Background. Immunization of healthy women before pregnancy is a potential approach to providing increased levels of maternal antibody to newborns to protect them from infections occurring during the perinatal period and first months of life. Methods. Healthy nonpregnant Pima Indian women of childbearing age were randomized to receive one of two Haemophilus influenzae type b (Hib) conjugate vaccines [HbOC or Hib-meningococcal outer membrane protein complex (OMP)] or a 23-valent pneumococcal polysaccharide vaccine (PnPs). Infants received Hib-OMP vaccine at 2, 4 and 12 months of age. Vaccine safety and immunogenicity was evaluated in the women and their infants. Results. Anti-polyribose ribitol phosphate antibody titers were significantly higher in women in both Hib conjugate vaccine groups than in the pneumococcal vaccine group throughout the 37-month observation period. Antibody responses to HbOC vaccine were significantly higher than those to Hib-OMP. A subsequent booster dose of each Hib conjugate vaccine induced reactions and antibody responses similar to those of the first dose. Infants born to mothers immunized with Hib vaccines compared with PnPs had significantly higher polyribose ribitol phosphate-specific IgG antibody titers at birth and 2 months of age but lower antibody responses to Hib-OMP at 6 months and similar titers before and after boosting with Hib-OMP at 1 year of age. By contrast women immunized with PnPs did not have significantly elevated concentrations of pneumococcal-specific antibody at delivery, and their infants had pneumococcal antibody titers similar to those of infants born to mothers who did not receive pneumococcal vaccine before pregnancy. Conclusion. Hib conjugate vaccine given to women before pregnancy significantly increased the proportion of infants who had protective Hib antibody levels at birth and 2 months of age.

OBJECTIVE:We developed an enzyme-linked immunosorbent assay (ELISA) for the quantitation of respiratory syncytial virus (RSV) in respiratory secretions in intubated patients infected with RSV. METHODS:We compared the quantitative ELISA and a standardized plaque assay in intubated children <2 years of age who were mechanically ventilated for severe RSV disease and enrolled in a randomized double blind placebo-controlled treatment trial of a monoclonal antibody to the F protein of RSV (palivizumab; Synagis). We also examined the relationship between the concentrations of virus as measured by ELISA and of three inflammatory indices in respiratory secretions (white blood cell count, myeloperoxidase and eosinophilic cationic protein). RESULTS:Quantitative ELISA and plaque assay were highly correlated for both tracheal aspirates (r = 0.67, P = 0.001) and nasal wash specimens (r = 0.75, P = 0.001). Treatment with palivizumab significantly neutralized RSV in tracheal aspirates as measured by plaque assay. In contrast quantitation of RSV by ELISA was not affected by palivizumab treatment. This finding is consistent with results that were obtained in preliminary studies of RSV-containing media treated with monoclonal antibody, where we found that the ELISA measured virus whether antibody-bound or not. The inflammatory indices were not correlated with RSV concentration measured by ELISA or plaque assay. CONCLUSIONS:We conclude that this quantitative ELISA is a potentially useful tool for measurement of RSV concentration in respiratory secretions that may help elucidate the pathophysiology of acute RSV infection. Specific antiviral strategies for the treatment of RSV disease could be evaluated by this method.

Infant rats were passively immunized to determine the protective capacity of pneumococcal anticapsular antibodies. Animal-passaged strains of Streptococcus pneumoniae serotypes 1, 4, 5, 6b, 7f, 9v, 14, 18c, 19f, and 23f were used as challenge inocula (1-1500 cfu) in a model of pulmonary infection that resulted in bacteremia, meningitis, and death. From untreated control animals, histologic sections of lung demonstrated infiltrative pneumonia and lung homogenate cultures grew S. pneumoniae at concentrations of 10(3)-10(8) cfu per gram of lung tissue. A type-specific anti-capsular antibody serum concentration of 0.1-1.15 microg/mL resulted in a statistically significant reduction in mortality compared with the reduction in untreated controls, except for serotype 14, which required 2.32 microg/mL for a significant reduction in mortality. The serum antibody level that provided 50% reduction in mortality ranged from 0.1-3.5 microg/mL for all serotypes.

