Glycoconjugate Vaccines Research Articles

Chemoenzymatic synthesis of conjugatable oligosialic acids

Bacterial polysaccharides are components of glycoconjugate vaccines against encapsulated bacterial pathogens. Because these vaccines are prepared by random coupling methods the structure of the immunogens is difficult to characterize. As a model for the development of well-defined glycoconjugates we have devised a chemoenzymatic method for preparation of conjugatable oligosialic acids. In this scheme, chemically synthesized azide or alkyne derivatives of lactosides are sialylated by bacterial sialyltransferases. An HPLC method to follow the progress of the enzymatic reaction was developed based on fluorogenic coupling partners of the Huisgen 1,3-cycloaddition reaction, since the azide and alkyne aglycons are not convenient chromaphores. We have used this chemoenzymatic scheme to synthesize oligosialic acids of Neisseria meningitidis group C polysaccharide with a conjugatable aglycon. The chemoenzymatically synthesized oligosialic acids were coupled to bovine serum albumin with the 1,3-cycloaddition reaction. The resulting glycoconjugate was reactive with N. meningitidis group C-specific monoclonal antibody, thus confirming this as a possible pathway to glycoconjugate antigens.

Immunogenicity and tolerability of a quadrivalent meningococcal glycoconjugate vaccine in children 2–10 years of age

Meningococcal disease incidence is highest in infants, but a significant burden of disease also occurs in children. In this Phase II, single-centre US study, 619 healthy children (2–10 years of age) received one dose of an investigational quadrivalent meningococcal CRM 197-conjugated vaccine (MenACWY-CRM) or a licensed quadrivalent meningococcal polysaccharide vaccine (MPSV4). Immunogenicity was assessed using the serum bactericidal assay with human complement (hSBA) at 1 and 12 months post-vaccination. Local and systemic reactions were recorded for 7 days, all adverse events (AEs) for 1 month, and medically significant and serious AEs (SAEs) for 12 months post-vaccination. For all four serogroups, more MenACWY-CRM recipients achieved an hSBA titre ≥1:4 at 1 month post-vaccination (A: 82%; C: 83%; W-135: 95%; Y: 91%) compared with the group that received MPSV4 (A: 45%; C: 66%; W-135: 71%; Y: 61%); this difference persisted through to 12 months post-vaccination. Both vaccines were well tolerated. In children 2–10 years of age, MenACWY-CRM induced a higher immune response than that of MPSV4, and was well tolerated.

Toll-like receptor 2 dependent immunogenicity of glycoconjugate vaccines containing chemically derived zwitterionic polysaccharides

Group B Streptococcus (GBS) causes serious infection in neonates and is an important target of vaccine development. Zwitterionic polysaccharides (ZPS), obtained through chemical introduction of positive charges into anionic polysaccharides (PS) from GBS, have the ability to activate human and mouse antigen presenting cells (APCs) through toll-like receptor 2 (TLR2). To generate a polysaccharide vaccine with antigen (Ag) and adjuvant properties in one molecule, we have conjugated ZPS with a carrier protein. ZPS-glycoconjugates induce higher T-cell and Ab responses to carrier and PS, respectively, compared to control PS-glycoconjugates made with the native polysaccharide form. The increased immunogenicity of ZPS-conjugates correlates with their ability to activate dendritic cells (DCs). Moreover, protection of mothers or neonate offspring from lethal GBS challenge is better when mothers are immunized with ZPS-conjugates compared to immunization with PS-conjugates. In TLR2 knockout mice, ZPS-conjugates lose both their increased immunogenicity and protective effect after vaccination. When ZPS are coadministered as adjuvants with unconjugated tetanus toxoid (TT), they have the ability to increase the TT-specific antibody titer. In conclusion, glycoconjugates containing ZPS are potent vaccines. They target Ag to TLR2-expressing APCs and activate these APCs, leading to better T-cell priming and ultimately to higher protective Ab titers. Thus, rational chemical design can generate potent PS-adjuvants with wide application, including glycoconjugates and coadministration with unrelated protein Ags.

