Chiayung Chu

Epitopic overload at the site of injection may result in suppression of the immune response to combined capsular polysaccharide conjugate vaccines.

Capsular polysaccharide (CP) conjugate vaccines targeting a variety of bacterial infections are currently under development and clinical evaluation. The inclusion of multiple CP serotypes combined in a single injection is an important maneuver being evaluated. The combination of CP conjugate vaccines into a single multivalent injection may result in competition among the different components and adversely affect the immunogenicity of any individual conjugate. We observed a reduction of 30-90% in antibody responses to several serotypes in mice when immunogenicity of a 12-valent Escherichia coli (E. coli) lipopolysaccharide (LPS) conjugate vaccine was compared to the immunogenicity of each monovalent vaccine evaluated separately. A reduction of 30% was observed in the Staphylococcus aureus (S. aureus) type 8 CP antibodies when a type 8-rEPA conjugate was combined with a type 5-rEPA conjugate. S. aureus types 5 and 8-rEPA conjugates were combined with 100 micrograms of either rEPA (homologous) or diphtheria toxoid (DT) (heterologous) carrier proteins, and evaluated in rEPA or DT primed mice. The addition of the homologous protein resulted in a 64% reduction in type 5 CP antibodies. The heterologous protein did not affect the immunogenicity of the type 5. We postulate that the free protein competed with the conjugate and recruited most of the rEPA primed T cells. In the case of the DT conjugates, the DT targeted different populations of the T cells, thus interference was not observed. These data suggested that the epitopic load rather than the antigenic load at the site of injection caused reduced immunogenicity of the conjugates. We theorize that individual components of multivalent CP vaccines conjugated to the same carrier proteins would compete for a limited number of specific carrier protein primed T cells. This would result in one or more components being unavailable in eliciting a sufficient immune response. The use of multiple carrier proteins should be considered as an approach to reduce interference when multivalent conjugate vaccines are to be formulated into a single injection.

Safety and Immunogenicity of Improved Shigella O-Specific Polysaccharide-Protein Conjugate Vaccines in Adults in Israel

ABSTRACT Data suggest that the O-specific polysaccharide (O-SP) domain of the lipopolysaccharide (LPS) of Shigella species is both an essential virulence factor and a protective antigen and that a critical level of serum immunoglobulin G (IgG) to this antigen will confer immunity to shigellosis. Because covalent attachment of polysaccharides to proteins increases their immunogenicity, especially in infants and in young children, the O-SP of Shigella species were bound to medically useful proteins, and the safety and immunogenicity of the resultant conjugates were confirmed in adults and 4- to 7-year-old children. Succinylation of the carrier protein improved the immunogenicity of Shigella conjugates in mice and increased their yield. Based on these results, a clinical trial of O-SP conjugates of Shigella sonnei and Shigella flexneri 2a bound to succinylated mutant Pseudomonas aeruginosa exotoxin A (rEPAsucc) or native or succinylated Corynebacterium diphtheriae toxin mutant (CRM9 or CRM9succ) was conducted in healthy adults. The conjugates were safe and immunogenic. S. sonnei-CRM9,S. sonnei-CRM9succ, and S. sonnei-rEPAsucc elicited significant rises of geometric mean (GM) IgG anti-LPS within 1 week of injection (P < 0.001). At 26 weeks, the GM anti-LPS levels elicited by these three conjugates were similar and higher than their prevaccination levels (P < 0.0001). GM IgG anti-LPS levels elicited by S. flexneri2a-rEPAsucc were significantly higher than those elicited by S. flexneri 2a-rCRM9succ at all intervals after injection. At 26 weeks, the levels of IgG anti-LPS in vaccinees were higher than their prevaccination levels (P < 0.0001). The serum antibody responses were specific, as there was no significant rise of anti-LPS to the heterologous O-SP in any vaccinee. Both conjugates elicited statistically significant rises of serum antibodies to the injected carrier protein. At 6 months, these five Shigellaconjugates elicited higher fold rises than similar conjugates (D. N. Taylor et al., Infect. Immun. 61:3678–3687, 1993). Based on these data, we chose S. sonnei-CRM9 and S. flexneri2a-rEPAsucc for evaluation in children.

The Vi conjugate typhoid vaccine is safe, elicits protective levels of IgG anti-Vi, and is compatible with routine infant vaccines.

