Production of Haemophilus influenzae b Meningitis in Infant Rats by Intraperitoneal Inoculation

Sprague-Dawley rats have a marked age-related susceptibility to Haemophilus influenzae type b that does not correlate with serum bactericidal activity. Eighty percent of 5-day-old animals that survive to 48 h after an intraperitoneal inoculation of a mean lethal dose of bacteria have histologically documented meningitis. Animals surviving the inoculations as infants manifest cerebral dysfunction as adults. This model should facilitate experimental study of bacterial meningitis.     

Age-Related Susceptibility to Haemophilus influenzae Type b Disease in Rabbits

Anti-type b antibodies were detected in newborns and in most 6- to 8-week-old rabbits, similar to those observed in humans. Culture of the pharynx and rectum of rabbits at varied ages failed to yield Haemophilus influenzae type b. Bacteria with cross-reacting antigens were observed in both the pharyngeal and rectal cultures from rabbits of varied ages. Rabbits without preexisting serum anti-type b antibodies were highly susceptible to septicemia and death after injection of small numbers of H. influenzae type b. Passively acquired antibody, whether observed in newborns or injected into animals without preexisting antibodies, conferred a high degree of protection against H. influenzae type b.     

School-Age Consequences of Haemophilus influenzae Type b Meningitis

Divided 126 school-age children who had been treated earlier in life for Haemophilus influenzae Type b (Hib) meningitis into subgroups with and without neurologic complications, and compared these groups to each other and to sibling controls. Outcomes assessed included neuropsychological abilities, behavioral adjustment, school performance, and adaptive behavior. Analyses revealed that outcomes were less favorable for the postmeningitis (PM) children with complications than for either PM children without complications or siblings. Differences were apparent in neuropsychological testing, teacher ratings of behavior, and a measure of adaptive behavior. PM children without complications were indistinguishable from siblings. Although analyses failed to show variation between groups as a function of sex or age at testing, differences on one verbal measure were found only at lower socioeconomic levels. Findings clarify the school-age consequences of Hib meningitis and document the need to consider disease and social variables in evaluating the impact of early neurologic disease on development.     

Human Antibody Response to Individual Outer Membrane Proteins of Haemophilus influenzae Type b

To evaluate the potential of outer membrane proteins of Haemophilus influenzae as a vaccine, sera from 11 healthy persons and from 23 patients convalescing from disease caused by Haemophilus influenzae type b were assayed for antibodies to individual outer membrane proteins of a single type b isolate, strain Eag, by a gel radioimmunoassay. All 23 patients, ranging in age from 2 months to 62 years, with 17 patients being 24 months or less, had antibodies to some of these proteins in their sera (range, antibodies to 4 to 17 proteins per patient). Although the intensity and spectrum of the response varied, all patients had antibodies to one particular outer membrane protein and 19 patients had antibodies to another, with those patients 5 years and older having antibodies to more proteins than did infants (24 months). In the two cases examined, convalescent sera had greater amounts and broader spectra of antibodies than did acute sera. In addition, 10 of 11 healthy subjects not known to have had systemic H. influenzae disease also had antibodies to individual outer membrane proteins, with older children having greater amounts than did their younger siblings and with children showing a different spectrum of response than that for adults. Thus, antibodies to outer membrane proteins are commonly found in humans. Also, these results and those demonstrating that hyperimmune rabbit antisera to strain Eag reacted with each of five type b substrains possessing some different outer membrane proteins indicate considerable cross-reactivity among these proteins. These results encourage continued consideration of outer membrane proteins in a vaccine.     