Recent development in carbohydrate-based cancer vaccines

Tumor-associated carbohydrate antigens (TACAs) are important molecular markers on the cancer cell surface, useful for the development of therapeutic cancer vaccines or cancer immunotherapies. However, because of their poor immunogenicity and/or immunotolerance, most TACAs fail to induce T cell-mediated immunity that is critical for cancer therapy. This review summarizes the recent effort to overcome this problem via constructing TACA conjugates with improved immunogenicity, such as by covalently coupling TACAs to proper carrier molecules to form clustered or multi-epitopic conjugate vaccines, coupling TACAs to a T cell peptide epitope and/or an immunostimulant epitope to form fully synthetic multi-component glycoconjugate vaccines, and developing vaccines based on chemically modified TACAs, which is combined with metabolic engineering of cancer cells.

Characterization of fHbp , nhba ( gna2132 ), nadA , porA , Sequence Type (ST), and Genomic Presence of IS 1301 in Group B Meningococcal ST269 Clonal Complex Isolates from England and Wales

Highly effective glycoconjugate vaccines exist against four of the five major pathogenic groups of meningococci: A, C, W-135, and Y. An equivalent vaccine against group B meningococci (menB) has remained elusive due to the poorly immunogenic capsular polysaccharide. A promising alternative, the investigational recombinant menB (rMenB)- outer membrane vesicle (OMV) vaccine, contains fHBP, NHBA (previously GNA2132), NadA, and outer membrane vesicles (OMVs) from the New Zealand MeNZB vaccine. MenB currently accounts for 90% of meningococcal disease in England and Wales, where the multilocus sequence type (ST) 269 (ST269) clonal complex (cc269) has recently expanded to account for a third of menB cases. To assess the potential cc269 coverage of the rMenB-OMV vaccine, English and Welsh cc269 isolates from the past decade were genetically characterized with respect to fHBP, NHBA, and NadA. All of the isolates harbored fHbp and nhba alleles, while 98% of the cc269 isolates were devoid of nadA. Subvariant profiling of fHbp, nhba, and porA against STs revealed the presence of two broadly distinct and well-defined clusters of isolates, centered around ST269 and ST275, respectively. An additional molecular marker, insertion sequence IS1301, was found to be present in 100% and <2% of isolates of the respective clusters. On the basis of the genetic data, the potential rMenB-OMV coverage of cc269 in England and Wales is high (up to 100%) within both clusters. Expression studies and serum bactericidal antibody assays will serve to enhance predictions of coverage and will augment ongoing studies regarding the significance of IS1301 within the ST269 cluster.

Vaccination-induced IgG response to Galα1–3GalNAc glycan epitopes in lambs protected against Haemonchus contortus challenge infection

Lambs vaccinated with Haemonchus contortus excretory/secretory (ES) glycoproteins in combination with the adjuvant Alhydrogel are protected against H. contortus challenge infection. Using glycan micro-array analysis we showed that serum from such vaccinated lambs contains IgG antibodies that recognise the glycan antigen Galα1–3GalNAc-R and GalNAcβ1–4(Fucα1–3)GlcNAc-R. Our studies revealed that H. contortus glycoproteins contain Galα1–3Gal-R as well as significant levels of Galα1–3GalNAc-R, which has not been previously reported. Extracts from H. contortus adult worms contain a galactosyltransferase acting on glycan substrates with a terminal GalNAc, indicating that the worms possess the enzymatic potential to synthesise terminal Gal-GalNAc moieties. These data illustrate that glycan micro-arrays constitute a promising technology for fast and specific analysis of serum anti-glycan antibodies in vaccination studies. In addition, this approach facilitates the discovery of novel, antigenic parasite glycan antigens that may have potential for developing glycoconjugate vaccines or utilization in diagnostics.

