Haemophilus influenzae infections in the H. influenzae type b conjugate vaccine era

The widespread use of Haemophilus influenzae type b (Hib) conjugate vaccines has nearly eradicated invasive Hib disease where the vaccines are used. This success was accompanied by a shift in capsular serotypes of invasive H. influenzae disease, with nontypeable strains replacing type b strains as the most common bloodstream isolate, but there is no convincing evidence of a true increase in the incidence of non-serotype b invasive infections. H. influenzae causes predominantly mucosal infections. The introduction of vaccines for otitis media and global shifts in antimicrobial susceptibility emphasize the importance of continued surveillance of H. influenzae colonization and disease patterns.     

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.     

Subclass distribution of IgA antibodies in saliva and serum after immunization with Haemophilus influenzae type b conjugate vaccines

IgA subclass distribution of antibodies against capsular polysaccharide (PS) of Haemophilus influenzae type b (Hib) was studied in saliva and serum samples of children vaccinated with two (n = 58) or three doses (n = 53) of Hib vaccine. One month after the second dose of Hib conjugate vaccine, at 7 months old, 40% of the children had IgA1 and 41% had IgA2 anti-Hib PS antibodies in saliva. One month after the third dose, at 15–25 months old, IgA1 was the predominating subclass; 72% of the children had IgA1, 26% had IgA2 anti-Hib PS in saliva. The mean concentration of IgA1 anti-Hib PS, expressed as optical density (OD) values, was significantly higher after three doses (OD 80.7) than after two doses (OD 18.9). The mean concentration of IgA2 did not change significantly after the third dose (OD 23.8 after two doses, OD 18.1 after three doses). In serum, IgA1 anti-Hib PS predominated both after two (17% had IgA1, none had IgA2) and three doses (72% had IgA1, 4% had IgA2) of Hib vaccine. In conclusion, both IgA1 and IgA2 anti-Hib PS were found in saliva of immunized children after two doses of Hib conjugate vaccine, whereas the third vaccine dose induced a shift towards IgA1 anti-Hib PS dominance in saliva.     

Haemophilus influenzae serotype b conjugate vaccine failure in twelve countries with established national childhood immunization programmes

The present study describes the clinical and immunological features of children with Hib vaccine failure, who were identified through national surveillance between 1996 and 2001 in Europe, Israel and Australia. True vaccine failure was defined as invasive Hib disease occurring ≥2 weeks after one dose, given after the first birthday, or ≥1 week after ≥2 doses, given at <1 year of age. Of the 423 cases (representing 0.2 cases per 100 000 child-years at risk) reported, 330 (78%) had received three doses in the first year of life and developed disease at a median age of 28 months. Of the remaining 93, 48 had received two doses in infancy, 34 had received four doses including a booster, and 11 had received a single dose after 12 months of age. These children developed disease at a median age of 12, 33 and 71 months, respectively. In total, 47 out of 258 children (18%) with available information had an underlying medical problem (including prematurity) and 53 out of 161 (33%) had immunoglobulin deficiency. Convalescent Hib antibody concentrations were above the putative protective concentration of 1.0 mg/L in 147/194 (76%) children; low concentrations were associated with both the presence of an underlying medical problem and young age at the time of Hib disease. Almost all children who received an additional vaccine dose developed antibodies at protective concentrations. Thus, Hib vaccine failure is rare, but can occur with any immunization schedule. Children with Hib vaccine failure should have immunoglobulin and convalescent Hib antibody concentrations measured after infection and receive additional vaccination, if required.     

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.     

Antigenicity and immunogenicity of a synthetic oligosaccharide-protein conjugate vaccine against Haemophilus influenzae type b

Polysaccharide-protein conjugates as vaccines have proven to be very effective in preventing Haemophilus influenzae type b infections in industrialized countries. However, cost-effective technologies need to be developed for increasing the availability of anti-H. influenzae type b vaccines in countries from the developing world. Consequently, vaccine production with partially synthetic antigens is a desirable goal for many reasons. They may be rigidly controlled for purity and effectiveness while at the same time being cheap enough that they may be made universally available. We describe here the antigenicity and immunogenicity of several H. influenzae type b synthetic oligosaccharide-protein conjugates in laboratory animals. The serum of H. influenzae type b-immunized animals recognized our synthetic H. influenzae type b antigens to the same extent as the native bacterial capsular polysaccharide. Compared to the anti-H. influenzae type b vaccine employed, these synthetic versions induced similar antibody response patterns in terms of titer, specificity, and functional capacity. The further development of synthetic vaccines will meet urgent needs in the less prosperous parts of the world and remains our major goal.     

