Glycoconjugate Vaccines Research Articles

Persistence of functional antibodies to group B streptococcal capsular polysaccharides following immunization with glycoconjugate vaccines

The duration of functional activity of group B streptococcal (GBS) glycoconjugate vaccine-induced capsular polysaccharide-specific (CPS) IgG was evaluated among healthy adult responders. Opsonophagocytic activity declined significantly from a 4-week post-immunization peak, but substantial functional activity, exceeding 1log10 reduction in GBS cfu/mL, was retained at 18 months to 2 years post-immunization for each GBS type assessed. The persistence of functional antibody activity when GBS CPS-specific IgG concentrations decline, although remaining significantly higher than pre-immunization levels, suggests that long-term protection may be expected from candidate GBS glycoconjugates administered to this population.

Towards a Well Defined Meningitis Vaccine: Chemoenzymatic Synthesis of Meningococcal Glycoconjugate Vaccine Candidates

Immunization efforts against meningitis have been effective at reducing incidence of the disease worldwide. Glycoconjugate vaccines are the most commonly used form of meningococcal vaccine. They are composed of capsular polysaccharides attached to a carrier protein. The chemical methods used to make these vaccines leads to heterogeneous mixtures. Well defined vaccines are needed to elucidate the relationship between vaccine structure and generated immune response. In this work we describe our efforts to produce these types of structures using chemoenzymatic synthesis. Chemically synthesized lactosides, containing azido groups at the aglycon were used as substrates for recombinant sialyltransferase enzymes from Camplyobacter jejuni and Neisseria meningitidis serogroup C. Optimization of this method led to glycans of varying chain lengths. These azido‐containing oligosialic acids, which mimic polysaccharides found in the capsule of Neisseria, were then conjugated to alkyne‐modified Hc fragment of the tetanus toxoid protein using the “click” chemistry method of 1,3‐cycloaddition. The resulting glycoconjugates were shown to be more immunogenic in mice than unconjugated oligosialic acids.

Determination of glycation sites by tandem mass spectrometry in a synthetic lactose-bovine serum albumin conjugate, a vaccine model prepared by dialkyl squarate chemistry.

Neoglycoconjugate vaccines synthesized by the squaric acid spacer method allow single point attachment of the carbohydrate antigen to the protein carrier. However, the localization of the carbohydrate antigen sites of conjugation on the protein carrier has been an elusive task difficult to achieve. Covalent attachment of the lactose antigen to the bovine serum albumin (BSA) was prepared by the squaric acid method using a hapten:BSA ratio of 20:1. Different reaction times were used during the conjugation reaction and two different lactose-BSA glycoconjugate vaccines were obtained. The carbohydrate antigen hapten:BSA ratios of these lactose-BSA glycoconjugate vaccines were determined by MALDI-TOF/RTOF-MS and the glycation sites in the neoglycoconjugates were determined using nano-LC/ESI-QqTOF-MS/MS analysis of the trypsin and GluC V8 digests of the conjugates. We have identified a total of 15 glycation sites located on the BSA lysine residues for the neoglycoconjugate vaccine formed with a hapten:BSA ratio of 5.1:1, However, the tryptic and GluC V8 digests of the hapten-BSA glycoconjugate with a hapten:BSA ratio of 19.0:1 allowed identification of 30 glycation sites located on the BSA. These last results seem to indicate that this conjugation results in formation of various glycoforms. It was observed that the number of identified glycation sites increased when the hapten:BSA ratio of glycoconjugate formation increased, and that the location of the glycation sites appears to be mainly on the outer surface of the BSA carrier molecule which is in line with the assumption that the sterically more accessible lysine residues, namely those located on the outer surface of the BSA, would be conjugated preferentially.

Correlation of Group C Meningococcal Conjugate Vaccine Response with B- and T-Lymphocyte Activity

Despite the success of conjugate vaccination against meningococcal group C (MenC) disease, post-vaccination, some individuals still exhibit rapid waning of initially protective bactericidal antibody levels. The mechanism of this relative loss of humoral protection remains undetermined. In this report we have investigated the relationship between T- and B-cell activation and co-stimulation and the loss of protective antibody titers. We have found that healthy volunteers who lose protective MenC antibody levels one year after receipt of glycoconjugate vaccine exhibit no detectable cellular defect in polyclonal B- or T-cell activation, proliferation or the B-memory pool. This suggests that the processes underlying the more rapid loss of antibody levels are independent of defects in either initial T- or B-cell activation.