ABSTRACT Typhoid fever remains a serious problem in developing countries. Current vaccines are licensed for individuals who are 5 years old or older. A conjugate of the capsular polysaccharide (CP) of Salmonella enterica serovar Typhi (Vi) bound to recombinant exoprotein A of Pseudomonas aeruginosa (Vi-rEPA) enhanced Vi immunogenicity and protected 2- to 5-year-olds in Vietnam. In this study, Vi-rEPA was evaluated for use in infants. A total of 301 full-term Vietnamese infants received Expanded Program on Immunization (EPI) vaccines alone or with Vi-rEPA or Haemophilus influenzae type b-tetanus toxoid conjugate (Hib-TT) at 2, 4, and 6 months and Vi-rEPA or Hib-TT alone at 12 months. Infants were visited 6, 24, and 48 h after each injection to monitor adverse reactions. Maternal, cord, and infant sera were assayed for IgG anti-Vi and for IgG antibodies to Hib CP and the diphtheria, tetanus, and pertussis toxins at 7, 12, and 13 months. No vaccine-related serious adverse reactions occurred. In the Vi-rEPA group, the IgG anti-Vi geometric mean (GM) increased from the cord level of 0.66 to 17.4 enzyme-linked immunosorbent assay units (EU) at 7 months, declined to 4.76 EU at 12 months, and increased to 50.1 EU 1 month after the 4th dose (95% of infants had levels of ≥3.5 EU, the estimated protective level). Controls had no increase of the IgG anti-Vi GM. Infants with cord anti-Vi levels of <3.5 EU responded with significantly higher IgG anti-Vi levels than those with levels of ≥3.5 EU. Anti-diphtheria, -tetanus, and -pertussis toxin levels were similar in all groups. Vi-rEPA was safe, induced protective anti-Vi levels, and was compatible with EPI vaccines, and it can be used in infants. High cord IgG anti-Vi levels partially suppressed infant responses to Vi-rEPA.

Age-related efficacy of Shigella O-specific polysaccharide conjugates in 1–4-year-old Israeli children

BACKGROUND Despite its high worldwide morbidity and mortality, there is yet no licensed vaccine for shigellosis. We reported the safety and immunogenicity of Shigella O-specific polysaccharide-protein conjugates in adults and young children and efficacy of Shigella sonnei conjugate in young adults. METHODS A double-blinded, randomized and vaccine-controlled Phase 3 evaluation of S. sonnei and Shigella flexneri 2a O-SP-rEPA conjugates, 25 microg, injected IM twice, 6 weeks apart, into healthy 1-4 years old, is reported. The children were followed for 2 years by telephone every other week and stool cultures were obtained for each episode of acute diarrhea (> or =3 loose stools/day or a bloody/mucous stool). Sera were taken randomly from 10% of the participants for IgG anti-LPS and anti-carrier levels. RESULTS Of the 2799 enrollees, 1433 received S. sonnei and 1366 S. flexneri 2a conjugates; 2699 (96.4%) completed the 2-year follow-up. Local reactions occurred in approximately 5% and approximately 4% had temperatures > or =38.0 degrees C lasting 1-2 days. There were no serious adverse events attributable to the vaccines. Of the 3295 stool cultures obtained, 125 yielded S. sonnei and 21 S. flexneri 2a. Immunogenicity and efficacy were age-related. The overall efficacy of the S. sonnei conjugate was 27.5%; 71.1% (P=0.043) in the 3-4 years old. The numbers for S. flexneri 2a were too few for meaningful analysis. Cross-protection by S. flexneri 2a for non-vaccine S. flexneri types was found, but the numbers were too few for statistical significance. There was an age-related rise of vaccine-specific IgG anti-LPS in both groups, peaking at about 10 weeks and declining thereafter, but remaining > or =4-fold higher than in the controls 2 years after the second dose. CONCLUSIONS Shigella conjugates are safe and immunogenic in 1-4 years old. The S. sonnei conjugate elicited 71.1% efficacy in the 3-4 years old and can be predicted to be efficacious in individuals older than 3 years of age. These results urge studies with our improved conjugates.

Safety and immunogenicity of Shigella sonnei-CRM9 and Shigella flexneri type 2a-rEPAsucc conjugate vaccines in one- to four-year-old children.

