Haemophilus influenzae type b immunization of children with sickle cell diseases.

Haemophilus influenzae type B vaccine is recommended for children 1.5 to 6 years of age with sickle cell anemia, but the adequacy of their response is unknown. A total of 69 children with sickle cell syndromes, 1.5 to 5.6 years of age, were immunized with two vaccines alternatively, single blind. PRP vaccine was given to 36 children and a diphtheria toxoid conjugated vaccine, PRP-D, was given to 36. Coded pre- and postvaccine sera were tested by radioimmunoassay for anti-PRP antibody. The groups did not differ in age distribution or type of sickle hemoglobinopathy. Preexisting antibody levels were low in both vaccine groups; 65% were less than 0.15 microgram/mL. The vaccines were safe but associated with frequent minor reactions. PRP-D gave higher geometric mean titers and mean fold titer increase than PRP in all children (15.58 micrograms/mL [234-fold] v 2.63 micrograms/mL [29-fold]) and in the subgroups 1.5 to 2.5 years of age or with pretiter values less than 0.15 microgram/mL. Titers for 64% of children receiving PRP and 94% receiving PRP-D were greater than or equal to 1.0 microgram/mL. Thus, both vaccines were useful in this population, but PRP-D was more immunogenic. Duration of antibody levels postvaccination, booster responses, and PRP-D immunogenicity in younger children with sickle cell syndromes all require further study.

Persistence of antibody and booster responses to reimmunization with Haemophilus influenzae type b polysaccharide and polysaccharide diphtheria toxoid conjugate vaccines in children initially immunized at 15 to 24 months of age.

To evaluate the persistence of antibody after Haemophilus influenzae type b polysaccharide vaccine (PRP) and H influenzae type b polysaccharide diphtheria toxoid conjugate vaccine (PRP-D), a group of 141 infants initially immunized between 15 and 24 months of age were studied 1 year later. One month after immunization with PRP, the man anti-PRP antibody level was 0.27 microgram/mL and 1 year later was 0.29 microgram/mL (not significant). In the group immunized with PRP-D, the levels were 1.34 micrograms/mL and 1.20 micrograms/mL (not significant), respectively. To evaluate immunogenicity and safety of a booster immunization 1 year after initial vaccination, subjects were randomly assigned to receive saline, PRP, or PRP-D. In addition, 73 age-matched previously unimmunized subjects were vaccinated with PRP or PRP-D. In all groups, adverse reactions were minor and resolved by 48 hours. Subjects receiving booster immunization with PRP or PRP-D had significantly greater antibody responses than children of the same age receiving their first dose of vaccine. The highest antibody levels were achieved in children initially immunized with PRP-D, regardless of whether the booster vaccine was PRP (112.8 micrograms/mL) or PRP-D (122.0 micrograms/mL) (not significant). Antibody levels after booster vaccine were significantly lower in those initially given PRP compared with those initially given PRP-D but significantly higher than in age-matched previously unimmunized control subjects (PRP booster 3.16 micrograms/mL vs control of 0.62 microgram/mL [P less than .05]; PRP-D booster 12.31 micrograms/mL vs control 2.31 micrograms/mL [P less than .01]).

Depression of anticapsular antibody after immunization with Haemophilus influenzae type b polysaccharide-diphtheria conjugate vaccine.

Invasive Haemophilus influenzae type b infections have been observed in the week after immunization with capsular polysaccharide vaccine. We sought to document depression of antibody concentrations after immunization of 18-month-old infants with H. influenzae type b capsular polysaccharide-diphtheria conjugate vaccine. All 9 infants with detectable preimmunization anticapsular antibody had depression of antibody concentrations on the second day after immunization (P = 0.002). By Day 7 all had achieved anticapsular antibody concentrations greater than 0.15 micrograms/ml, a level believed to provide protection to immediate challenge with H. influenzae type b. Of those without detectable preimmunization antibody, 7 of 21 (33%; 95% confidence interval, 11 to 56%) had not achieved concentrations of greater than 0.15 mg/ml 1 week after immunization. We conclude that there is depression of anticapsular antibody concentrations during the first week after immunization with H. influenzae type b capsular polysaccharide-diphtheria conjugate vaccine. We speculate that H. influenzae type b infections after immunization with H. influenzae type b vaccines may be the result of: (1) low antibody concentrations because of either depression of antibody concentrations or failure to develop antibody; and (2) exposure to H. influenzae type b. Depression of antibody concentrations could be explained by binding of in vivo antibody to the vaccine.