Pneumococcal polysaccharide vaccine at 12 months of age produces functional immune responses

Infections with Streptococcus pneumoniae (pneumococcus) are a cause of significant child mortality. Pneumococcal glycoconjugate vaccines are expensive and provide limited serotype coverage. The 23-valent pneumococcal polysaccharide vaccine (Pneumovax) might provide wider serotype coverage but is reported to be weakly immunogenic in children less than 2 years of age. We have previously reported that Pneumovax administered to healthy 12-month-old Fijian infants elicits significant serotype-specific IgG responses. However, the functional capacity of these responses in 12-month-old infants is not known. We sought to assess the functional, serotype-specific immune response of 12-month-old infants after immunization with Pneumovax. Functional responses of 12-month-old infants were assessed by using the opsonophagocytic and antibody avidity assay against 8 serotypes and 23 serotypes, respectively. Seventy-one percent of infants produced strong opsonophagocytic activity against 4 of 8 serotypes, and 30% produced high-avidity serotype-specific IgG antibodies to 10 of 23 serotypes at 2 weeks after Pneumovax. Responses were protective for most serotypes that cause disease in Western countries, whereas responses to most of the epidemiologically relevant serotypes for developing countries were low. This is the first comprehensive study evaluating the functional antibody response to Pneumovax in 12-month-old infants. Pneumovax induced functional antibody responses to several serotypes causing disease in Western countries but induced poorer responses to serotypes that are responsible for the majority of disease in developing countries. Pneumovax might be of benefit in some populations, but further studies are required before this can be recommended in developing countries.

Quantifying uncertainty in determination of polysaccharides in glycoconjugate vaccines based on in-house validation data

The measurement uncertainty in quantifying polysaccharides in glycoconjugate vaccines was estimated in order to ascertain compliance of these products in the batch release procedure of an Official Medicines Control Laboratory. A single-laboratory validation approach of an internal analytical procedure to quantify the total saccharide by the way of the sialic acid (N-Acetylneuraminic acid) concentration in a meningococcal glycoconjugate vaccine by High Performance Anion Exchange Chromatography coupled with Pulsed Amperometric Detection is reported. Uncertainty components from precision, bias and other sources were evaluated. It turned out that uncertainty is dominated by the precision contribution, while bias was mainly due to the non-recovery from hydrolysis. As a consequence, a correction factor was considered. At a confidence level of approximately 95%, the expanded uncertainty of the total polysaccharide concentration in a vaccine, when measured using the internal method, was found to be 11.5%.

Immunogenicity and tolerability of a multicomponent meningococcal serogroup B (4CMenB) vaccine in healthy adolescents in Chile: a phase 2b/3 randomised, observer-blind, placebo-controlled study

Effective glycoconjugate vaccines against Neisseria meningitidis serogroups A, C, W-135, and Y have been developed, but serogroup B remains a major cause of severe invasive disease in infants and adolescents worldwide. We assessed immunogenicity and tolerability of a four-component vaccine (4CMenB) in adolescents. We did a randomised, observer-blind, placebo-controlled, study at 12 sites in Santiago and Valparaíso, Chile. Adolescents aged 11-17 years received one, two, or three doses of 4CMenB at 1 month, 2 month, or 6 month intervals. Immunogenicity was assessed as serum bactericidal activity using human complement (hSBA) against three reference strains for individual vaccine antigens, and assessed by ELISA against the fourth strain. Local and systemic reactions were recorded 7 days after each vaccination, and adverse events were monitored throughout the study. Participants were initially randomised to five groups (3:3:3:3:1) during the primary phase to receive either one dose, two doses 1 or 2 months apart, or three doses of 4CMenB, or three doses of placebo, with an additional three groups generated for the booster phase. All subjects received at least one dose of 4CMenB. Geometric mean titres, proportions of participants with serum bactericidal antibody titres of 4 or more, and Clopper-Pearson 95% CIs were calculated. The study is registered with ClinicalTrials.gov, number NCT00661713. Overall, 1631 adolescents (mean age 13·8 [SD 1·9] years) received at least one dose of 4CMenB. After two or three doses, 99-100% of recipients had hSBA titres of 4 or more against test strains, compared with 92-97% after one dose (p<0·0145) and 29-50% after placebo. At 6 months 91-100% of participants still had titres of 4 or more for each strain after two or three doses, but only 73-76% after one dose; seroresponse rates reached 99-100% for each strain after second or third doses at 6 months. Local and systemic reaction rates were similar after each 4CMenB injection and did not increase with subsequent doses, but remained higher than placebo. No vaccine-related serious adverse events were reported and no significant safety signals were identified. On the basis of immunogenicity responses this study provides evidence for an adolescent 4CMenB vaccine schedule of two doses, 1-6 months apart, to provide protection against meningococcal B infection. The extent of this protection against meningococcus B variants circulating worldwide will be determined by national surveys. Novartis Vaccines and Diagnostics.

