Harm Snippe

Gold nanoparticles as carriers for a synthetic Streptococcus pneumoniae type 14 conjugate vaccine.

AIMS Coupling of capsular polysaccharides of pathogens to immunogenic protein carriers (conjugate vaccines) improves carbohydrate immune response. Our idea is to explore gold nanoclusters as carriers to prepare fully synthetic carbohydrate vaccines. MATERIALS & METHODS Gold glyconanoparticles bearing a synthetic tetrasaccharide epitope related to the Streptococcus pneumoniae type 14 capsular polysaccharide (Pn14PS), the T-helper ovalbumin 323-339 peptide (OVA(323-339)), and D-glucose were prepared by a one-pot method. Their immunogenicity was tested in mice. Cytokine levels after spleen cell stimulation with OVA(323-339) were analyzed using a luminex-multiplex cytokine assay. The capacity of the evoked antibodies to promote the uptake of S. pneumoniae type 14 by leukocytes was assessed. RESULTS & DISCUSSION Glyconanoparticles containing 45% of tetrasaccharide and 5% OVA(323-339) triggered specific anti-Pn14PS IgG antibodies. Cytokine levels confirmed that glyconanoparticles led to T-helper cell activation. The anti-saccharide antibodies promoted the phagocytosis of type 14 bacteria by human leukocytes, indicating the functionality of the antibodies. CONCLUSION Gold nanoparticles have great potential as carriers for the development of a great diversity of fully synthetic carbohydrate-based vaccines.

Activation and Cell Cycle Antigens in CD4+ and CD8+ T Cells Correlate with Plasma Human Immunodeficiency Virus (HIV-1) RNA Level in HIV-1 Infection

The relationship between T cell activation and human immunodeficiency virus type 1 (HIV-1) replication was studied in HIV-infected subjects, 20 with and 10 without anti-HIV treatment. Expression of Ki-67 proliferation-associated antigen was increased in CD4+ and CD8+ T cells and correlated with HLA-DR. In subjects without anti-HIV treatment, the plasma HIV-1 RNA level correlated with HLA-DR in CD4+ T cells, with Ki-67 in CD8+ T cells, and with expression of CD38 in both T cell subsets. A proportion of treated subjects had increased T cell activation despite 4 months of highly active antiretroviral treatment (HAART). In subjects receiving HAART, a high percentage of HLA-DR+ CD4+ T cells was associated with signs of opportunistic infections. This work supports the concept that, in the natural course of HIV-1 infection, HIV replication itself leads to general T cell activation and that opportunistic infections generate additional CD4+ T cell activation and HIV replication.

Synthetic Polysaccharide Type 3-Related Di-, Tri-, and Tetrasaccharide–CRM197 Conjugates Induce Protection against Streptococcus pneumoniae Type 3 in Mice

ABSTRACT Di-, tri-, and tetrasaccharides, synthesized according to the chemical structure of pneumococcal polysaccharide type 3 (PS3), were coupled to the cross-reactive material (CRM197) of modified diphtheria toxin in different molar carbohydrate/protein ratios using the squarate coupling method. To study protective immunity, female BALB/c mice were subcutaneously immunized twice (with a 3-week interval) using the amount of conjugates corresponding to 2.5 μg of oligosaccharide per mouse. The conjugates evoked PS3 binding immunoglobulin G antibodies that lasted for at least 7 weeks after the booster. Immunogenicity was not influenced by the carbohydrate/protein ratio. All mice with PS3-specific antibodies survived the intraperitoneal challenge with Streptococcus pneumoniaetype 3. Therefore, synthetic oligosaccharide-protein conjugates might have potential as vaccines.

Th1-directing adjuvants increase the immunogenicity of oligosaccharide-protein conjugate vaccines related to Streptococcus pneumoniae type 3.