Influence of prevaccination immunity on the human B-lymphocyte response to a Haemophilus influenzae type b conjugate vaccine.

The purpose of this study was to investigate whether preexisting immunity to components of a polysaccharide-protein conjugate influences the B-lymphocyte response to vaccination with the conjugate. Thirty-two healthy adults were vaccinated once or twice with a conjugate (PRP-D) consisting of Haemophilus influenzae type b capsular polysaccharide (PRP) and diphtheria toxoid (DT), and the response was related to the prevaccination levels of PRP and DT antibodies. Positive correlations were found between increases in plasma PRP (median, 32.0 micrograms/ml) and DT (1.14 IU/ml) antibodies and numbers of circulating PRP and DT antibody-secreting cells (AbSC) (postvaccination days 6 to 9). The B-cell responses (antibody response and AbSC) to both PRP and DT correlated positively with prevaccination levels of anti-DT. DT AbSC appeared earlier (peak, day 7) than PRP AbSC (peak, day 8). Individuals whose PRP AbSC peaked early (day 7) had higher prevaccination anti-DT levels than those who peaked later (P less than 0.05). In contrast, the prevaccination levels of anti-PRP did not correlate significantly with the magnitude of the antibody or AbSC response and did not affect the kinetics of the AbSC. Following revaccination with PRP-D, small increases in the level of PRP antibodies (median, 2.9 micrograms/ml; n = 11) were found; no significant increase in the level of DT antibodies was seen. The numbers of PRP AbSC were lower (P = 0.04) and peaked earlier (day 7) than after the first vaccination. The isotype pattern of PRP AbSC, which was dominated by immunoglobulin A (IgA) after the first vaccination, now showed a more equal distribution between IgG and IgA AbSC. It is concluded that after immunization with PRP-D both the magnitude and the kinetics of the antipolysaccharide B-cell response are influenced by prevaccination immunity to the carrier molecule.     

The regulation of the immune response of mice to Haemophilus influenzae type b capsular polysaccharide.

The regulation of age-related antibody response to Haemophilus influenzae type b polysaccharide (HITB-PS) was studied by measuring the splenic plaque forming cells (PFC) following immunization with this capsular polysaccharide. The magnitude of PFC response to HITB-PS was found to be dose-related, enhanced by Freund's complete adjuvant and influenced by the genetic strain of mice. Priming with a low dose of HITB-PS did not induce a state of immunological unresponsiveness. Treatment with antilymphocyte serum significantly increased the PFC response to HITB-PS. Athymic nude mice showed an enhanced ability to induce both IgG and IgA-PFC responses as well as a significant increase in the biosynthesis of protein and mitogenicity in spleen cells. These findings suggest that the immune response to HITB-PS is regulated by the suppressor T cell. The magnitude of the IgM-PFC response induced by HITB-PS in mice increased gradually from two weeks of age and reached a plateau at 8 weeks. Treatment with fetuin resulted in the inhibition of direct IgM and IgG-PFC responses to HITB-PS; the suppressive effect on the immune response was more profound and lasting in young than in adult mice.     

Molecular epidemiology of Haemophilus influenzae type b isolated from children with clinical cases of conjugate vaccine failures

We analyzed eight invasive Haemophilus influenzae strains isolated from individual children vaccinated appropriately for their ages. Five of the strains were type b and three were nontypeable strains. Children infected with nontypeable strains had a protective level of anti-H. influenzae type b antibodies, while only one of the children whose cases represented true vaccine failure did.     