In Vitro Activation of Human T Lymphocytes by Haemophilus influenzae Type b Capsular Polysaccharides

Polyribosilribitolphosphate (PRP), the capsular polysaccharide from Haemophilus influenzae type b, is a T-cell-independent type 2 antigen. In vitro culture of adult peripheral blood T cells with 15 micrograms/ml PRP leads to induction of interleukin-2 receptor (IL-2R) expression on up to 10% of T cells. These cells are CD4+ and carry the alpha beta T-cell receptor. PRP, at concentrations above 1-5 micrograms/ml, can also induce in vitro proliferation of both adult and neonatal T cells. We conclude that PRP acts as a human T-cell mitogen. The in vitro proliferative response as well as IL-2R expression was studied in T cells derived from adults after vaccination with native PRP, with PRP conjugated to a carrier protein, or with diphtheria toxoid. Vaccination with conjugated PRP decreased the doses of PRP required for in vitro induction of IL-2R expression and T-cell proliferation. This indicates that vaccination with PRP conjugated to a carrier protein improves the in vitro T-cell response to PRP activation.     

Efficacy of Haemophilus influenzae type b polysaccharide-diphtheria toxoid conjugate vaccine in infancy

Haemophilus influenzae type b capsular polysaccharide-diphtheria toxoid conjugate vaccine has recently been shown to be capable of inducing antibodies to H. influenzae in infants. In an evaluation of its clinical efficacy, 60,000 children were enrolled in an open trial in Finland. Children born on odd-numbered days between October 1, 1985, and September 30, 1986, received the vaccine at 3, 4, 6, and 14 months; those born on even-numbered days served as controls. The geometric mean antibody titer measured in a cohort of 99 children rose from a prevaccination level of 0.08 microgram per milliliter at three months of age to 0.42 microgram per milliliter at seven months. Only minor adverse reactions were reported. Up to February 1987, two cases of invasive H. influenzae infection had occurred among the children who had received three doses of vaccine, whereas 12 cases had occurred among the controls (P = 0.0005 by Poisson one-tailed test). The rate of short-term (average follow-up time, five months) protection provided by this conjugate vaccine in infancy was thus 83 percent.     

Differential Complement Resistance Mediates Virulence of Haemophilus influenzae Type b

Studies were undertaken to gain insight into the virulence of type b in contrast to the other Haemophilus influenzae capsular types. A relationship was found between the comparative virulence of H. influenzae types in humans and their resistance to the bactericidal effect of antibody-free complement. Type b was most resistant to the bactericidal effect of complement. The other types could be divided into three groups based upon their susceptibility to complement; this grouping was also related to their structural similarities. No association between virulence and either the biotype, source of isolate, in vitro association with peripheral polymorphonuclear leukocytes, or the total amount of capsular polysaccharide was found. However, among the type b strains, higher levels of cell-associated polysaccharide were associated with increased resistance to complement. The relative virulence of the six H. influenzae types in the infant rat model was generally similar to that in humans. After intraperitoneal challenge, type b and type a strains had the lowest 50% effective doses for bacteremia, removed by several logs from the values of the other types. By intranasal challenge, type b strains produced higher rates and levels of bacteremia than did type a strains. High levels of natural bactericidal antibodies to types c and e were found in adult female rats; this finding alone could not account for the differences in virulence among the H. influenzae types in the infant rat model. We propose that the virulence of type b strains is due to their greater resistance to the bactericidal activity of serum complement alone. Resistance to type b disease requires serum antibody to induce the complement-mediated reaction.     

Avidity Determinations for Haemophilus influenzae Type b Anti-Polyribosylribitol Phosphate Antibodies

Determination of antibody avidity measurements can be difficult in human serum depending on the population evaluated. We evaluated three approaches for the determination of antibody avidity for immunoglobulin G (IgG). These approaches were (i) elution of bound antibody with increasing concentrations of a chaotropic agent using a single serum dilution, (ii) binding interference of multiple serum dilutions by a single concentration of a chaotrope, and (iii) elution of multiple serum dilutions by a single concentration of a chaotrope. Parameters that affect the determination of avidity measurements and their limitations were evaluated with pre- and post-Haemophilus influenzae type b conjugate vaccination sera (n = 89). We determined that elution of low-avidity antibodies present in multiple dilutions of the serum sample by a single concentration of a chaotrope (0.15 M sodium thiocyanate [NaSCN]) was optimal for the determination of avidity measurements throughout a wide range of IgG concentrations (0.94 to 304.6 μg/ml). The percent reduction in concentration as determined by the elution assay with 0.15 M NaSCN correlated highly (r = 0.84) with weighted averages obtained by an elution assay with multiple solutions of NaSCN. The correlation (r = 0.57) between elution and binding interference, when a single concentration of a chaotrope was used, was lower than the correlation between the two elution methods (r = 0.84). We found that the serum dilution, the heterogeneity of the antibody population, and the concentration of the chaotrope were the primary variables affecting avidity determinations. In this study, we present multiple analysis methods depending on the methodology used. We also present the factors that affect the analysis of avidity determinations given the polyclonal nature of human sera. This experimental approach should benefit the evaluation of similar antibodies induced by other bacterial polysaccharide vaccines.     

