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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.

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.

Codelivery of adjuvants at the primary immunization site is essential for evoking a robust immune response to neoglycoconjugates.

A series of nonformulated adjuvants, i.e. Quil-A, MPL, DDA, CpG and Alum were codelivered with a synthetic branched tetrasaccharide fragment corresponding to Streptococcus pneumoniae type 14 polysaccharide (Pn14PS) conjugated to CRM(197) in order to investigate antibody- and cell-mediated immune responses in a mouse model. The first immunization was performed intracutaneously in the presence of adjuvants. Booster injections which were given without adjuvant dramatically enhanced and expanded the immune response from IgM to IgG1 and other IgG subclasses. Codelivery of the neoglycoconjugate with CpG or Alum had no additional effect over vaccine in saline, and elicited mainly IgG1 antibodies against Pn14PS. CpG even has a modest suppressive effect on the antibody response. Codelivery of the neoglycoconjugate with Quil-A, MPL, DDA alone or in combination resulted in both IgG1, IgG2a, IgG2b and IgG3 antibodies. Quil-A alone or in combination with MPL induced systemic IL-5 and IL-6 6h after primary immunization. This adjuvant combination also increased CD4/CD8 T cells ratio in lymph nodes and peripheral blood on day 1 after immunization. Seven days later, the ratios in blood and lymph nodes had returned to normal. In conclusion, codelivery of Quil-A alone or in combination with MPL had the most dramatic effect on antibody- and cell-mediated immune response to neoglycoconjugate of Pn14PS.

DNA analysis in perpetrator identification of terrorism-related disaster: Suicide bombing of the Australian Embassy in Jakarta 2004

We report the strategy that we employed to identify the perpetrator of a suicide car bombing in front of the Australian Embassy in Jakarta, Indonesia, on 9 September 2004. The bomb was so massive that only small tissue pieces of the perpetrator could be recovered, preventing conventional approach to the identification of the bomber, necessitating the introduction of DNA analysis as the primary means for perpetrator identification. Crime scene investigation revealed the trajectory of the bomb blast, which was used to guide the collection of charred tissue fragments of the perpetrator. Mitochondrial DNA analysis was first conducted on 17 tissue fragments, recovered over large areas of the trajectory to, (a) confirm that they are of a common source, i.e. the perpetrator, and thus (b) establish the mtDNA HV1 sequence profile of the perpetrator. The mtDNA of the perpetrator matches that of a maternally related family member of one of four suspects. Standard autosomal STR analysis confirmed the identification. This case is of interest as an illustration of a successful application of DNA analysis as the primary means of disaster perpetrator identification.

Serotype Distribution and Antibiotic Susceptibility of Streptococcus pneumoniae Strains Carried by Children Infected with Human Immunodeficiency Virus

Abstract Background We studied the serotype distribution and antibiotic susceptibility of Streptococcus pneumoniae isolates carried by children infected with HIV in Jakarta, Indonesia. Methods Nasopharyngeal swabs were collected from 90 HIV infected children aged 4 to 144 months. S. pneumoniae was identified by conventional and molecular methods. Serotyping was performed with sequential multiplex PCR and antibiotic susceptibility with the disk diffusion method. Results We identified S. pneumoniae carriage in 41 children (46%). Serotype 19F was most common among 42 cultured strains (19%) followed by 19A and 6A/B (10% each), and 23F (7%). Most isolates were susceptible to chloramphenicol (86%), followed by clindamycin (79%), erythromycin (76%), tetracycline (43%), and sulphamethoxazole/trimethoprim (41%). Resistance to penicillin was most common with only 33% of strains being susceptible. Strains of serotypes targeted by the 13-valent pneumococcal conjugate polysaccharide vaccine (PCV13) were more likely to be multidrug resistant (13 of 25 or 52%) compared to non-PCV13 serotype isolates (3 of 17 or 18%; Fisher exact test p = 0.05). Conclusion Our study provides insight into the epidemiology of pneumococcal carriage in young HIV patients in Indonesia. These findings may facilitate potential preventive strategies that target invasive pneumococcal disease in Indonesia.

Antibody- and cell-mediated immune responses to a synthetic oligosaccharide conjugate vaccine after booster immunization

Memory formation to CRM-neoglycoconjugate, a synthetic branched tetrasaccharide of Streptococcus pneumoniae type 14 polysaccharide (Pn14PS) that is conjugated to a CRM197 protein, was investigated using mice models. Mice were first immunized with the CRM-neoglycoconjugate and then boosted with either the same neoglycoconjugate or a native Pn14PS in order to investigate the effect of booster immunization. Boosting with the CRM-neoglycoconjugate resulted in increased levels of interleukin 5 (IL-5) in the serum on Day 1, followed by the appearance of high levels of specific anti-Pn14PS IgG antibodies on Day 7. Boosting with native Pn14PS resulted in neither IL-5 induction nor the generation of anti-Pn14PS IgG antibodies. In vitro (re)stimulation of spleen cells after booster injection with the neoglycoconjugate revealed the presence of IL-4 and IL-5. This was not seen in spleen cells obtained from mice boosted with the polysaccharide. When stimulated with heat-inactivated bacteria, however, the polysaccharide-boosted mice did have higher levels of IFN-γ and lower levels of IL-17 than both the CRM-neoglycoconjugate-boosted mice and the mock-immunized mice. In conclusion, neoglycoconjugate boosting is responsible for the activation of memory cells and the establishment of sustained immunity. Not only is a booster with native polysaccharide ineffective in inducing opsonic antibodies, but it also interferes with several immunoregulatory mechanisms.