Characterization of the antibody response to a Haemophilus influenzae type b conjugate vaccine in children with recurrent lower respiratory tract infection

Children with recurrent lower respiratory tract infection (RLRI) may respond poorly to polysaccharide antigens. To examine how such children respond to a polysaccharide coupled to a protein carrier, we immunized 15 children with RLRI aged 8–69 months and 15 carefully age-matched healthy controls once with a Haemophilus influenzae type b (Hib) conjugate vaccine. Total IgG subclasses, total antipolysaccharide Hib antibodies, and antipolysaccharide Hib antibodies of IgM, IgG, IgA, and IgG 1–4 specificity were determined by ELISA. There were no significant differences between the two groups in any single total IgG subclass, but total IgG measured as the sum of all four subclasses was significantly lower in the children with RLRI than in the controls ( P = 0.036). Before vaccination, the children with RLRI had significantly less IgG antipolysaccharide Hib antibody than the controls ( P = 0.005), whereas 1 month later they had significantly more IgM antibody (P = 0.038). No other significant differences were found between the groups before or after immunization with respect to antipolysaccharide Hib antibodies. Since naturally occurring IgG antibodies are thought to be aquired partly as a consequence of antigenic stimulation on mucosal surfaces, we hypothesize that the low level of specific IgG found before immunization, as well as the low total IgG in the children with RLRI, may reflect an impaired ability to prime through mucosal surfaces. This is supported by our finding of an increased IgM response to Hib conjugate vaccine in these children, since this isotype predominates in the primary immune response, i.e., in the absence of immunologic memory. In conclusion, children with RLRI can be protected against invasive Hib infection as well as healthy children, but may have an immunodeficiency characterized by defective ability to respond to antigenic stimulation on mucosal surfaces.     

Functional antibody activity elicited by fractional doses of Haemophilus influenzae type b conjugate vaccine (polyribosylribitol phosphate-tetanus toxoid conjugate).

We evaluated the functional activities of antibodies, serum bactericidal activity (SBA), and immunoglobulin G (IgG) antibody avidity indices, using sodium thiocyanate (NaSCN) elution, elicited after vaccination with fractional doses of the Haemophilus influenzae type b conjugate (polyribosylribitol phosphate [PRP] conjugated to tetanus toxoid [PRP-T]) vaccine. A cohort of 600 infants from the Dominican Republic were randomized to receive one of three regimens of the PRP-T vaccine at ages 2, 4, and 6 months: full doses (10 microg of PRP antigen), one-half doses (5.0 microg), and one-third doses (3.3 microg) (J. Fernandez et al., Am. J. Trop. Med. Hyg. 62:485-490, 2000). Sixty serum samples, collected at age 7 months, with > or =2.0 microg of anti-PRP IgG per ml were randomly selected for avidity determinations. Geometric mean IgG concentrations were 13, 14, and 17 microg/ml for infants who received the full-dose (n = 19), one-half-dose (n = 19), and one-third-dose (n = 22) regimens, respectively. SBA geometric mean titers (1/dilution) were 85.0, 82.0, and 76.1 in sera from infants receiving the full-, one-half-, and one-third-dose regimens, respectively. Avidity indices (mean +/- standard error weighted average of NaSCN molar concentration x serum dilution factor) were 71.9 +/- 9.4, 123.6 +/- 26.8, and 150.9 +/- 24.9 for the full-, one-half-, and one-third-dose regimens, respectively. Upon comparison, the only significant difference (P = 0.024) found was a greater avidity index for sera from infants receiving the one-third-dose regimen than for sera from infants receiving the the full-dose regimen. We conclude that fractional doses elicit similar functional antibody activities in infants with > or = 2 microg of anti-PRP IgG per ml, corresponding to 89, 90, and 97% of infants receiving three doses of either the full concentration or one-half or one-third of the labeled concentration, respectively. This approach offers an alternative strategy for the prevention of H. influenzae type b disease in countries with limited resources.     

