An ELISA employing a Haemophilus influenzae type b oligosaccharide-human serum albumin conjugate correlates with the radioantigen binding assay.

An ELISA measuring total Ig antibodies to the capsular polysaccharide of Haemophilus influenzae type b (HbPs) in human sera using an antigen composed of Haemophilus b oligosaccharides conjugated to human serum albumin (HbO-HA) was shown to have an excellent correlation to the radioantigen binding assay (RABA). When 214 sera with different anti-HbPs levels were assayed for total Ig by HbO-HA ELISA and by RABA the correlation coefficient was 0.917 and the paired t test p value was 0.575. Use of competitive ELISA employing soluble HbPs, HbO-HA and human albumin as competitors, showed that the HbO-HA ELISA was specific for antibodies to HbPs. The HbO-HA ELISA yielded reproducible results both within and between laboratories. The HbO-HA ELISA can also be used to determine the isotype of anti-HbPs antibodies by using isotype specific enzyme conjugates. The sum of the IgG, IgA and IgM HbO-HA ELISA results had excellent correlation to the RABA results (correlation coefficient = 0.976). Thus, the HbO-HA ELISA can be substituted for the classical RABA and also be utilized for quantitating the isotype of the anti-HbPs antibodies.

Safety and immunogenicity of Haemophilus influenzae type b oligosaccharide-CRM197 conjugate vaccine in infants aged 15 to 23 months.

A total of 268 infants aged 15 to 23 months received one dose of a vaccine composed of Haemophilus influenzae type b oligosaccharides covalently linked to the nontoxic diphtheria toxin variant CRM197 (HbOC; HibTITER). Side effects associated with vaccination were infrequent, transient, and mild. One month after a single vaccination, the anti-H influenzae type b capsular polysaccharide antibody concentration rose from a geometric mean prevaccination level of 0.20 microgram/mL to 13.77 micrograms/mL. Of these infants, 99% had a postvaccination level greater than or equal to 1.00 microgram/mL, a level associated with long-term protection. The immune response was long-lived: all of the children who were monitored 17 to 27 months after vaccination had concentrations greater than or equal to 1.00 microgram/mL. The anti-H influenzae type b capsular polysaccharide antibody generated was predominantly of the IgG isotype and IgG1 subclass. The immune sera had bactericidal activity in vitro and conferred passive protection in the infant rat meningitis model.

Safety and immunologic response to Haemophilus influenzae type b oligosaccharide-CRM197 conjugate vaccine in 1- to 6-month-old infants.

A Haemophilus influenzae type b oligosaccharide-CRM197 conjugate (HbOC) vaccine was evaluated for safety and immunogenicity in 432 infants 1 to 6 months of age. In a multicenter study involving 10 sites in six states, infants were vaccinated with three 10-micrograms doses of HbOC at 2-month intervals. Side effects associated with vaccination were mild, transient, and occurred in fewer than 2% of the subjects. More than 90% of infants of all ages responded after two doses, and more than 98% had anti-H influenzae type b capsular polysaccharide (HbPs) antibody levels greater than or equal to 1 microgram/mL after three doses. One month after the third vaccination, the geometric mean anti-HbPs antibody levels were 16.84, 26.23, and 29.11 in infants initially vaccinated at 1 to 2, 3 to 4, and 5 to 6 months of age, respectively. A long-term antibody response was observed; more than 80% of these infants had anti-HbPs levels greater than or equal to 1 microgram/mL at 2 years of age. The HbOC generated an immune response characteristic of a protein antigen; IgG anti-HbPs antibodies of IgG1 subclass predominated and the response could be boosted. The immune sera killed H influenzae type b when evaluated in an in vitro bactericidal assay. The data indicate that HbOC safely primed and boosted the immune system of young infants, providing long-lasting protective levels of anti-HbPs antibodies.

Safety and immunogenicity of a combined diphtheria, tetanus, pertussis and Haemophilus influenzae type b vaccine in young infants.