Chemistry of a new investigational quadrivalent meningococcal conjugate vaccine that is immunogenic at all ages

Meningococcal disease is a serious medical condition that can prove fatal within hours in otherwise healthy individuals. Disease incidence is highest in infants, yet there is no broadly protective quadrivalent vaccine that covers this age group. A new investigational quadrivalent meningococcal glycoconjugate vaccine against meningococcal serogroups A, C, W-135, and Y (MenACWY-CRM, Novartis Vaccines, Siena, Italy), has been developed to meet this medical need. This article discusses the vaccine technology behind MenACWY-CRM, focusing on the heritage of CRM 197, the conjugation chemistry, the sizing of the oligosaccharides, and the advantages that these may confer on the vaccine. We highlight the differences between available vaccines and look at the clinical experience with vaccines against other diseases, demonstrating the importance of each component to the immunogenicity of conjugate vaccines. The specific technological approach, including conjugation of meningococcal oligosaccharides of defined length to the CRM 197 protein, has led to a vaccine that has the potential to provide broad meningococcal protection against serogroups A, C, W-135, and Y for all ages.

Group B streptococcal conjugate vaccines elicit functional antibodies independent of strain O-acetylation

Recently, it was discovered that sialic acid residues on group B streptococcal (GBS) capsular polysaccharides (CPS) are O-acetylated. Since GBS vaccine development has focused on de- O-acetylated CPS, it became germane to investigate the influence of de- O-acetylated GBS vaccine formulations on functional activity of sera against strains that bear the O-acetyl modification. Post-immunization sera from healthy adult recipients of de- O-acetylated GBS CPS-tetanus toxoid conjugate vaccines were evaluated in opsonophagocytosis assays using 20 GBS clinical isolates representing type Ia, Ib, II, III, or V CPS that varied in amount of O-acetylation from 2% to 40%. Ninety percent or greater opsonophagocytosis and killing of all strains were achieved, using CPS type-specific post-immunization sera. These data indicate that de- O-acetylated CPS-conjugate vaccines contain immunogenic epitopes that offer protection against GBS, independent of O-acetyl CPS modifications. Thus, presence of O-acetyl groups on the GBS CPS is not essential for functional antibodies to be elicited by GBS glycoconjugate vaccines.

Quadrivalent meningococcal ACYW-135 glycoconjugate vaccine for broader protection from infancy

Invasive meningococcal disease is a global public-health concern, with infants and adolescents bearing the majority of the disease burden. Vaccination is the most rational strategy to prevent meningococcal disease. Control of serogroup C disease has largely been achieved by the introduction of glycoconjugate meningococcal C vaccines, initially in the UK in 1999, and subsequently in several other countries. The recent licensure of a quadrivalent glycoconjugate vaccine against serogroups A, C, Y and W-135 in the USA and Canada has broadened protection against Neisseria meningitidis in 2–55 year olds. The investigational quadrivalent meningococcal serogroup A, C, Y and W-135 glycoconjugate vaccine (MenACYW–CRM197), which is immunogenic from infancy, has the potential to extend protection to the most vulnerable age group. This article discusses this novel quadrivalent vaccine formulation and its potential to control invasive disease caused by N. meningitidis serogroups A, C, Y and W-135.

N -Linked Glycosylation in Bacteria: An Unexpected Application

Traditionally, glycoproteins have been considered the exclusive property of eukaryotes and archaea, but it is now evident that glycoproteins are found in all domains of life. In recent years N-linked glycosylation among some epsilon-proteobacteria has emerged as a new and exciting research area and represents a useful model to understand this complex process in simple, genetically tractable bacteria. Above all, the transfer of N-linked glycosylation systems to the work-horse bacterium, Escherichia coli, has enabled, for the first time, the production of recombinant glycoproteins. This has potentially provided the option for tailor-made glycoproteins and has opened up the field of glycoengineering, particularly with respect to the development of glycoconjugate vaccines.