Role of toll like receptors in the antibody response to encapsulated bacteria

Encapsulated bacteria are major pathogens in humans. The capsular polysaccharides (PS) of these bacteria are T-independent type 2 antigens, are not processed by antigen presenting cells and do not induce T cell help. PS antigens are poor immunogens in children less than two years, the peak age incidence of encapsulated bacterial infection. The TNF family receptors BAFFR and TACI interaction with the cytokines BAFF and APRIL are essential co-stimulatory factors for humoral responses to PS. Linkage of PS to a carrier protein to make glycoconjugate vaccines, enhances the immune response to PS similar to a T cell dependent antigen. Multiple doses of glycoconjugate vaccines are required to elicit protection, making their use in the developing world problematic. TLR engagement augments BAFF mediated PS antibody responses and TLR ligands serve as adjuvants for induction of anti-PS antibodies either for pure PS or for PS-protein conjugate vaccines. A variety of TLR ligands stimulate increased production of antibodies directed to both PS and protein components of encapsulated bacteria, and glycoconjugate vaccines, suggesting their future role in immunization strategies.

Synthesis and preliminary biological evaluation of carba analogues from Neisseria meningitidis A capsular polysaccharide

The Gram-negative encapsulated bacterium Neisseria meningitidis type A (MenA) is a major cause of meningitis in developing countries, especially in the sub-Saharan region of Africa. The development and manufacture of an efficient glycoconjugate vaccine against MenA is greatly hampered by the poor hydrolytic stability of its capsular polysaccharide, consisting of (1→6)-linked 2-acetamido-2-deoxy-α-d-mannopyranosyl phosphate repeating units. The replacement of the ring oxygen with a methylene group to get a carbocyclic analogue leads to the loss of the acetalic character of the phosphodiester and consequently to the enhancement of its chemical stability. Here we report the synthesis of oligomers (mono-, di- and trisaccharide) of carba-N-acetylmannosamine-1-O-phosphate as candidates for stabilized analogues of the corresponding fragments of MenA capsular polysaccharide. Each of the synthesized compounds contains a phosphodiester-linked aminopropyl spacer at its reducing end to allow for protein conjugation. The inhibition abilities of the synthetic molecules were investigated by a competitive ELISA assay, showing that only the carba-disaccharide is recognized by a polyclonal anti-MenA serum with an affinity similar to a native MenA oligosaccharide with average polymerization degree of 3.

Immunization with the RrgB321 fusion protein protects mice against both high and low pilus-expressing Streptococcus pneumoniae populations