Capsular polysaccharide Haemophilus influenzae type b vaccine: clinical and immunologic responses to two vaccines.

The clinical reactions and immunologic responses to two Haemophilus influenzae type b capsular polysaccharide vaccines were studied in 24- to 36-month-old children. A candidate vaccine (Connaught) induced greater than or equal to 1.0 microgram/ml of antipolysaccharide antibody in 37 (61.7%) of 60 infants 24 to 26 months of age; this compared with 8 (38.1%) of 21 infants 24 to 36 months of age given a licensed vaccine (Praxis). Reactions to both vaccines within 24 hours of administration were minimal; a smaller percentage of recipients of the candidate vaccine were asymptomatic (P less than 0.05). The lower levels of antibody production than previously reported may necessitate a reevaluation of current recommendations.

Vaccination of 18-month-old children with conjugated polyribosyl ribitol phosphate stimulates production of functional antibody to Haemophilus influenzae type b.

Eighteen-month-old children were immunized with polyribosyl ribitol phosphate (PRP) of Haemophilus influenzae type b or with PRP that had been conjugated to diphtheria toxoid. Conjugated vaccine stimulated significant mean increases in antibody titer as measured by radioimmunoassay and bactericidal effect, as well as a modest increase in opsonizing activity. In contrast unconjugated vaccine caused lesser albeit significant rises in antibody titer, but a negligible antibacterial effect. These results suggest that vaccinating infants with conjugated PRP is more likely to stimulate production of antibodies that are protective against systemic infection caused by H. influenzae type b than vaccinating with unconjugated PRP.

Analysis of lipidation of recombinant Lyme vaccine protein (rOspA) by electrospray mass spectroscopy.

Lipidation at the N-terminal of recombinant outer surface protein A (rOspA) is an important determinant of its immunogenicity. Lipidation patterns of rOspA can be sensitive to processing environments and storage conditions. In order to assure product consistency and stability. it is essential to characterize and monitor lipidation patterns of rOspA through its life-cycle. Electrospray mass spectroscopy combined with maximum entropy calculation was employed to analyze the lipidation of rOspA. The results revealed that more than 90% of protein is a tri-lipidated rOspA and the remainder is di-lipidated. It was demonstrated that the method is both sensitive and quantitative and has the potential to be used for routine quality control and stability testing.

Characterization of post-translationally modified recombinant protein using liquid chromatography/mass spectrometry.

Post-translational modification plays an important role in the biological functions of many proteins. Characterization of such modifications is crucial in the development of recombinant protein-based vaccines. Liquid Chromatography coupled with Mass Spectrometry (LC/MS) provides a powerful tool with high resolution and accuracy to characterize proteins and their post-translational modifications. The technique was applied to characterization of a Lyme vaccine protein rOspA (recombinant Outer surface protein A). The results demonstrate that LC/MS could be used to resolve and characterize various forms of post-translational lipidation of the protein. It was this detailed structural information that made it possible to validate a method for quantitative determination of the lipidation forms of rOspA.

Immunogenicity of overlapping synthetic peptides covering the entire sequence of Haemophilus influenzae type b outer membrane protein P2.

Haemophilus influenzae type b is a major cause of bacterial meningitis in young children. Antibodies against the outer membrane protein P2 are protective in the infant rat model of bacteremia. To identify conserved, surface-exposed, and protective epitopes of P2, 17 overlapping peptides covering the entire sequence of the protein were synthesized. Antisera from mice, guinea pigs, and rabbits raised against chromatographically purified P2 were tested for their reactivities to the peptides by enzyme-linked immunosorbent assays (ELISA). Three major linear immunodominant B-cell epitopes were mapped to residues 53 to 81, 241 to 265, and 314 to 341 of mature P2. Human convalescent-phase antisera also reacted strongly with these three epitopes. Rabbit antisera against all peptide-keyhole limpet hemocyanin conjugates except two peptides containing residues 8 to 19 and 302 to 319 recognized the corresponding peptides in ELISA and reacted with P2 on immunoblots. Immunization with all unconjugated peptides, except the 19 N-terminal residues, induced very strong peptide-specific antibody responses, and these antisera reacted with P2 on immunoblots. Rabbit antisera raised against peptides corresponding to residues 1 to 14, 125 to 150, 193 to 219, and 241 to 319 also recognized P2 purified from H. influenzae nontypeable isolates. Identification of these immunodominant B-cell epitopes and conserved regions is a first step toward the rational design of a universal H. influenzae vaccine.