Antigen processing of glycoconjugate vaccines; the polysaccharide portion of the pneumococcal CRM 197 conjugate vaccine co-localizes with MHC II on the antigen processing cell surface

Pneumococcal (Pn) polysaccharides (PS) are T-independent (TI) antigens and do not induce immunological memory or antibodies in infants. Conjugation of PnPS to the carrier protein CRM 197 induces PS-specific antibody in infants, and memory similar to T-dependent (Td) antigens. Conjugates have improved immunogenicity via antigen processing and presentation of carrier protein with MHC II and recruitment of T cell help, but the fate of the PS attached to the carrier is unknown. To determine the location of the PS component of PnPS-CRM 197 in the APC, we separately labeled PS and protein and tracked their location. The PS of types 14-CRM 197 and 19F-CRM 197 was specifically labeled by Alexa Fluor ® 594 hydrazide (red). The CRM 197 was separately labeled red in a reaction that did not label PS. Labeled antigens were incubated with APC which were fixed, permeabilized and incubated with anti-MHC II antibody labeled green by Alexa Fluor ® 488, followed by confocal microscopy. Labeled CRM 197 was presented on APC surface and co-localized with MHC II (yellow). Labeled unconjugated 14 or 19F PS did not go to the APC surface, but PS labeled 14-CRM 197 and 19F-CRM 197 was internalized and co-localized with MHC II. Monoclonal antibody to type 14 PS bound to intracellular type 14 PS and PS-CRM 197. Brefeldin A and chloroquine blocked both CRM 197 and PS labeled 14-CRM 197 and 19F-CRM 197 from co-localizing with MHC II. These data suggest that the PS component of the CRM 197 glycoconjugate enters the endosome, travels with CRM 197 peptides to the APC surface and co-localizes with MHC II.

Immunogenicity and Immune Memory of a Nonadjuvanted Quadrivalent Meningococcal Glycoconjugate Vaccine in Infants

The highest rate of invasive meningococcal disease is among children under 2 years of age. There is currently no licensed quadrivalent (serogroups A, C, W-135, and Y) meningococcal glycoconjugate vaccine approved for infants. We evaluated the immunogenicity and reactogenicity of a novel quadrivalent nonadjuvanted meningococcal glycoconjugate vaccine (MenACWY-CRM) in healthy infants. One hundred eighty infants (90 in Canada and 90 in the United Kingdom) received 2 doses of MenACWY-CRM at 2 and 4 months of age administered concomitantly with routine infant vaccines. At 12 months of age, the Canadian infants received either MenACWY-CRM or a reduced dose of a licensed meningococcal polysaccharide vaccine. In the United Kingdom, all infants received a further dose of MenACWY-CRM. The serological marker of protection was a titer of > or =1:4 using a serum bactericidal assay with human complement (hSBA). Two doses of MenACWY-CRM induced hSBA titers > or =1:4 in 57% (95% confidence interval [CI]: 45-67) and 50% (95% CI: 38-62) of infants against serogroup A in Canada and the United Kingdom, respectively, 93% (95% CI: 85-97) and 86% (95% CI: 46-93) against serogroup C, 95% (95% CI: 87-99) and 82% (95% CI: 71-90) against serogroup W-135, and 91% (95% CI: 82-96) and 74% (95% CI: 63-83) against serogroup Y. After a booster dose of MenACWY-CRM at 12 months, at least 94% of participants achieved hSBA titers > or =1:4 against each of the serogroups C, W-135, and Y and more than 79% against serogroup A. The vaccine was well tolerated. The nonadjuvanted MenACWY-CRM is immunogenic and well tolerated in infancy and could provide broad protection against meningococcal disease in this vulnerable age group.

Demonstration of Immunologic Memory Using Serogroup C Meningococcal Glycoconjugate Vaccine

Studies of glycoconjugate vaccines have traditionally used an immune challenge with a plain polysaccharide vaccine to demonstrate immunologic memory. Plain polysaccharide vaccines are poorly immunogenic in children and can induce subsequent immunologic hyporesponsiveness. We therefore assessed the use of glycoconjugate vaccines as an alternative method of demonstrating immunologic memory. Children immunized with hepatitis B vaccine or serogroup C meningococcal glycoconjugate vaccine (MenCC) at age 2, 3, 4 months received a plain polysaccharide meningococcal serogroup A/C vaccine (MenACP) or MenCC at age 12 months. A post hoc analysis of serum bactericidal activity responses to MenCC assessed whether this differed in MenCC primed and MenCC naive infants. MenCC primed children displayed higher geometric mean serum bactericidal titers than MenCC naive children following MenACP (1518 compared with 30; P = 0.003). A similar difference was seen after a dose of MenCC to toddlers (MenCC primed: 8663, MenCC naive: 710; P < 0.001). The latter comparison became a borderline significance after adjusting for higher pretoddler immunization serum bactericidal geometric mean titers in the MenCC primed group (P = 0.068). Administration of glycoconjugate vaccines provides an important alternative method of demonstrating immunologic memory, avoiding the use of plain polysaccharide vaccines that are potentially deleterious in children. This has implications for the design of all future clinical trials of glycoconjugate vaccines.