OBJECTIVE: To study the safety and immunogenicity of a combined diphtheria-tetanus-pertussis (DTP)-Haemophilus influenzae type b (HbOC) vaccine (TETRAMUNE) in infants as young as 2 months of age as compared to separate administration of DTP and HbOC. METHODS: Two-month-old infants were randomized to receive three doses 2 months apart of either DTP-HbOC as a single 0.5-mL injection or to receive 0.5 mL of DTP and HbOC concurrently in separate legs. Local and systemic adverse reactions were monitored within 72 hours of each immunization, and immunogenicity of each of the four vaccine components was measured. RESULTS: The incidence of both local and systemic adverse events following the tetravalent vaccine was similar to the incidence following separate vaccine administration. After three doses of vaccine, the response to each of the vaccine components was higher in the combined vaccine when compared to separate administration. In the case of the Haemophilus influenzae type b component, this enhancement was also seen after two doses. The response to the combined vaccine was consistent among the three lots tested as was the enhancement over separate administration. CONCLUSIONS: The DTP-HbOC vaccine was safe and immunogenic in young infants and was generally more immunogenic than separate vaccination with DTP and HbOC. The use of such a combined vaccine reduces the number of injections given to young infants by half and is an important step toward improving vaccine delivery.

Safety and immunogenicity of heptavalent pneumococcal vaccine conjugated to CRM197 in United States infants.

OBJECTIVE: To determine the safety and immunogenicity of heptavalent pneumococcal saccharide vaccine (serotypes 4, 6B, 9V, 14, 18C, 19F, 23F) individually conjugated to CRM197 (PNCRM7), administered at 2, 4, 6, and 12 to 15 months of age. DESIGN: Two hundred twelve healthy 2-month-old infants were equally randomized to receive four consecutive doses of PNCRM7 or an investigational meningococcal group C conjugate vaccine, which served as a control. Concomitantly administered routine vaccines were oral polio vaccine and combined diphtheria toxoid, tetanus toxoid, and whole cell pertussis vaccine/Haemophilus influenzae type b vaccine consisting of capsular oligosaccharides conjugated to CRM197 (DTP/HbOC) at 2, 4, and 6 months, and either measles-mumps-rubella vaccine or HbOC at 12 to 15 months. Active safety surveillance was conducted for 3 days after each dose. Antibody concentrations to each of the 7 pneumococcal serotypes were measured by enzyme-linked immunosorbent assay prevaccination, after doses two and three, prebooster, and postbooster. RESULTS: Significantly fewer children experienced local reactions at the PNCRM7 injection site than at the DTP/HbOC site. There was no increase in the incidence or severity of local reactions at the PNCRM7 site with increasing doses of vaccine. Mild to moderate postvaccination fever was common in both the PNCRM7 and control vaccine groups, however DTP/HbOC was administered concurrently. All 7 vaccine serotypes were immunogenic. The kinetics of the immune responses were serotype-specific. After three doses of PNCRM7, between 92% to 100% of children had >/=0.15 microg/mL of antibody, and 51% to 90% achieved a level of >/=1 microg/mL against specific serotypes. A booster dose of PNCRM7 resulted in a brisk anamnestic response to all 7 vaccine serotypes, demonstrating effective stimulation of T-cell memory by the primary series of vaccinations. CONCLUSION: Primary immunization followed by a booster dose of PNCRM7 seemed to be acceptably safe and resulted in significant rises in antibody to all 7 serotypes. Implications. Studies to assess vaccine efficacy of PNCRM7 for prevention of systemic disease, nasopharyngeal colonization, and acute otitis media are in progress. If PNCRM7 proves to be protective, there is the potential to prevent up to 85% of invasive pneumococcal disease occurring in US children.

Characterization of immune response as an indicator of Haemophilus influenzae type b vaccine efficacy.

Quantitation of antibodies to Haemophilus influenzae type b (Hib) polysaccharide has been an active area of investigation associated with the development of polysaccharide and subsequently polysaccharide-protein conjugate vaccines. These clinical studies indicate that there are several serologic parameters associated with Hib vaccine efficacy in infants. Efficacious vaccines elicit polysaccharide-specific antibodies in infants; they prime the immune system for an anamnestic response; the immune response is long-lived through the period of greatest risk for disease; and the elicited antibodies have functional activity as demonstrated in bactericidal and opsonophagocytic assays or protection in an infant rat challenge model. The immune response to different Hib vaccines varies both quantitatively and qualitatively. With the introduction of routine Hib vaccine immunization, vaccine performance can rely on these serologic parameters. Quantitative serologic assays, the radio-antigen binding and enzyme-linked immunosorbent assays, have been developed and standardized. The quality of the antigen as well as optimization of all assay steps and reagents are key to ensuring specific and reproducible antibody quantitation.