Avidity determinations for Haemophilus influenzae Type b anti-polyribosylribitol phosphate antibodies

Determination of antibody avidity measurements can be difficult in human serum depending on the population evaluated. We evaluated three approaches for the determination of antibody avidity for immunoglobulin G (IgG). These approaches were (i) elution of bound antibody with increasing concentrations of a chaotropic agent using a single serum dilution, (ii) binding interference of multiple serum dilutions by a single concentration of a chaotrope, and (iii) elution of multiple serum dilutions by a single concentration of a chaotrope. Parameters that affect the determination of avidity measurements and their limitations were evaluated with pre- and post-Haemophilus influenzae type b conjugate vaccination sera (n=89). We determined that elution of low-avidity antibodies present in multiple dilutions of the serum sample by a single concentration of a chaotrope (0.15 M sodium thiocyanate [NaSCN]) was optimal for the determination of avidity measurements throughout a wide range of IgG concentrations (0.94 to 304.6 microg/ml). The percent reduction in concentration as determined by the elution assay with 0.15 M NaSCN correlated highly (r=0.84) with weighted averages obtained by an elution assay with multiple solutions of NaSCN. The correlation (r=0.57) between elution and binding interference, when a single concentration of a chaotrope was used, was lower than the correlation between the two elution methods (r=0.84). We found that the serum dilution, the heterogeneity of the antibody population, and the concentration of the chaotrope were the primary variables affecting avidity determinations. In this study, we present multiple analysis methods depending on the methodology used. We also present the factors that affect the analysis of avidity determinations given the polyclonal nature of human sera. This experimental approach should benefit the evaluation of similar antibodies induced by other bacterial polysaccharide vaccines.     

Efficacy of Haemophilus influenzae type b vaccination of children: a meta-analysis

Haemophilus influenzae type b (Hib) infection is a leading cause of meningitis and pneumonia in infants and children in developing countries, and yet the implementation of routine Hib vaccination is very slow. The aim of the present study was to quantify the protective efficacy of H. influenzae type b vaccination of young children. MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials Register were searched. References of selected articles were also reviewed and experts contacted. Eight randomized trials were found that compared the efficacy of H. influenzae type b conjugate vaccine to placebo or no vaccine. Information on study design, patients enrolled, age, vaccine type, cases of invasive H. influenzae type b disease, adverse events, and items to assess potential for bias was recorded. The incidence of invasive H. influenzae type b infection formed the primary outcome. The odds ratio (OR) of developing Hib infection was combined using a random effects model to provide a measure of vaccine efficacy. The protective effect, defined as the relative risk reduction, was estimated as (1-OR). From eight trials, the protective efficacy of the Hib conjugate vaccine was 84% (OR 0.16; 95%CI 0.08–0.30) against invasive Hib disease, 75% (OR 0.25; 95%CI 0.08–0.84) against meningitis, and 69% (OR 0.31; 95%CI 0.10–0.97) against pneumonia. Serious adverse events were rare. The results provide firm evidence that Hib conjugate vaccines are safe and effective in reducing the risk of all forms of invasive Hib disease, further establishing that vaccination of children in developing countries can protect them from a potentially fatal yet preventable disease.     