Augmentation of HIV-1 subtype C vaccine constructs induced immune response in mice by CpG motif 1826-ODN

The greatest biomedical challenge of this century is to develop a preventive vaccine against Human Immunodeficiency Virus (HIV-1). For an HIV vaccine to be effective, it appears logical to develop new strategies that enhance the level of the immune response as well as steer it towards the desirable cellular type. In view of this, there is a need for rational inclusion of biological adjuvants into the HIV-1 vaccination strategies that could potentiate the immune responses both qualitatively and quantitatively. The adjuvant may include the immunostimulatory oligonucleotides containing CpG motifs, whose immunomodulatory characters are well established and represent the basis for an effective vaccine adjuvant. In our study, we investigated the use of an immunostimulatory oligonucleotide (or CpG motif), 1826-ODN to augment the immune response elicited by plasmid DNA vaccine constructs containing Indian subtype C HIV-1 envelope gp120 and gag-protease genes in Balb/c mouse model system. A dose of 2-microg CpG motifs/mouse was found to be optimum when co-administered with the DNA vaccine constructs with the peak level of humoral and cell mediated immune responses at 6 weeks post immunization. Murine IFN-gamma ELISpot assay demonstrated that the use of 1826-ODN led to a broad based and long term recognition of the subtype C envelope and gag peptides. The use of CpG motifs has been effective in augmenting the immune responses generated by the DNA constructs. Taken together, these results are an important advancement towards the design of future preclinical and clinical trials of these vaccine constructs.     

Immunoglobulin G avidities in infants in Mexico after primary immunization with three doses of polyribosylribitol phosphate-tetanus toxoid Haemophilus influenzae type b vaccine

Serum immunoglobulin G concentrations and avidities specific to Haemophilus influenzae type b (Hib) were measured in 208 children living in Guadalajara and Mexico City. Protective concentrations were found in 98.9% and 100.0% of participants, respectively. Geometric mean concentrations differed between both populations and/or among age groups. Mean avidities differed only among the 7- to 12-month-old children. Diphtheria-tetanus-whole-cell pertussis-hepatitis B-Hib primary vaccination seems to induce protection in Mexican children.     

Induction of an immune response to the porin of Haemophilus influenzae type b by monoclonal anti-idiotypic antibodies.

Monoclonal anti-idiotypes were generated against monoclonal antibody (mAb) Hb-2 which recognized a highly conserved epitope on the outer membrane porin protein from Haemophilus influenzae type b (Hib). Four hybridomas reacting with F(ab') 2 fragments of Hb-2 were selected and characterized. Inhibition studies using syngeneic anti-anti-idiotypic antisera suggested that at least three different antigenic determinants on Hb-2 were recognized by these monoclonal anti-idiotypes. The binding of each anti-idiotype to Hb-2 was inhibited by Hb-2 whereas the reaction was not affected by any other anti-Hib mAb. Complete inhibition of the binding of anti-idiotype to the idiotype could be achieved with 10 micrograms of total outer membrane protein (OMP) from Hib suggesting that the anti-idiotypes might be directed against paratope-associated idiotypes. Outer membrane antigens not recognized by mAb Hb-2 did not inhibit the reaction. Furthermore, the pre-incubation of Hb-2 with each anti-idiotype specifically prevented the reaction of Hb-2 with its antigen. Antibodies with specificity for the porin were generated in guinea pigs immunized with anti-idiotypes AHb-22 and AHb-23. This study indicates that these particular monoclonal anti-idiotypes may be used as an antigen substitute for the porin of Hib in a xenogeneic species.     

Effect of systemic immunization on pulmonary clearance of Haemophilus influenzae type b.

The effect of systemic immunization on pulmonary clearance of Haemophilus influenzae type b (Hib) was studied in a mouse model system. Immunization of mice by intraperitoneal injection of viable Hib cells resulted in the appearance of Hib-directed antibodies in both serum and bronchoalveolar lavage fluid. The development of this Hib-directed antibody activity was associated with significant enhancement of early pulmonary clearance of Hib. Systemic immunization did not affect the recruitment of polymorphonuclear leukocytes to the alveoli, suggesting that the enhanced clearance of Hib observed in immunized animals was due to specific antibodies which promote either phagocytosis or extracellular killing of Hib. The spectrum of Hib-directed antibody specificities detected in sera from immunized animals was essentially identical to that detected in bronchoalveolar lavage fluids from these same animals. Similarly, intravenous administration of an immunoglobulin G monoclonal antibody specific for Hib lipopolysaccharide resulted in the subsequent appearance of this antibody in the alveolar spaces where it enhanced pulmonary clearance of Hib. This study shows that this mouse model system can be used to measure the effect of both active and passive immunization on the clearance of Hib from the lower respiratory tract.     