Safety of combined oligosaccharide conjugate Haemophilus influenzae type b (HbOC) and whole cell diphtheria-tetanus toxoids-pertussis vaccine in infancy. The Kaiser Permanente Pediatric Vaccine Study Group.

The safety of the combined oligosaccharide conjugate Haemophilus influenzae (Hib) type b (HbOC) and whole cell diphtheria-tetanus toxoids-pertussis (DTP) vaccine (Tetramune, HbOC-DTP; Lederle) in infancy was evaluated in 6644 recipients of this vaccine and compared with 3914 recipients of separate injections of whole cell DTP and HbOC vaccines when given as a three dose regimen to infants at 2, 4 and 6 months of age in each group. Of the total number of infants in the study, a subset of 1435 were enrolled into the study and then randomly assigned to receive either the Hib-DPT combined vaccine or the separate components. This subset was used to assess local and systemic side effects which were evaluated utilizing telephone interviews 48 to 72 hours after vaccine. The remaining children in the study population were enrolled in a nonrandomized manner. For these children parents were offered the experimental Hib-DPT vaccine and refusers were given HbOC and DTP. Both of these groups of children as well as the randomized subset described above were used to assess rates of episodes of hospitalization, emergency room utilization and sudden infant death syndrome in HbOC-DTP recipients and children who received HbOC and DTP separately. Immunogenicity was evaluated in 123 children by collection of a single serum sample 30 days after the third dose of HbOC-DTP. The observed immunogenicity was comparable to that observed in other recent studies for HbOC and DTP component antigens. The profile of local and systemic side effects observed was virtually identical to that observed after DTP plus HbOC given separately.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of antigenuria after immunization with three Haemophilus influenzae type b conjugate vaccines.

The incidence of antigenuria was documented after vaccination of 75 children 15 to 60 months of age with one of three Haemophilus influenzae type b conjugate vaccines, PRP-D (ProHIBiT), PRP-T and HbOC (HibTITER). Unconcentrated and concentrated urine was tested on the first, third, fifth and seventh days after vaccination. Antigenuria occurred on Day 1 in 100% of PRP-D, 43% of PRP-T and 12% of HbOC recipients. The percentages of children excreting antigen on Day 3 were 95, 17 and 8%; on Day 5 they were 36, 4 and 12%; and on Day 7 they were 14, 0 and 18% for PRP-D, PRP-T and HbOC, respectively. The difference in the occurrence of antigenuria resulting from each vaccine was statistically significant on Day 1 and for PRP-D compared with PRP-T or HbOC on Day 3. It is important for clinicians to be aware of the frequency with which antigenuria occurs after these vaccines so that appropriate therapeutic decisions can be made.

Safety and immunogenicity of four doses of Neisseria meningitidis group C vaccine conjugated to CRM197 in United States infants.

BACKGROUND: Following widespread use of conjugate pneumococcal vaccine, Neisseria meningitidis likely will become the leading cause of bacterial sepsis and meningitis in US children. This report describes the safety and immunogenicity in US children of four consecutive doses of a meningococcal group C vaccine conjugated to CRM197 via reductive amination (MnCC). METHODS: One hundred six healthy 2-month-old infants received MnCC at 2, 4 and 6 months of age in a randomized, controlled double blind study; children in the other treatment arm were given a 7-valent conjugate pneumococcal vaccine. Parents reenrolled 64 of these children at 12 to 15 months to receive a fourth dose of MnCC. Routine childhood vaccines, including DTP, were coadministered. Temperatures and symptoms were recorded for 3 days after each immunization. Serum enzyme-linked immunosorbent assay IgG and bactericidal antibodies were measured prevaccination and before and 1 month after Doses 3 and 4. RESULTS: Moderate to severe local reactions, defined as erythema or induration > or =2.4 cm or pain that interfered with limb movement was reported after 0 to 3.2% of MnCC injections, depending on the reaction and dose. Fever occurred in 23 to 37% of children, but the contribution of MnCC to the febrile reactions is unknown. Geometric mean concentrations of IgG antibody to group C meningococcal polysaccharide were 3.72 microg/ml after Dose 3 and 8.03 microg/ml after the booster. Geometric mean functional serum bactericidal antibody titers after Doses 3 and 4 were 1:463 and 1:2341, respectively. One hundred percent of children had a serum bactericidal antibody titer of > or =1:64 after three doses and > or = 1:128 after the booster. CONCLUSIONS: The MnCC vaccine had an acceptable safety profile and generated high titers of bactericidal antibody in immunized US infants and toddlers. It appears to be an attractive candidate vaccine for the prevention of serogroup C meningococcal disease in young children.