Prevention of Haemophilus influenzae type b disease: past success and future challenges

Haemophilus influenzae type b (Hib) is responsible for significant morbidity and mortality worldwide, particularly in children under 5 years of age. In countries where the Hib conjugate vaccine is not routinely used, Hib is a leading cause of childhood pneumonia and meningitis. Routine use of the Hib conjugate vaccines has resulted in a remarkable decline in Hib disease in developed and developing countries. However, Hib conjugate vaccines are not routinely available in most developing countries, many of which have high burdens of Hib disease. This review outlines the pathogenesis and epidemiology of Hib disease, and the various options for prevention.     

Separation of Serum Bactericidal and Opsonizing Activities for Haemophilus influenzae Type b

When assay conditions permitted bacterial growth, immune serum had no bactericidal activity for Haemophilus influenzae but promoted rapid phagocytic killing of this organism by human leukocytes.     

Regulatory T Cells in the Antibody Response to Haemophilus influenzae Type b Polysaccharide

An in vitro culture system for the induction of an antipolysaccharide response was used to study the cellular interactions which determine the magnitude and nature of this B-lymphocyte response. Healthy adult volunteers were vaccinated with the Haemophilus influenzae type b polysaccharide (PRP)-tetanus toxoid (TT) conjugate vaccine. Optimal in vitro anti-PRP and anti-TT antibody responses were obtained when B cells were cultured with equal amounts of T cells. The in vitro response is antigen dependent and antigen specific. Culturing with PRP mixed with TT in the presence of T cells induces the highest number of anti-PRP antibody-secreting cells (ASC) (128.4 ×/÷ 15.9 [geometric mean ×/÷ standard deviation] immunoglobulin M [IgM] anti-PRP ASC/10⁶ cells; 9.3 ×/÷ 7.6 IgG anti-PRP ASC/10⁶ cells). Culturing without T cells induced no anti-PRP ASC; culturing with only PRP, in the presence of T cells, yielded low numbers of anti-PRP ASC (3.7 ×/÷ 5.2 IgM anti-PRP ASC/10⁶ cells and 1.2 ×/÷ 2.2 IgG anti-PRP ASC/10⁶ cells). Transwell studies showed that the requirements for the antibody response against the polysaccharide are different from those of an antiprotein response. Cytokines formed as a consequence of contact between protein-specific B and T cells were on their own not sufficient to activate TT-specific B cells (8.4 ×/÷ 1.4 anti-TT ASC/10⁶ cells); direct contact between T and B cells appeared to be an absolute requirement. However, physical contact between B and T cells in one compartment of the Transwell system resulted in the release of soluble factors able to stimulate B cells in the other compartment to secrete antipolysaccharide antibodies (164 ×/÷ 1.6 anti-PRP ASC/10⁶ cells).     

Hyperimmune Products in the Prevention and Therapy of Infectious Disease

This paper reviews a meeting at which basic pathophysiology of infections, mechanisms of action of hyperimmune products and pharmacokinetic and pharmacodynamic parameters, as well as currently available hyperimmunes and their potential new targets and uses, were discussed. A hyperimmune product was defined as either a monoclonal antibody or a polyclonal preparation enriched with antibody directed against one or more particular targets. A number of issues were emphasised, including: resistant bacterial pathogens, such as Staphylococcus aureus and Streptococcus pyogenes; the role of hyperimmune intravenous globulins in the prevention of sepsis in low birthweight infants; hepatitis B virus infection associated with liver transplantation; combination therapy; the potential role of hyperimmunes in the prevention and treatment of hepatitis C virus; and the use of immunoglobulins for the prophylaxis of Epstein-Barr virus-related lymphoproliferative disease. Routes of administration were also discussed. It was concluded that the development of hyperimmunes faces numerous obstacles. It was agreed that the use of hyperimmunes in clinical trials must be standardised; clinical trials must be large enough to have sufficient power to demonstrate efficacy with clear-cut end-points, and means need to be developed, in conjunction with regulatory agencies, for the feasible evaluation of combination products. However, progress in all these aspects will provide a wide range of hyperimmunes for future use.     

Haemophilus influenzae type b antigenuria in children.