ABSTRACT Oligosaccharide (OS)-protein conjugates are promising candidate vaccinesagainst encapsulated bacteria, such as Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae. Although the effects of several variables such as OS chain length and protein carrier have been studied, little is known about the influence of adjuvants on the immunogenicity of OS-protein conjugates. In this study, a minimal protective trisaccharide epitope of Streptococcus pneumoniae type 3 conjugated to the cross-reacting material of diphtheria toxin was used for immunization of BALB/c mice in the presence of different adjuvants. Subsequently, half of the mice received a booster immunization with conjugate alone. Independent of the use and type of adjuvant, all mice produced long-lasting anti-polysaccharide type 3 (PS3) antibody levels, which provided full protection against challenge with pneumococcal type 3 bacteria. All adjuvants tested increased the anti-PS3 antibody levels and opsonic capacities as measured by an enzyme-linked immunosorbent assay and an in vitro phagocytosis assay. The use of QuilA or a combination of the adjuvants CpG and dimethyl dioctadecyl ammonium bromide resulted in the highest phagocytic capacities and the highest levels of Th1-related immunoglobulin G (IgG) subclasses. Phagocytic capacity correlated strongly with Th1-associated IgG2a and IgG2b levels, to a lesser extent with Th2-associated IgG1 levels, and weakly with thiocyanate elution as a measure of avidity. Thus, the improved immunogenicity of OS-protein conjugates was most pronounced for Th1-directing adjuvants.

Identification of the Smallest Structure Capable of Evoking Opsonophagocytic Antibodies against Streptococcus pneumoniae Type 14

ABSTRACT Synthetic overlapping oligosaccharide fragments of Streptococcus pneumoniae serotype 14 capsular polysaccharide (Pn14PS), {6)-[β-d-Galp-(1→4)-]β-d-GlcpNAc-(1→3)-β-d-Galp-(1→4)-β-d-Glcp-(1→}n, were conjugated to CRM197 protein and injected into mice to determine the smallest immunogenic structure. The resulting antibodies were then tested for Pn14PS specificity and for their capacity to promote the phagocytosis of S. pneumoniae type 14 bacteria. Earlier studies have reported that the oligosaccharide corresponding to one structural repeating unit of Pn14PS, i.e., Gal-Glc-(Gal-)GlcNAc, induces a specific antibody response to Pn14PS. The broader study described here, which evaluated 16 oligosaccharides, showed that the branched trisaccharide element Glc-(Gal-)GlcNAc is essential in inducing Pn14PS-specific antibodies and that the neighboring galactose unit at the nonreducing end contributes clearly to the immunogenicity of the epitope. Only the oligosaccharide conjugates that produce antibodies recognizing Pn14PS were capable of promoting the phagocytosis of S. pneumoniae type 14. In conclusion, the branched tetrasaccharide Gal-Glc-(Gal-)GlcNAc may be a serious candidate for a synthetic oligosaccharide conjugate vaccine against infections caused by S. pneumoniae type 14.

A Genome Wide Association Study of Hepatitis B Vaccine Response in an Indonesian Population Reveals Multiple Independent Risk Variants in the HLA Region

We performed a two-stage genome-wide association study (GWAS) of antibody titer in 3614 hepatitis B vaccine recipients from Indonesias Riau Archipelago, leading to the identification of at least three independent signals within the human leukocyte antigen (HLA) complex. These appear to implicate HLA-DR [rs3135363; P= 6.53 × 10(-22); odds ratio (OR) = 1.53, 95% confidence interval (CI) = 1.35-1.74]; HLA-DP, previously associated with the risk of chronic hepatitis B infection (rs9277535; P= 2.91 × 10(-12); OR = 0.72, 95% CI = 0.63-0.81); and a gene rich HLA Class III interval (rs9267665; P = 1.24 × 10(-17); OR = 2.05, CI = 1.64-2.57). The substantial overlap of these variants and those identified by GWAS of chronic hepatitis B infection confirms vaccine response as a model for infection, while suggesting that the vaccine is least effective in those most at risk of lifelong infection, following exposure to the virus.

Synthetic 6B Di-, Tri-, and Tetrasaccharide-Protein Conjugates Contain Pneumococcal Type 6A and 6B Common and 6B- Specific Epitopes That Elicit Protective Antibodies in Mice