A Randomized Trial to Determine the Tolerability and Immunogenicity of a Quadrivalent Meningococcal Glycoconjugate Vaccine in Healthy Adolescents

Because of the well-documented increased risk of meningococcal disease among adolescents, vaccination is recommended for this population in many countries, including the United States. This study compared the tolerability and immunogenicity in adolescents of a candidate quadrivalent meningococcal CRM197 glycoconjugate vaccine against serogroups A, C, W-135, and Y (MenACWY-CRM) with that of the licensed unconjugated quadrivalent polysaccharide vaccine (MPSV4). This phase II study was conducted in the United States among 524 adolescents aged 11-17 years in 2 stages, with different randomization schemes. The first 334 participants, enrolled in Stage 1, were randomized (1:1) to receive either MenACWY-CRM(+) (with adjuvant) or MPSV4. The next 190 participants, enrolled in Stage 2, were randomized (4:1) to receive either MenACWY-CRM(-) (without adjuvant) or MPSV4. Safety data were collected using diary cards and active surveillance. Human complement serum bactericidal activity (hSBA) titers were measured 1 and 12 months postvaccination. MenACWY-CRM and MPSV4 vaccines were well tolerated (local reactions, 63%-71% vs. 60%-62%; systemic reactions, 44%-56% vs. 46%-59%, respectively). One month postvaccination, similar hSBA titers were observed with the adjuvanted and nonadjuvanted MenACWY-CRM. The immunogenicity of MenACWY-CRM(-), measured by geometric mean titer, was significantly (P < 0.05) greater than that of MPSV4 for all 4 vaccine serogroups at 1 month. The percentage of subjects with hSBA titers > or =1:4 was also significantly greater (P < 0.01) for MenACWY-CRM(-) recipients for serogroups A, C, and Y and noninferior for W-135. The proportions of MenACWY-CRM(-) recipients with hSBA titers > or =1:4 to the vaccine serogroups at 1 month were 84% to 96% and geometric mean titers were 34 to 100. The percentage of subjects with hSBA titers > or =1:4 was significantly (P < 0.01) greater than MPSV4 for serogroups C, W-135, and Y 12 months postvaccination. MenACWY-CRM was well tolerated and immunogenic, with evidence of persistence of bactericidal antibodies for at least 12 months postvaccination.

Immunogenicity of a Tetravalent Meningococcal Glycoconjugate Vaccine in Infants: A Randomized Controlled Trial

Immunogenicity of a Tetravalent Meningococcal Glycoconjugate Vaccine in Infants: A Randomized Controlled Trial

Synthesis of a monophosphoryl lipid A derivative and its conjugation to a modified form of a tumor-associated carbohydrate antigen GM3.

An efficient synthesis of a derivative of monophosphoryl lipid A suitable for coupling to various structures for the construction of glycoconjugate vaccines and its conjugation with an N-modified form of the tumor-associated antigen GM3 is presented.

Synthetic and immunological studies of 5'-N-phenylacetyl sTn to develop carbohydrate-based cancer vaccines and to explore the impacts of linkage between carbohydrate antigens and carrier proteins.