To evaluate the effect of passive immunization with anticapsular antibodies on nasopharyngeal carriage, two models of Streptococcus pneumoniae colonization were developed in infant rats. In a direct inoculation model, 3- to 4-day-old infant rats were intranasally inoculated with 2 x 10(5) cfu of S. pneumoniae type 3 or 6 x 10(3) cfu of S. pneumoniae type 23F. In an intralitter transmission model, 2 infant rats were intranasally inoculated with 10(3) cfu of pneumococcus type 3 or type 19F and placed in a cage containing 10 infant rats. Pretreatment with bacterial polysaccharide immune globulin led to a significant reduction in colonization of contact animals with S. pneumoniae type 3 or 19F in the intralitter transmission model (P < .05). No effect of immune globulin could be demonstrated in the direct inoculation model. These results indicate that systemic anticapsular antibodies conferred significant protection against nasopharyngeal acquisition by intralitter spread of S. pneumoniae type 3 and 19F.

We have developed a model of low-inoculum Streptococcus pneumoniae infection in infant rats. We challenged 4-day-old Sprague-Dawley pups via intraperitoneal or intrapulmonary injection of S. pneumoniae serotypes 1, 3, 4, 5, 6b, 7f, 9v, 14, 19f, and 23f. To achieve bacteremia with low inocula, it was necessary to passage the isolates in rats. Inocula of the 10 S. pneumoniae serotypes producing bacteremia in 50% or more animals ranged from 1 to 400 CFU. Virulence was similar by intraperitoneal and intrapulmonary routes. Lung specimens from animals challenged by the intrapulmonary route grew S. pneumoniae and demonstrated histologic evidence of focal infection. Meningitis was detected in 20 to 50% of bacteremic animals, and mortality invariably followed bacteremia within 24 to 48 h. This model of intrapulmonary infection uses low inocula of S. pneumoniae and results in bacteremia, meningitis, and death in infant rats.

BACTERIAL POLYSACCHARIDE IMMUNE GLOBULIN (BPIG) DOES NOT PROTECT INFANT RATS FROM NASAL COLONIZATION WITH STREPTOCOCCUS PNEUMONIAE (SP). • 1055

We challenged 4 day old Sprague-Dawley infant rats with SP serotypes 6B, 14, or 19 using an intrapulmonary injection model which produces histologic evidence of pneumonia. 1 day prior to challenge, treated animals were given low or high doses of subcutaneous (SC) BPIG (10 or 50 μg), while control animals received SC saline. Just prior to bacterial challenge, blood was obtained for measurement of serum antibody concentrations. Blood and cerebrospinal fluid were obtained for culture daily for 3 days; lung homogenate was cultured in a subset of animals. Mortality was monitored for 7 days. Mean serum anti-SP IgG concentrations for low and high dose BPIG were approximately 2.0 and 10.0 μg/mL, respectively, for types 6B and 19. Representative data for S. pneumoniae serotype 6B are:Table (*Denominators reduced after animals sacrificed for lung culture and histology). Lung homogenate culture were positive forS. pneumoniae. Similar data were obtained using serotypes 14 and 19. Preliminary conclusions are that anticapsular antibodies of 2-20 μg/mL provide 80-90% protection against lung challenge, independent of inoculum from 3 to 300 cfu.

Our preliminary studies suggested that infant rats exposed to SP via the intranasal route develop persistent nasal colonization with SP. In these experiments, 10μL of 0.5% low-gelling-point agarose containing approximately 2×105 SP serotypes 3, 4, 6 or 19 were placed onto the nares of infant rats (n=12 for each group). All pups became colonized with SP, without bacteremia. To develop this intranasal model of SP colonization further and attempt to induce invasive pneumococcal disease, 4 day old Sprague-Dawley infant rats received an intranasal inoculation of 2 × 105cfu SP serotype 3 in 10μL of 0.5% agarose. Six days following intranasal challenge, animals were randomized to either intrapulmonary injection of 0.025 ml of 0.5% agarose to produce lung injury or to no injection. Nasal aspirates (NA) and blood cultures (BCx) were monitored for SP just prior to the intrapulmonary injection (Day 6) and daily thereafter for three days (Days 7-9). (+NA and +BCx indicate positive nasal aspirates and blood cultures for SP, respectively): Table Two animals with bacteremia after lung puncture died one day after the bacteremia was detected. We conclude that: 1)this intranasal inoculation model produces reliable and persistent SP nasal colonization in the infant rat and 2)intranasal colonization followed by intrapulmonary puncture with agarose resulted in pneumococcal bacteremia and death in 20% of animals. Therefore, intranasal inoculation with SP serotype 3 together with intrapulmonary injection of agarose may become a suitable model of invasive pneumococcal disease following SP colonization in the infant rat.