Safety and immunogenicity of heptavalent pneumococcal CRM197 conjugate vaccine in infants and toddlers.

OBJECTIVES: The objectives of this study were (1) to determine the safety and immunogenicity of heptavalent pneumococcal CRM197 conjugate (PNCRM7) vaccine in infants and (2) to determine the effect of concurrent hepatitis B immunization during the primary series and the effect of concurrent diphtheria and tetanus toxoid and acellular pertussis [DTaP (ACEL-IMUNE)] and conjugate CRM197 Haemophilus influenzae type b [HbOC (HibTITER) immunization at time of the booster dose on the safety and immunogenicity of PNCRM7and these other concurrently administered vaccines. METHODS: This was a randomized double-blinded study in 302 healthy infants in the Northern California Kaiser Permanente (NCKP) Health Plan. Infants received either PNCRM7 vaccine or meningococcal group C conjugate vaccine as a control at 2, 4 and 6 months of age and a booster at 12 to 15 months of age. Study design permitted the evaluation of immunology and safety of concurrent administration of routine vaccines. Antibody titers were determined on blood samples drawn before and 1 month after the primary series and the booster dose. RESULTS: After the third dose of PNCRM7 geometric mean concentrations (GMCs) ranged from 1.01 for serotype 9V to 3.72 microg/ml for serotype 14. More than 90% of all subjects had a post-third dose titer of > or =0.15 microg/ml for all serotypes, and the percentage of infants with a post-third dose titer of > or =1.0 microg/ml ranged from 51% for type 9V to 89% for type 14. After the PNCRM7 booster dose, the GMCs of all seven serotypes increased significantly over both post-Dose 3 and pre-Dose 4 antibody levels. In the primary series there were no significant differences in GMCs of pneumococcal antibodies between the subjects given PN-CRM7 alone or concurrently with hepatitis B vaccine. At the toddler dose concurrent administration of PNCRM7 and DTaP and HbOC resulted in a near conventional threshold for statistical significance of a post-Dose 4 GMC for serotype 23F [alone 6.75 mirog/ml vs. concurrent 4.11 microg/ml (P = 0.057)] as well as significantly lower antibody GMCs for H. influenza polyribosylribitol phosphate, diphtheria toxoid, pertussis toxin and filamentous hemagglutinin. For all antigens there were no differences between study groups in defined antibody titers that are considered protective. CONCLUSION: We conclude that PNCRM7 vaccine was safe and immunogenic. When this vaccine was administered concurrently at the booster dose with DTaP and HbOC vaccines, lower antibody titers were noted for some of the antigens when compared with the antibody response when PNCRM7 was given separately. Because the GMCs of the booster responses were all generally high and all subjects achieved similar percentages above predefined antibody titers, these differences are probably not clinically significant.

Kinetics of antibody response to Haemophilus influenzae type b vaccines. Pennridge Pediatric Associates.

Serum antibodies to the capsular polysaccharide of Haemophilus influenzae type b (Hib) are effective in preventing or ameliorating invasive disease caused by this human pathogen. Polysaccharide and conjugate (saccharide covalently linked to protein carrier) vaccines have been developed which stimulate the production of such antibodies. The polysaccharide-specific antibody concentrations in the sera of vaccine-naïve adults and toddlers on days 1, 3, 7, 14 and 28 following immunisation with one dose of the Hib polysaccharide vaccine (PRP, polyribosylribitol phosphate) or an oligosaccharide-CRM197 conjugate vaccine (HbOC, HibTITER) were determined. Antibody responses occurred within 7 days of immunisation with the maximum response usually occurring 14 days post-immunisation, irrespective of vaccine or subject age. In this small study, a significant transient decline in pre-existing antibodies was observed only in the groups receiving the polysaccharide vaccine and not in the groups receiving HbOC vaccine. Because of the small magnitude of antigen-specific antibody decline and its transient nature, it is unlikely that this observation has clinical significance.