5'- N-Phenylacetyl sTn (sTnNPhAc), an unnatural derivative of sTn antigen expressed by many tumors, and its alpha-linked protein conjugates were prepared and investigated to explore glycoconjugate cancer vaccines. sTnNPhAcalpha-KLH elicited a robust T cell dependent immunity. The antiserum derived from sTnNPhAcalpha- or sTnNPhAcbeta-KLH-inoculated mice was similarly reactive to sTnNPhAcalpha and sTnNPhAcbeta but showed very little reactivity to sTn, NeuNPhAcalpha(2,3)GalNAc--a regioisomer of sTnNPhAc, isolated phenylacetyl group, and the linker employed to conjugate sTnNPhAc and carrier protein. It was concluded that the sTnNPhAc-elicited immunity was specific for the whole antigen rather than the phenylacetyl group or other partial structures of sTnNPhAc and that the reducing end configuration or linkage of sTnNPhAc did not affect its immunological identity. It was also concluded that a new linker designed to conjugate carbohydrates and proteins did not provoke any immune reaction and that the linker, as well as the associated new and convenient coupling strategy, can be safely used for the development of glycoconjugate vaccines.

A novel combined Hib-MenC-TT glycoconjugate vaccine as a booster dose for toddlers: a phase 3 open randomised controlled trial

Objective:To study the immunogenicity and reactogenicity of a combined Haemophilus influenzae type b and Neisseria meningitidis serogroup C tetanus toxoid conjugate vaccine (Hib-MenC-TT) when administered as a booster dose in...

Rational chemical design of the carbohydrate in a glycoconjugate vaccine enhances IgM-to-IgG switching

Many pathogens are sheltered from host immunity by surface polysaccharides that would be ideal as vaccines except that they are too similar to host antigens to be immunogenic. The production of functional IgG is a desirable response to vaccines; because IgG is the only isotype that crosses the placenta, it is of particular importance in maternal vaccines against neonatal disease due to group B Streptococcus (GBS). Clinical studies found a substantially lower proportion of IgG-relative to IgM-among antibodies elicited by conjugates prepared with purified GBS type V capsular polysaccharide (CPS) than among those evoked by CPSs of other GBS serotypes. The epitope specificity of IgG elicited in humans by a conjugate prepared with type V CPS is for chemically desialylated type V CPS (dV CPS). We studied desialylation as a mechanism for enhancing the ability of type V CPS to induce IgM-to-IgG switching. Desialylation did not affect the structural conformation of type V CPS. Rhesus macaques, whose isotype responses to GBS conjugates match those of humans, produced functionally active IgG in response to a dV CPS-tetanus toxoid conjugate (dV-TT), and 98% of neonatal mice born to dams vaccinated with dV-TT survived lethal challenge with viable GBS. Targeted chemical engineering of a carbohydrate to create a molecule less like host self may be a rational approach for improving other glycoconjugates.

Microbial carbohydrate depolymerization by antigen-presenting cells: Deamination prior to presentation by the MHCII pathway

After uptake by the endosome of an antigen-presenting cell (APC), exogenous proteins are known to be degraded into peptides by protease digestion. Here, we report the mechanism by which pure carbohydrates can be depolymerized within APC endosomes/lysosomes by nitric oxide (NO)-derived reactive nitrogen species (RNSs) and/or superoxide-derived reactive oxygen species (ROSs). Earlier studies showed that depolymerization of polysaccharide A (PSA) from Bacteroides fragilis in the endosome depends on the APC's having an intact inducible nitric oxide synthase (iNOS) gene; the chemical mechanism underlying depolymerization of a carbohydrate within the endosome/lysosome is described here. Examining the ability of the major RNSs to degrade PSA, we determined that deamination is the predominant mechanism for PSA processing in APCs and is a required step in PSA presentation to CD4(+) T cells by MHCII molecules. Structural characterization of the NO-derived product PSA-NO indicates that partial deaminative depolymerization does not alter the zwitterionic nature of PSA. Unlike native PSA, PSA-NO is presented by iNOS-deficient APCs to induce CD4(+) T cell proliferation. Furthermore, metabolically active APCs are required for PSA-NO presentation. In contrast to PSA degradation by RNSs, dextran depolymerization in the endosome depends on ROSs, including hydrogen peroxide- and superoxide-derived ROSs. This study provides evidence that MHCII pathway-mediated carbohydrate antigen processing in APCs is achieved by chemical reactions. RNSs and ROSs may be involved in the presentation of glycopeptides by MHC molecules via the processing of other carbohydrate-containing antigens, such as bacterial or viral glycoproteins or glycoconjugate vaccines.