OBJECTIVE:To test the efficacy of a recombinant endotoxin neutralizing protein as compared with saline in rats with Escherichia coli sepsis. DESIGN:Prospective, controlled animal trial. SETTING:Hospital animal research laboratory. SUBJECTS:Male Wistar rats challenged with intraperitoneal E. coli, O18ac K1, and treated 1 hr later with ceftriaxone and gentamicin. INTERVENTIONS:Recombinant endotoxin neutralizing protein, 50 mg/kg, was administered to rats 1, 2, or 3 hrs after E. coli challenge; saline was administered to control animals. MEASUREMENTS AND MAIN RESULTS:Quantitative bacteremia, 1 hr after challenge and before antibiotic administration, was not significantly different between treatment groups (range geometric mean 451 to 621 colony-forming units [cfu]/mL). The endotoxin concentration, measured immediately before recombinant endotoxin neutralizing protein administration, was significantly higher in animals sampled and treated at 2 hrs (geometric mean 260 EU/mL; 95% confidence interval 140 to 480 EU/mL), or 3 hrs (geometric mean 697 EU/mL; 95% confidence interval 307 to 1585 EU/mL) after E. coli challenge, compared with animals sampled and treated at 1 hr (geometric mean 17 EU/mL; 95% confidence interval 7 to 69 EU/ mL). Survival rate was significantly greater in rats treated with recombinant endotoxin neutralizing protein at 1 hr (23/27; p < .001) or 2 hrs (8/30; p < .01) after E. coli challenge than in controls (1/32). CONCLUSION:Administration of recombinant endotoxin neutralizing protein delayed up to 2 hrs after challenge with E. coli improves survival in antibiotic-treated rats with Gram-negative sepsis.

OBJECTIVE A recombinant endotoxin neutralizing protein was evaluated for its ability to ameliorate the effects of Escherichia coli sepsis in rats. DESIGN Prospective, controlled animal trial. SETTING Hospital animal research laboratory. SUBJECTS Wistar rats, treated with gentamicin 1 hr after challenge with intraperitoneal E. coli O18ac. INTERVENTIONS The animals received a recombinant endotoxin neutralizing protein, in doses of 5, 25, or 50 mg/kg, either 30 or 60 mins after challenge; controls received saline. MEASUREMENTS AND MAIN RESULTS Geometric mean serum endotoxin concentrations in endotoxin neutralizing protein-treated animals did not differ from control animals. Tumor necrosis factor concentrations in animals treated with endotoxin neutralizing protein 30 mins after challenge were significantly lower than controls. Animals treated with 25 or 50 mg/kg of endotoxin neutralizing protein 30 mins after E. coli challenge had significant improvements in survival compared with controls. Animals treated with 50 mg/kg of endotoxin neutralizing protein 60 mins after E. coli challenge had significant improvements in survival compared with controls. CONCLUSION Endotoxin neutralizing protein significantly reduces mortality from Gram-negative sepsis in an antibiotic-treatment model of E. coli peritonitis and bacteremia in rats, mediated by a neutralization of the biological effects of endotoxin.

A recombinant endotoxin-neutralizing protein (ENP) from Limulus polyphemus and a monoclonal IgM anti-lipid A antibody (HA-1A) were compared in a rat model of Escherichia coli sepsis. One hour after intraperitoneal challenge with 10(6) cfu of E. coli O18ac K1, animals were sensitized to endotoxin with lead acetate and treated with ENP, HA-1A, or saline, followed by ceftriaxone and gentamicin. Before treatment, 95% of rats had high-grade bacteremia and high serum endotoxin concentrations, which were similar in all treatment groups (P > .60). One hour after treatment, there was no bacterial growth in any blood sample, and endotoxin concentrations were significantly lower in the ENP group than in the HA-1A and saline groups (P < .01). At 24 h after challenge, survival in the ENP group was significantly higher than in the HA-1A saline group (P < .001). ENP improved survival in a rat model of E. coli sepsis with high mortality despite effective antibiotic therapy.