Impact of immunization on Haemophilus influenzae type b disease.

Epidemiological surveillance programs have shown that before the introduction of effective vaccines, Haemophilus influenzae type b (Hib) was the primary pathogen associated with bacterial meningitis in children. Vaccines composed of the bacterium's polysaccharide conjugated onto protein carriers began to be introduced into routine health care practices for infants as early as 1989 in some European countries. Continued introduction in industrialized nations, including the United States in late 1990, has resulted in the rapid decline in the incidence of reported invasive Hib disease. Follow-up surveillance studies show that (a) the decline in the incidence of Hib disease is temporally related to the introduction of effective vaccines, (b) the decline in Hib epiglottitis preceded the decline in meningitis in the United States, (c) the incidence of disease declined in children under the age of 5 years but remained constant in older children and adults, (d) other bacterial pathogens are now the primary causative agents of infant meningitis and epiglottitis even though the incidence of disease caused by these other pathogens has not changed, and (e) the pharyngeal carriage rate of Hib in children has declined without any evidence of an increase in the carriage of non-type b strains or other pathogens. The introduction of effective conjugate vaccines appears to protect at-risk children from invasive Hib disease as well as reduce the opportunities for interpersonal transmission of this bacterium. In addition, Hib conjugate vaccine utilization has benefited society through economic savings.

Use of animal testing for evaluating glycoconjugate vaccine immunogenicity.

Most animal species respond with high antibody levels to polysaccharide antigens after they have been covalently linked to a protein carrier, converting a T-cell independent to a T-cell dependent antigen. This chemical modification has enabled the development of glycoconjugate vaccines for Haemophilus influenzae type b, Neisseria meningitidis, and multivalent Streptococcus pneumoniae. This new generation of vaccines can be well characterized physically and chemically to ensure consistent vaccine manufacture. Such analytical tests provide an alternative to animal models for Quality Control Laboratories; biological models can be difficult and costly to develop and use on a routine basis. If animal tests are used, they need to be refined, defined, and validated for their intended purpose.

Response to a Haemophilus influenzae type b diphtheria CRM197 conjugate vaccine in children with a defect of antibody production to Haemophilus influenzae type b polysaccharide.

A defect in antibody response to immunization with Haemophilus influenzae type b (Hib) capsular polysaccharide vaccine has been reported in children with recurrent infections and normal immunoglobin levels. We identified 15 children, aged 2 to 6 years, with this defect, and we evaluated their response to immunization with an Hib capsular oligosaccharide diphtheria CRM197 protein-conjugate vaccine (HbOC). The children received a series of three vaccines: HbOC at 0 and 8 weeks, and the Hib polysaccharide vaccine at 16 weeks. Levels of antibody to the Hib capsular polysaccharide (polyribosyl ribitol phosphate, PRP) and to diphtheria toxoid were obtained before and 4 weeks after each vaccination. The geometric mean serum anti-PRP concentration was 0.17 microgram/ml before immunization and 29.3 micrograms/ml after the second HbOC immunization (week 12). All 15 children had postvaccination anti-PRP antibody levels greater than 1.0 microgram/ml after receiving the second HbOC (week 12). In addition, booster responses were observed after the second Hib conjugate in 13 of the patients and after the Hib polysaccharide in four of the patients. All patients with low preimmunization diphtheria titers had a response to the diphtheria toxoid. These results suggest that conjugation of Hib polysaccharide with diphtheria CRM197 overcomes the defective antibody response to Hib oligosaccharide in children who are initially observed with recurrent infections and inability to respond to the Hib polysaccharide vaccine.

Use of highly encapsulated Streptococcus pneumoniae strains in a flow-cytometric assay for assessment of the phagocytic capacity of serotype-specific antibodies.

