestive disorders, it is important to know the relative secretory and metabolic responses to enteral and parenteral feeding. Twenty-seven healthy volunteers were studied while receiving either oral drinks or duodenal infusions of a complex formula diet, duodenal or intravenous infusions of elemental (protein as free amino acids, low fat) formulae, or saline. Pancreaticobiliary secretory responses were measured by nasoduodenal polyethylene glycol perfusion and aspiration, while monitoring blood hormone and nutrient levels. Diets were matched for protein (1.5 g x kg(-1) x d(-1)) and energy (40 kcal x kg(-1) x d(-1)). Compared with placebo, all oroenteral diets stimulated amylase, lipase, trypsin, and bile acid secretion and increased plasma concentrations of gastrin and cholecystokinin, whereas intravenous feeding did not. The complex formula produced a similar response whether given as drinks or duodenal infusions. Changing the duodenal formula to elemental reduced enzyme secretion by 50%, independently of CCK. Higher increases in plasma insulin, glucose, and amino acids were noted with intravenous feeding. Delivering food directly to the intestine by a feeding tube does not reduce pancreaticobiliary secretion. Enteral "elemental" formulae diminish, but only intravenous feeding avoids pancreatic stimulation. Intravenous administration impairs metabolic clearance.