ABSTRACT The immunogenicity and protective capacity of Streptococcus pneumoniae 6B capsular polysaccharide (PS)-derived synthetic phosphate-containing disaccharide (Rha-ribitol-P-), trisaccharide (ribitol-P-Gal-Glc-), and tetrasaccharide (Rha-ribitol-P-Gal-Glc-)-protein conjugates in rabbits and mice were studied. In rabbits, all saccharides conjugated to keyhole limpet hemocyanin (KLH) evoked high levels of pneumococcal (Pn) type 6B antibodies that facilitated type-specific phagocytosis. Unlike the disaccharide rabbit antisera, tri- and tetrasaccharide rabbit antisera also reacted with 6A PS in an enzyme-linked immunosorbent assay (ELISA) and promoted phagocytosis of 6A pneumococci. All these rabbit antisera passively protected mice against a Pn 6B challenge. The disaccharide conjugate-induced antiserum, however, failed to protect mice against a 6A challenge. In mice, phagocytic and protective anti-Pn 6B antibodies were only induced by the tetrasaccharide conjugate and not by PS 6B or PS 6B-protein conjugates. These antibodies did not cross-react with 6A PS in ELISA and were unable to phagocytize 6A pneumococci. In conclusion, the disaccharide and tetrasaccharide conjugates already contain epitopes capable of inducing 6B-specific, fully protective antibodies in rabbits and mice, respectively.

Neutralizing and non-neutralizing monoclonal antibodies to the E2 glycoprotein of Semliki Forest virus can protect mice from lethal encephalitis.

Two monoclonal antibodies (UM 4.2 and UM 5.1) directed against the glycoprotein E2 of Semliki Forest virus (SFV) are described; both belong to the IgG2a isotype but are of different idiotype. Analysis employing isoelectric focusing resulted in different focusing patterns for both monoclonals (UM 4.2, pI 8; UM 5.1, pI 7.2). They further differed in their ability to neutralize virus. The UM 4.2 antibodies were inactive in neutralization, while the UM 5.1 antibodies exceeded conventional mouse hyperimmune serum in this respect. Both monoclonal antibodies, however, were able to protect mice passively from a lethal infection with SFV. Based on the amount of protein, the UM 5.1 antibodies were 100-fold more effective than the UM 4.2 antibodies in mouse protection tests.

New genetic associations detected in a host response study to hepatitis B vaccine

The immune response to hepatitis B vaccination differs greatly among individuals, with 5–10% of healthy people failing to produce protective levels of antibodies. Several factors have been implicated in determining this response, chiefly individual genetic variation and age. Aiming to identify genes involved in the response to hepatitis B vaccination, a two-stage investigation of 6091 single-nucleotide polymorphisms (SNPs) in 914 immune genes was performed in an Indonesian cohort of 981 individuals showing normal levels of anti-HBs versus 665 individuals displaying undetectable levels of anti-HBs 18 months after initial dose of the vaccine. Of 275 SNPs identified in the first stage (476 normal/372 nonresponders) with P<0.05, significant associations were replicated for 25 polymorphisms in 15 genes (503 normal/295 nonresponders). We validated previous findings (HLA-DRA, rs5000563, P-value combined=5.57 × 10−10; OR (95%CI)=0.61 (0.52–0.71)). In addition, we detected a new association outside of the human leukocyte antigen loci region that passed correction for multiple testing. This SNP is in the 3′ downstream region of FOXP1, a transcription factor involved in B-cell development (P-value combined=9.2 × 10−6; OR (95%CI)=1.38 (1.2–1.6)).These findings might help to understand the biological reasons behind vaccine failure and other aspects of variation in the immune responses of healthy individuals.

Adjuvant Effect of Nonionic Block Polymer Surfactants in Humoral and Cellular Immunity

The adjuvant activities of four chemically similar, but physicochemically different nonionic surface-active agents called pluronic polyols F 68, L 31, L 101 and L 121 were studied. These four agents were tested in mice using an experimental model developed for studying the adjuvant activity of the cationic surface-active agent dimethyl dioctadecyl ammonium bromide (DDA). L 121 and DDA enhanced the primary antibody response to sheep red blood cells (SRBC) while F 68, L 31 and L 101 suppressed this response. The secondary humoral response to SRBC was enhanced by the polyol L 121 while the secondary response to dinitrophenylated bovine serum albumin (DNP22-BSA) was enhanced by both L 121 and L 101. DDA and the polyol L 101 were very effective adjuvants for induction of delayed-type hypersensitivity (DTH) to SRBC and DNP22-BSA after intracutaneous immunization of mice with a mixture of antigen and adjuvant. Since the four pluronic polyols were composed of identical chemical constituents, we proposed that difference in their activities as adjuvants were due to variation in their physicochemical properties. A correlation was found between a physicochemical parameter, the hydrophilelipophile balance (HLB), and the adjuvant activities of the pluronic polyols and several other types of nonionic surface-active agents. The agents which were strong adjuvants all had HLB values within a narrow range which classified them as spreading agents.