Background and objective. Shigella conjugate vaccines have been shown to be safe, immunogenic and efficacious in adult volunteers. We have now investigated the safety and immunogenicity of investigational Shigella sonnei and Shigella flexneri 2a conjugate vaccines in 1- to 4-year-old children, the age group at greatest risk for shigellosis. Methods. The O-specific polysaccharides of S. sonnei and S. flexneri 2a, the two most common shigellae from patients in Israel, were bound to medically useful carrier proteins to form conjugates. Eighty healthy 1- to 4-year-olds were randomized to receive two 0.5-ml im injections 6 weeks apart of either S. sonnei-CRM9 or S. flexneri 2a-r EPAsucc. Blood was taken before, 6 weeks after the first injection, 4 weeks after the second injections and 2 years after immunization for assay of IgG anti-lipopolysaccharide, diphtheria toxin and Pseudomonas aeruginosa exotoxin A antibodies by enzyme-linked immunosorbent assay. Results. During an 8-day surveillance period after each immunization, low fever (37.8–39.0°C) lasting only 24 to 48 h occurred in 2 of 40 recipients after the first injection and 4 of 40 recipients after the second injection of S. flexneri 2a-r EPAsucc and in 2 of 38 of S. sonnei-CRM9 after the second injection; no fever was detected after the first injection. Liver function tests were normal in all vaccinees. S. sonnei-CRM9 elicited a >4-fold rise in IgG anti-LPS in 92.1% and S. flexneri 2a-r EPAsucc in 85% (P < 0.0001) after the second injection; both conjugates elicited type-specific booster responses. At 2 years the geometric mean concentrations of both IgG anti-lipopolysaccharides were significantly higher than preimmunization levels. A >4-fold rise of IgG anti-diphtheria (65.8%) and IgG anti-ETA (77.5%) was observed. Conclusion. These experimental Shigella conjugate vaccines were safe and immunogenic in 1- to 4-year-old children.

Phase I study of a lipooligosaccharide-based conjugate vaccine against nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) accounts for about one-third of purulent otitis media (OM) in children and is a common cause of pulmonary infection in adults with decreased resistance. Based upon sero-epidemiological data in humans and immunochemical data in laboratory animals, a lipooligosaccharide (LOS)-tetanus toxoid (TT) conjugate was prepared and evaluated for its safety and immunogenicity in a Phase I study of 40 healthy adults. The conjugate was injected intramuscularly into all volunteers: 28 of them received a second injection 14 weeks later. Local and systemic reactions were monitored and sera, taken before and 2, 6, 14, 16, and 38 weeks after injection, were assayed for IgG, IgA, and IgM antibodies to the LOS by ELISA and for bactericidal activity. The results indicate that there were no significant local or systemic reactions after either injection. All volunteers had pre-existing IgG anti-LOS. The geometric mean (GM) level rose from 14 to 40 at 2 weeks, remained at 35 at 6 weeks (40 or 35 versus 14, P<0.01) and dropped to 27 at 14 weeks after the first injection. There was also a rise 2 weeks after the second injection (27 versus 37, P<0.05). A total of 52.5% of subjects showed serum-conversion (greater than four-fold increase) after one and two injections. At 38 weeks, the GM IgG anti-LOS was still higher than before initial injection (20 versus 14, P<0.05). A similar pattern of reactivity was observed for IgA and IgM anti-LOS. Similar to that observed in mice, but not in rabbits, the conjugate-induced antibodies did not yield significant bactericidal activity in vitro. The LOS-TT conjugate is well tolerant in adults and a Phase II evaluation of the conjugate in children is planned.

Prevention of invasive bacterial diseases by immunization with polysaccharide-protein conjugates.

Covalent binding of CPS to T cell-dependent carrier proteins to form conjugates can be done by clinically acceptable methods. As a component of a conjugate, two immunologic properties of CPS are changed: 1) their immunogenicity is increased and; 2) reinjection induces a booster response in the young (T cell-dependence). Serum antibodies induced by the CPS alone, or as a component of a conjugate, are qualitatively similar: the difference between antibodies elicited by the CPS or the conjugate is quantitative. A clinical trial with a Hib-DT conjugate showed that conjugates could confer immunity in an age group not protected by the CPS alone. (table; see text) Induction of serum CPS antibodies confers protection against capsulated bacteria in the bloodstream: their role in the interaction of these pathogens on the mucous membranes has not been characterized. Preliminary in vitro experiments suggest that secretory antibodies to non-capsular structures may also exert protective immunity.