RrgB321, a fusion protein of the three Streptococcus pneumoniae pilus-1 backbone RrgB variants, is protective in vivo against pilus islet 1 (PI-1) positive pneumococci. In addition, antibodies to RrgB321 mediate a complement-dependent opsonophagocytosis of PI-1 positive strains at levels comparable to those obtained with antisera against glycoconjugate vaccines. In the pneumococcus, pilus-1 displays a biphasic expression pattern, with different proportions of two bacterial phenotypes, one expressing and one not expressing the pilus-1. These two populations can be stably separated in vitro giving rise to the enriched high (H) and low (L) pilus expressing populations. In this work we demonstrate that: (i) the opsonophagocytic killing mediated in vitro by RrgB321 antisera is strictly dependent on the pilus expression ratio of the strain used; (ii) during the opsonophagocytosis assay pilus-expressing pneumococci are selectively killed, and (iii) no switch towards the pilus non-expressing phenotype can be observed. Furthermore, in sepsis and pneumonia models, mice immunized with RrgB321 are significantly protected against challenge with either the H or the L pilus-expressing population of strains representative of the three RrgB variants. This suggests that the pilus-1 expression is not down-regulated, and also that the expression of the pilus-1 could be up-regulated in vivo. In conclusion, these data provide evidence that RrgB321 is protective against PI-1 positive strains regardless of their pilus expression level, and support the rationale for the inclusion of this fusion protein into a multi-component protein-based pneumococcal vaccine.

Rational design of glycoconjugate vaccines

Rational design of glycoconjugate vaccines

A mechanism for glycoconjugate vaccine activation of the adaptive immune system and its implications for vaccine design

Although glycoconjugate vaccines have provided enormous health benefits globally, they have been less successful in significant high-risk populations. Exploring novel approaches to the enhancement of glycoconjugate effectiveness, we investigated molecular and cellular mechanisms governing the immune response to a prototypical glycoconjugate vaccine. In antigen-presenting cells, a carbohydrate epitope is generated upon endolysosomal processing of group B streptococcal type III polysaccharide coupled to a carrier protein. In conjunction with a carrier protein-derived peptide, this carbohydrate epitope binds to major histocompatibility class II (MHCII) and stimulates carbohydrate-specific CD4+ T-cell clones to produce interleukins 2 and 4—cytokines essential for providing T-cell help to antibody-producing B cells. An archetypical glycoconjugate vaccine constructed to maximize the presentation of carbohydrate epitopes recognized by T cells is 50–100 times more potent and significantly more protective in an animal model of infection than is a currently used vaccine construct.

RrgB321, a Fusion Protein of the Three Variants of the Pneumococcal Pilus Backbone RrgB, Is ProtectiveIn Vivoand Elicits Opsonic Antibodies

Streptococcus pneumoniae pilus 1 is present in 30 to 50% of invasive disease-causing strains and is composed of three subunits: the adhesin RrgA, the major backbone subunit RrgB, and the minor ancillary protein RrgC. RrgB exists in three distinct genetic variants and, when used to immunize mice, induces an immune response specific for each variant. To generate an antigen able to protect against the infection caused by all pilus-positive S. pneumoniae strains, we engineered a fusion protein containing the three RrgB variants (RrgB321). RrgB321 elicited antibodies against proteins from organisms in the three clades and protected mice against challenge with piliated pneumococcal strains. RrgB321 antisera mediated complement-dependent opsonophagocytosis of piliated strains at levels comparable to those achieved with the PCV7 glycoconjugate vaccine. These results suggest that a vaccine composed of RrgB321 has the potential to cover 30% or more of all pneumococcal strains and support the inclusion of this fusion protein in a multicomponent vaccine against S. pneumoniae.

Persistence of Serum Bactericidal Antibody One Year After a Booster Dose of Either a Glycoconjugate or a Plain Polysaccharide Vaccine Against Serogroup C Neisseria meningitidis Given to Adolescents Previously Immunized With a Glycoconjugate Vaccine