Detection of Haemophilus influenzae type b (HIb) antigenuria by latex agglutination has been shown to be sensitive, specific, and rapid. In children, antigenuria persisted for a mean duration of 10 days and a maximum of 18 days. Antigenuria was demonstrated in 25 of 30 patients with HIb infection but not in 62 with other types of infection. In five children, antigenuria confirmed the diagnosis in the absence of bacteriological confirmation. In five other children, antigenuria was not found, but in this group the antigen was detected in another body fluid or HIb was recovered.     

Immunogenicity of Haemophilus influenzae Type b Conjugate Vaccines in Korean Infants: A Meta-analysis

A meta-analysis was performed on the immunogenicity of Haemophilus influenzae type b (Hib) conjugate vaccines after 2 (2 and 4 months) and 3 doses (2, 4, and 6 months) in Korean infants. A database search of MEDLINE, KoreaMed, and Korean Medical Database was done. The primary outcome measure was the proportion of infants with anti-polyribosylribitol phosphate (PRP) concentrations ≥1.0 µg/mL. Eight studies including eleven trials were retrieved. One trial reported on the diphtheria toxoid conjugate vaccine (PRP-D) and 2 trials each on the mutant diphtheria toxin (PRP-CRM) and Neisseria meningitidis outer-membrane protein (PRP-OMP) conjugate vaccine. Heterogeneity in study designs between trials on PRP-CRM was noted and one trial reported on a monovalent and another on a combination PRP-OMP vaccine. Thus, a meta-analysis was conducted only on the tetanus toxoid conjugate vaccine (PRP-T). After a primary series of 2 doses and 3 doses, 80.6% (95% confidence interval [CI]; 76.0-85.1%) and 95.7% (95% CI; 94.0-98.0%) of infants achieved an antibody level ≥1.0 µg/mL, respectively. The immunogenic response to the PRP-T vaccine was acceptable after a primary series of 3 doses and also 2 doses. A reduced number of doses as a primary series could be carefully considered in Korean infants.     

Risk of Invasive Haemophilus influenzae Type b (Hib) Disease in Adults with Secondary Immunodeficiency in the Post-Hib Vaccine Era

Prior to the introduction of Haemophilus influenzae type b (Hib) conjugate vaccines, invasive Hib disease affected almost exclusively children. According to some recent studies, in the postvaccine era, adults, the elderly, and immunocompromised persons can be affected more often than children. As the production of type-specific anti-capsular polysaccharide antibodies is the major defense mechanism against Hib, individuals with defects in humoral immune responses have high susceptibility to infections caused by Hib. We hypothesized that nonvaccinated adults with chronic conditions causing immunosuppression may lack protective antibody to Hib. We assessed serum anti-Hib IgG levels and bactericidal activity in 59 patients with chronic renal failure, 30 patients with type 2 diabetes mellitus, 28 patients with chronic obstructive pulmonary disease (COPD), and 20 patients with multiple myeloma compared to 32 healthy controls of similar age. Considering antibody at >0.15 μg/ml as the protective correlate in unvaccinated individuals, we detected subprotective Hib antibody levels in 29% of chronic renal failure, 20% of diabetes, 14% of COPD, and 55% of myeloma patients compared to 3% of healthy controls. Additionally, 70% of myeloma and 58% of chronic renal failure patients did not have detectable serum bactericidal activity against Hib. Among individuals with severe diseases causing secondary immunodeficiency, patients with multiple myeloma and chronic renal failure are at an increased risk of invasive Hib disease. Considering that Hib continues to circulate in the population, this study provides a rationale for the immunization of some adult patients with secondary immunodeficiency with the pediatric Hib vaccine to achieve protective immunity.     

Immune Response to Haemophilus influenzae Type b Vaccination in Patients with Chronic Renal Failure

Adult chronic renal failure patients undergoing hemodialysis are at an increased risk of invasive Haemophilus influenzae type b (Hib) disease due to the lack of functionally active anti-Hib antibodies. The pediatric Hib polysaccharide-protein conjugate vaccine is highly immunogenic in these patients and can provide protection against invasive Hib infection for at least 1 year.