Phase I clinical evaluation of a synthetic oligosaccharide-protein conjugate vaccine against Haemophilus influenzae type b in human adult volunteers

Since 1989, we have been involved in the development of a vaccine against Haemophilus influenzae type b. The new vaccine is based on the conjugation of synthetic oligosaccharides to tetanus toxoid. Our main goals have been (i) to verify the feasibility of using the synthetic antigen and (ii) to search for new production alternatives for this important infant vaccine. Overall, eight trials have already been conducted with adults, children (4 to 5 years old), and infants. We have described herein the details from the first two phase I clinical trials conducted with human adult volunteers under double blind, randomized conditions. The participants each received a single intramuscular injection to evaluate safety and initial immunogenicity. We have found an excellent safety profile and an antibody response similar to the one observed for the control vaccine.     

CpG寡脱氧核苷酸增强免疫抑制小鼠对乙肝疫苗的免疫应答

目的 :探讨CpG寡脱氧核苷酸 (ODN)增强免疫抑制小鼠对乙肝疫苗的免疫应答效果。方法 :选用环磷酰胺 (CTX)所致免疫抑制模型小鼠 ,以乙肝疫苗和 2 0 μgCpGODN联合或单独左胫前肌肌注免疫C5 7BL/6小鼠。 2wk后以同样剂量加强免疫 1次 ,再过 3wk后摘除眼球取血 ,用ELISA法检测抗 HBsIgG抗体和IL 12的水平。同时 ,无菌取脾脏做HE染色 ,观察脾脏淋巴细胞的数量和细胞核的变化。结果 :CpGODN与疫苗联合注射组产生的绝对抗体量比单独注射疫苗组提高 1倍 ;注射组产生IL 12的水平较单独注射疫苗组也有明显升高。光镜下各组脾脏淋巴细胞的变化如下 :正常对照组脾脏可见大量的淋巴细胞 ;与正常对照组相比较 ,CTX组的淋巴细胞明显稀少 ;而CTX加CpG组的淋巴细胞数明显增多 ,细胞核也明显增大。结论 :CpGODN能增强免疫抑制小鼠对乙肝疫苗的免疫应答     

Limited efficacy of a Haemophilus influenzae type b conjugate vaccine in Alaska Native infants. The Alaska H. influenzae Vaccine Study Group

BACKGROUND. The prevention of invasive Haemophilus influenzae type b disease requires a vaccine that is effective when administered during the first six months of life. The infants of Alaska Natives are at particularly high risk of invasive H. influenzae type b disease. METHODS. To evaluate the protective efficacy of a H. influenzae type b polysaccharide-diphtheria toxoid conjugate vaccine (polyribosylribitol phosphate-diphtheria toxoid [PRP-D]), we enrolled 2102 Alaska Native infants in a randomized, double-blind, placebo-controlled trial in which either the vaccine or a saline placebo was administered at approximately two, four, and six months of age. RESULTS. After 3969 subject-years of follow-up and 32 episodes of H. influenzae type b disease, the overall incidence of invasive disease was not reduced significantly in the vaccinated subjects (6.0 cases per 1000 patient-years), as compared with the placebo controls (9.6) or with other Alaska Native infants (6.0). After one, two, or three doses there was no significant protective efficacy with the vaccine; after three doses the efficacy was only 35 percent (95 percent confidence interval, -57 to 73). The lack of efficacy was not related to the age at onset of disease, age at immunization, type of disease, degree of Alaska Native heritage, time after immunization, or year of the study. Levels of H. influenzae type b anticapsular antibody in recipients of the vaccine became significantly higher than levels in those who received placebo only after the second and third doses. Even after the third dose, only 48 percent of the vaccinated infants had antibody levels of more than 0.1 microgram per milliliter (geometric mean titer, 0.18). Antibody responses did not vary with the level of maternally acquired antibody, degree of Alaska Native ancestry, or age at time of the first or second immunizations, but they increased with increasing age at time of the third dose (P less than 0.001). CONCLUSIONS. We found no evidence that the PRP-D vaccine provides significant protection, at least for Alaska Native infants, against invasive diseases caused by H. influenzae type b. The ineffectiveness of the vaccine paralleled its limited immunogenicity.