Immunogenicity of Haemophilus influenzae type b conjugate vaccine in children with congenital asplenia.

The immunogenicity of Haemophilus influenzae type b polysaccharide-protein conjugate vaccines in congenitally asplenic children is unknown. The short-term immunogenicity of the H. influenzae type b polysaccharide-diphtheria toxoid conjugate vaccine was therefore assessed in 10 children with congenital asplenia by measuring antipolyribosyl-ribitol phosphate antibody titers. An excellent antibody response was seen in 9 children (mean geometric titer in responders after immunization 44.7 micrograms/mL; range, 2.59-402). The remaining child responded to a booster dose. Further studies are required to assess whether H. influenzae type b conjugate vaccines are immunogenic in infancy in the presence of congenital asplenia.

A randomized, prospective field trial of a conjugate vaccine in the protection of infants and young children against invasive Haemophilus influenzae type b disease.

BACKGROUND: Haemophilus influenzae type b is the leading cause of invasive bacterial disease in young children. The capsular polysaccharide vaccine does not protect children at greatest risk (those under the age of 18 months), but a polysaccharide-protein conjugate vaccine has proved to be more immunogenic in this age group. METHODS: We enrolled 114,000 infants in Finland in an open, prospective, randomized trial of a H. influenzae type b capsular polysaccharide-diphtheria toxoid conjugate vaccine (polyribosylribitol phosphate-diphtheria toxoid [PRP-D]). Children born on odd-numbered days were vaccinated at the ages of 3, 4, 6, and 14 to 18 months; those born on even-numbered days formed the control group and received the same vaccine at the age of 24 months. RESULTS: After three doses of the vaccine there were 4 cases of verified bacteremic H. influenzae type b disease in the group receiving early vaccination, as compared with 64 cases in the control group, between the ages of approximately 7 and 24 months. The protective efficacy of the vaccine was thus 94 percent (95 percent confidence interval, 83 to 98). No serious adverse effects were reported. The immune response to the conjugate vaccine was characteristic of a T-cell-dependent response when studied in a cohort of 120 infants. The primary immunization series resulted in a geometric mean concentration of anticapsular antibody of 0.53 micrograms per milliliter at the age of seven months, and the fourth dose evoked an anamnestic response, with a mean antibody concentration of 45.22 micrograms per milliliter. CONCLUSIONS: A new conjugate vaccine consisting of the capsular polysaccharide of H. influenzae type b covalently linked to a protein carrier (PRP-D), administered to infants beginning at the age of 3 months, is highly effective in protecting young Finnish children (7 to 24 months old) against invasive H. influenzae type b infections.

Safety and immunogenicity of Haemophilus influenzae type b polysaccharide-diphtheria toxoid conjugate vaccine (PRP-D) in infants.

Safety and immunogenicity of a Haemophilus influenzae type b polysaccharide-diphtheria toxoid conjugate vaccine (PRP-D) was evaluated in infants seven to 14 months of age. PRP-D (80% of subjects) or saline placebo (20%) was randomly and blindly administered (two doses separated by two months). Incidence of mild reactions lasting less than 48 hr did not differ significantly between the placebo and vaccine recipients. Preimmunization levels of antibody to PRP were less than or equal to 0.15 micrograms/ml in 97% of subjects. A twofold increase in antibody concentration occurred in 88% of subjects following the first dose and in 99% following the second dose of vaccine. No change occurred in placebo recipients. Mean level of antibody and percentage of subjects with levels of antibody greater than or equal to 1 microgram/ml after vaccination increased with increasing age. Responses were related to vaccine lot but not to sex, race, or geographic location. Two doses of PRP-D in infants seven months of age and older induced antibody levels equal to or greater than levels in infants 24 months of age given the polysaccharide alone.