Adjuvant activity of QS-21 for experimental E. coli 018 polysaccharide vaccines

Three types of experimental vaccines containing O-side-chain polysaccharide from the enterotoxigenic strain Escherichia coli 018 were evaluated. The immunogenicity of free O-polysaccharide (PS), a polysaccharide-diphtheria toxoid conjugate (PS-conj), and detoxified lipopolysaccharide (dLPS) was tested in female ICR mice, either alone or in combination with QS-21, a purified saponin adjuvant derived from the bark of the tree Quillaja saponaria Molina. Both the number of individual mice responding and the titres of O-polysaccharide specific antibodies in pools of sera were increased by the addition of QS-21. The immune response to both O-specific polysaccharide and carrier was primarily IgM and IgG1. The addition of QS-21 not only increased the level of IgG1, but also had a significant adjuvant effect on antigen-specific IgG2a, IgG2b and IgG3.

Preparation and Characterization of Group A Meningococcal Capsular Polysaccharide Conjugates and Evaluation of Their Immunogenicity in Mice

ABSTRACT Epidemic and endemic meningitis caused by group A Neisseria meningitidis remains a problem in sub-Saharan Africa. Although group A meningococcal capsular polysaccharide (GAMP) vaccine confers immunity at all ages, the improved immunogenicity of a conjugate and its compatibility with the World Health Organizations Extended Program on Immunization offers advantages over GAMP alone. Conjugates of GAMP bound to bovine serum albumin (BSA) were synthesized, characterized, and evaluated for their immunogenicities in mice. Two methods, involving adipic acid dihydrazide (ADH) as a linker, were used. First, ADH was bound to GAMP activated with cyanogen bromide (CNBr) or with 1-cyano-4(dimethylamino)-pyridinium tetrafluoroborate (CDAP) to form GAMPCNBrAH and GAMPCDAPAH. These derivatives were bound to BSA by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form GAMPCNBrAH-BSA and GAMPCDAPAH-BSA. Second, ADH was bound to BSA with EDC to form AHBSA. AHBSA was bound to activated GAMP to form GAMPCNBr-AHBSA and GAMPCDAP-AHBSA. The yield of GAMPCDAP-AHBSA (35 to 40%) was higher than those of the other conjugates (5 to 20%). GAMP conjugates elicited immunoglobulin G (IgG) anti-GAMP in all mice after three injections of 2.5 or 5.0 μg of GAMP: the geometric mean (GM) was highest in recipients of GAMPCDAP-AHBSA (11.40 enzyme-linked immunosorbent assay units). Although the difference was not statistically significant, the 5.0-μg dose elicited a higher GM IgG anti-GAMP than the 2.5-μg dose. Low levels of anti-GAMP were elicited by GAMP alone. GAMPCDAP-AHBSA elicited bactericidal activity roughly proportional to the level of IgG anti-GAMP.

Phase 1 Study of a Recombinant Mutant Protective Antigen of Bacillus anthracis

ABSTRACT A phase 1 study of a recombinant mutant protective antigen (rPA) vaccine was conducted in 186 healthy adults aged 18 to 45 years. Volunteers were randomized to receive one of three formulations of rPA (formalin treated, alum adsorbed, or both), in 10- or 20-μg dosages each, or the licensed vaccine, AVA. Three injections were given at 2-month intervals and a 4th 1 year after the 3rd. Vaccinees were examined at the clinic once following each injection, at 48 to 72 h postinjection. Adverse reactions were recorded in diaries for 7 days. Sera were collected before each injection and 1 week after the 1st, 2 weeks after the 3rd and 4th, and 1 year after the 4th. Serum anti-PA IgG was assayed by enzyme-linked immunosorbent assay (ELISA) and toxin neutralization assay (TNA). All formulations at both dosages were safe and immunogenic, inducing booster responses, with the highest antibody levels following the 4th injection (354 to 732 μg/ml). The lowest levels were induced by the formalin-only-treated rPA; there was no statistical difference between levels induced by alum-adsorbed and formalin-treated/alum-adsorbed rPA or by the two dosages. The antibody levels declined in all groups during the 1-year intervals after the 3rd and 4th injections but less so during the 2nd year, after the 4th injection (fold decreases were 10 to 25 versus 3.4 to 7.0, P < 0.001). There were too few AVA recipients for statistical comparisons, but their antibody levels followed those of rPA. Anti-rPA measured by ELISA correlated with TNA titers (r = 0.97). These data support studying alum-adsorbed rPA in children.