A phagocytosis assay for Streptococcus pneumoniae based on flow cytometry (FACS) with human polymorphonuclear cells and human complement was developed for the study of human vaccination antisera. Human prevaccination sera already contain high levels of C-polysaccharide (C-PS) antibodies, which are not protective in humans but which might give false positive results in a flow-cytometry-based assay. Cultures of S. pneumoniae grown to log phase on three consecutive days, followed by heat inactivation, yielded stable and highly encapsulated strains for serotypes 6A, 6B, 14, 19F, and 23F. As a result, only serotype-specific antibodies were able to facilitate phagocytosis of these strains, whereas no phagocytosis was observed with antibodies against C-PS or pneumococcal surface proteins. No, or weak, phagocytosis was observed with human prevaccination sera, whereas in general, postvaccination antisera facilitated phagocytosis. A highly significant correlation was observed between enzyme-linked immunosorbent assay titers and FACS phagocytosis titers (r = 0.98, P < 0.001) for serotype 23F pneumococci with human vaccination antisera. For all serotypes, interassay variation was below 10%. Major advantages of this assay over the classical killing assay are that (i) limited amounts of sera are required (10 microliter per titration curve), (ii) 600 samples can be processed in one day by one person, and (iii) cells can be fixed and measurement of the samples can be performed up to 1 week later.

Relationship between serum bactericidal activity and serogroup-specific immunoglobulin G concentration for adults, toddlers, and infants immunized with Neisseria meningitidis serogroup C vaccines.

A new meningococcal group C-CRM(197) conjugate vaccine (MnCC; Meningitec) has been evaluated in multiple clinical trials in the United States and most recently has been approved for routine administration in the United Kingdom. Meningococcal serogroup C (MnC)-specific immunoglobulin G (IgG) antibodies in pre- and postimmunization sera obtained from healthy U.S. adults, toddlers, and infants were quantitated by enzyme-linked immunosorbent assay (ELISA) and by an antibody-dependent, complement-mediated serum bactericidal assay (SBA). Serogroup-specific IgG antibody (micrograms per milliliter) in adults immunized either with the quadrivalent polysaccharide (A, C, Y, and W-135) vaccine or with MnCC showed a strong correlation (r = 0.848 and 0.934, respectively) by linear regression analysis with SBA. Sera from infants immunized with the MnCC (n = 30) and an age-matched unimmunized control group (n = 15) were also analyzed. Linear regression analysis of serum bactericidal and IgG ELISA data from sera obtained at 2 months of age (preimmunization) showed no correlation; however, a high degree of correlation was observed at time points after two (r = 0.877) and three (r = 0.951) immunizations, where significant rises in anti-MnC polysaccharide antibodies occurred relative to the age-matched control group. Infants previously primed with 3 doses of MnCC were given a booster dose of conjugate vaccine at 12 to 15 months of age. The correlation coefficient of ELISA to SBA for combined pre- and postbooster data was r = 0.836 (n = 48 pairs). In conclusion, increases in serum bactericidal activity in immunized adult, toddler, and infant populations were found to correlate very well with increases in serogroup-specific IgG concentrations, whereas the correlation between these two assays in nonimmunized 2-month-old infants was poor. Characterizing the relationship between these methods is important for understanding the significance of antigen-specific antibody concentrations relative to vaccine performance and protection from disease.

Assignment of weight-based antibody units to a human antipneumococcal standard reference serum, lot 89-S.

A human reference serum pool, lot 89-S, has been developed for use in quantitating concentrations of antibody to Streptococcus pneumoniae. Weight-based units have been assigned to antibodies to 11 pneumococcal polysaccharide (PnPs) serotypes (1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F) by using enzyme-linked immunosorbent assay methodology and a human standard reference serum, USNRP IS 1644. The experimentally derived assignments for anti-PnPs antibodies of the immunoglobulin G (IgG), IgM, and IgA isotypes in lot 89-S correlate well to the separately determined immunoglobulin assignment. These assignments for this antipneumococcal standard serum were used to quantitate IgG, IgM, and IgA isotype levels and the total immunoglobulin level in pediatric samples from a pneumococcal conjugate vaccine trial. The data indicate that these assignments may be used to assess levels of antibody to PnPs serotypes in human serum.