Bactericidal antibody induced by immunization of infants with serogroup C Neisseria meningitidis (MenC) vaccines wanes rapidly during childhood. Adolescents are at particular risk from meningococcal disease, therefore they might benefit from a booster dose of vaccine. The duration of serologic response to such a booster in adolescents is unknown. In a previous study, English schoolchildren, aged 9 to 12 years, who had received a monovalent MenC glycoconjugate vaccine in 1999-2000, were given either a plain polysaccharide vaccine (MenC-PS group, n = 150) or a glycoconjugate vaccine (MenC-CRM group, n = 95) at 13 to 15 years of age. In this follow-up study, serum bactericidal antibody titers and specific immunoglobulin G concentrations were assessed 1 year later. Results were compared with unboosted controls of similar age (control group, n = 298). Compliance with study protocol was achieved for 146 of the MenC-PS group, 92 of the MenC-CRM group, and 293 of the control group. Compared with the control group, both the MenC-PS and MenC-CRM groups had a significantly higher (P < 0.0001) geometric mean serum bactericidal antibody titers 1 year after the booster dose (geometric mean titers for MenC-PS group 3388 [95% confidence interval {CI}: 2460-4665]; MenC-CRM group 4417 [95% CI: 2951-6609]; control group 316 [95% CI: 252-396]). Specific immunoglobulin G concentration also rose and remained elevated 1 year after the booster. A booster dose of MenC vaccine given to adolescents produced a marked rise in bactericidal antibody, which remained elevated 1 year later. Introduction of an adolescent booster of MenC vaccine might provide enhanced long-term population control of the disease.

Glycoconjugate vaccines and immune interactions, and implications for vaccination schedules

Conjugate vaccines using diphtheria toxoid variant (CRM197), diphtheria toxoid and tetanus toxoid (TT) as carrier protein may induce immune interactions (interference or impairment as measured by lower antibody levels, or enhancement [higher antibody levels]) when coadministered with other vaccines. Immune enhancement occurs when two TT conjugates are coadministered. CRM197 conjugate vaccines induce immune bystander interference when given with diphtheria-tetanus-acellular pertussis vaccines, which reduces responses to coadministered Haemophilus influenzae type b vaccine conjugated to TT. These bystander effects are greater as the amount of CRM197 administered increases. When large amounts of either TT or CRM197 are coadministered, dose-related carrier-induced epitopic suppression may occur, affecting immune responses to meningococcal or pneumococcal polysaccharides. These observations have implications for vaccine scheduling. The range of available alternative vaccines means that specific vaccine coadministrations can avoid or reduce CRM197-induced interference. Potential interactions arising from new CRM197 or TT conjugates will need to be thoroughly examined.

Carbohydrate-Monophosphoryl Lipid A Conjugates Are Fully Synthetic Self-Adjuvanting Cancer Vaccines Eliciting Robust Immune Responses in the Mouse

Tumor-associated carbohydrate antigens (TACAs) are useful targets in the development of therapeutic cancer vaccines. However, a serious problem with them is the poor immunogenicity. To overcome the problem, a monophosphorylated derivative of Neisseria meningitidis lipid A was explored as a potential carrier molecule and built-in adjuvant for the construction of structurally defined fully synthetic glycoconjugate vaccines. Some paradigm-shifting discoveries about the monophosphoryl lipid A (MPLA)-TACA conjugates were that they elicited robust IgG antibody responses, indicating T cell-mediated immunity, without an external adjuvant and that an external adjuvant, e.g., Titermax Gold, actually reduced rather than promoted the immunological activity of the conjugates. The induced antibodies were proved to bind selectively to target tumor cells. MPLA was therefore demonstrated to be a powerful built-in immunostimulant and adjuvant for an all new design of fully synthetic glycoconjugate cancer vaccines.

Meningitis and Encephalitis

: Neurologists have a vital role in the recognition of meningitis and encephalitis, the accurate evaluation and interpretation of CSF studies, and the management and prevention of the neurologic complications of CNS infectious diseases. ;: Although the tetravalent meningococcal glycoconjugate vaccine has decreased the incidence of meningococcal meningitis, the vaccine does not contain serogroup B, which is responsible for one-third of cases of meningococcal disease. Thus, meningitis due to Neisseria meningitidis is still a concern in a vaccinated individual. Empiric therapy for meningitis associated with sinusitis, otitis, or mastoiditis should include antibiotic therapy for anaerobes. An organism that classically causes a subacute or chronic meningitis, such as Mycobacterium tuberculosis, may on occasion present with an acute onset of symptoms. ;: Unlike most other diseases, the management of patients with suspected meningitis or encephalitis begins with empiric therapy. The etiologic organism cannot always be identified. The goal is to identify those that are treatable, provide supportive care for those that are not, and, when possible, prevent the neurologic complications of these infections.

Improved immune responses in mice using the novel chitosan adjuvant ViscoGel, with a Haemophilus influenzae type b glycoconjugate vaccine

An immune response to an antigen is more efficiently induced in combination with an adjuvant. Chitosan has due to documented immunostimulatory characteristics been proposed as an adjuvant candidate. However, a disadvantage with chitosan is its poor solubility at physiological pH. We have circumvented this obstacle by using a soluble type of chitosan (Viscosan), with a degree of deacetylation (DD) of 50% and a random distribution of acetyl groups. A hydrogel, ViscoGel, was made from Viscosan which was further mechanically processed into gel particles of predefined size. The first cells to infiltrate ViscoGel in mice, were identified mainly as neutrophils, detected already after 4 h. ViscoGel's impact on the immune response in mice together with a commercial vaccine against Haemophilus influenzae type b (Act-HIB) was then studied. Mixing Act-HIB with ViscoGel, induced significantly enhanced IgG1 and IgG2a titers in serum (p<0.05). We could reduce the antigen dose ten-fold in combination with ViscoGel and still obtain antibody titers similar to 2 μg Act-HIB administered alone. In addition, the Act-HIB specific cellular response was stronger in mice vaccinated together with ViscoGel (p<0.05). The cytokine response after vaccination with Act-Hib together with ViscoGel was of a mixed type. We found elevated levels of the Th1 associated cytokine INF-γ, the Th2-cytokine IL-4, the proinflammatory IL-6 and IL-17A, and the regulatory cytokine IL-10. Similar effects were seen when the adjuvant was administered either subcutaneously or intramuscularly. Taken together, using vaccination against H. influenzae type b as a model, we here show proof of concept for the novel vaccine adjuvant candidate, ViscoGel.

The design of semi-synthetic and synthetic glycoconjugate vaccines

Introduction:Glycoconjugate vaccines are among the safest and most efficacious vaccines developed during the last 30 years. They are a potent tool for prevention of life-threatening bacterial infectious diseases like meningitis and pneumonia. The concept of hapten–carrier conjugation is now being extended to other disease areas.Areas covered:This is an overview of the history and current status of glycoconjugate vaccines. The authors discuss the approaches for their preparation and quality control as well as those variables which might affect their product profile. The authors also look at the potential to develop fully synthetic conjugate vaccines based on the progress of organic chemistry. Additionally, new applications of conjugate vaccines technology in the field of non-infectious diseases are discussed. Through this review, the reader will have an insight regarding the issues and complexities involved in the preparation and characterization of conjugate vaccines, the variables that might affect their immunogenicity and the potential for future applications.Expert opinion:The immunogenicity of weak T-independent antigens can be increased in quantity and quality by conjugation to protein carriers, which provide T-cell help. Glycoconjugate vaccines are among the safest and most efficacious vaccines developed so far. Various conjugation procedures and carrier proteins can be used. Many variables impact on the immunogenicity of conjugate vaccines and a tight control through physicochemical tests is important to ensure manufacturing and clinical consistency. New and challenging targets for conjugate vaccines are represented by cancer and other non-infectious diseases.

Recombinant protein meningococcal serogroup B vaccine combined with outer membrane vesicles

Introduction: Meningococcal infection is a major cause of morbidity and mortality worldwide. Infection with Neisseria meningitidis is most common in young children, teenagers and people with certain medical conditions. Effective polysaccharide and glycoconjugate vaccines for serogroups A, C, W135 and Y have been developed. A similar capsular polysaccharide approach for serogroup B (MenB) has by most been judged as unsuitable, hence, no broad coverage vaccine has been licensed to date. The novel vaccine Bexsero (previously 4CMenB) has been developed and proven safe and immunogenic in clinical trials.Areas covered: The authors outline the constituents of Bexsero and immunogenicity and safety data from preclinical and clinical trials published in peer-reviewed literature, meeting proceedings and publicly-available clinical trial websites from 2000 to 2010.Expert opinion: Bexsero is well tolerated with a proven safety profile, and has demonstrated a robust immune response across different age groups against a range of diverse MenB strains. These data suggest that Bexsero has the ability to provide protection in infants, who are at the greatest risk of developing meningococcal disease.