The immune response to group B streptococcus type III capsular polysaccharide is directed to the -Glc-GlcNAc-Gal- backbone epitope.

The structures of the branched capsular polysaccharides of group B streptococcus type III (GBSIIIPS) and Streptococcus pneumoniae type 14 (Pn14PS) are identical apart from the (α2→3)-linked sialic acid in the side chains of GBSIIIPS. The present study tries to determine the minimal epitope in GBSIIIPS, using both a panel of anti-Pn14PS mouse sera and sera of humans vaccinated with either Pn14PS or GBSIIIPS. Type-specific Pn14PS antibodies that recognize the branched structure of Pn14PS have a low affinity for the native GBSIIIPS. Desialylation of GBSIIIPS results in dramatically higher affinity of anti-Pn14PS antibodies. Epitope specific anti-Pn14PS mouse antibodies and human sera of PCV7 vaccinees only recognized structures with the branching element -Glc-(Gal-)GlcNAc-, in particular -Gal-Glc-(Gal-)GlcNAc- in Pn14PS. On the other hand anti-GBSIIIPS human antibodies recognize predominantly the linear structure in the backbone of Pn14PS or GBSIIIPS, i.e., -Glc-GlcNAc-Gal-. This difference in antigenicity of Pn14PS and GBSIIIPS is in agreement with the difference in flexibility of the two polysaccharides caused by the presence or absence of sialic acid.

Detection and identification of coxsackievirus B3 from sera of an Indonesian patient with undifferentiated febrile illness

INTRODUCTION Coxsackievirus B3 (CVB3) virus has been implicated as the causative agent of various outbreaks of clinical disease, including hand, foot, and mouth diseases, aseptic meningitis, acute myocarditis, and inflammatory cardiomyopathy. METHODOLOGY Two hundred and nine undiagnosed cryopreserved specimens obtained from factory workers in Bandung, Indonesia, who displayed symptoms of acute febrile illness were gathered. Total RNA was isolated from serum and tested by conventional polymerase chain reaction (PCR) using Enterovirus genus-level primers and confirmed by sequencing. Concurrently, the virus was isolated in LLC-MK2 cells. RESULTS CVB3 virus was identified in an archived specimen from a patient who presented with symptoms of fever, headache, myalgia, and nausea. Sequencing results of the VP1 region from both the clinical sample and tissue culture supernatant showed 97% homology to a CVB3 virus isolate from Taiwan. Virus propagation in LLC-MK2 cell culture exhibited severe cytopathic effects two days post-inoculation. CONCLUSIONS We report the first case of CVB3 from an undifferentiated febrile illness specimen from Indonesia.

Nasopharyngeal carriage of Streptococcus pneumoniae in adults infected with human immunodeficiency virus in Jakarta, Indonesia.

This study investigated the distribution of serotype and antimicrobial susceptibility of Streptococcus pneumoniae carried by adults infected with human immunodeficiency virus (HIV) in Jakarta, Indonesia. Specimens of nasopharyngeal swab were collected from 200 HIV infected adults aged 21 to 63 years. Identification of S. pneumoniae was done by optochin susceptibility test and PCR for the presence of psaA and lytA genes. Serotyping was performed with sequential multiplex PCR and antibiotic susceptibility with the disk diffusion method. S. pneumoniae strains were carried by 10% adults with serotype 6A/B 20% was common serotype among cultured strains in 20 adults. Most of isolates were susceptible to chloramphenicol (80%) followed by clindamycin (75%), erythromycin (75%), penicillin (55%), and tetracycline (50%). This study found resistance to sulphamethoxazole/trimethoprim was most common with only 15% of strains being susceptible. High non-susceptibility to sulphamethoxazole/trimethoprim was observed in S. pneumoniae strains carried by HIV infected adults in Jakarta, Indonesia.

Effect of Chitosan and Liposome Nanoparticles as Adjuvant Codelivery on the Immunoglobulin G Subclass Distribution in a Mouse Model

Background We investigate the immunogenic properties of chitosan and liposome nanoparticles as adjuvant codelivery against a commercial pneumococcal conjugate vaccine (PCV) in an animal model. Methods The chitosan and liposome nanoparticles were prepared by ionic gelation and dry methods, respectively. The PCV immunization was performed intradermally in the presence of adjuvants and booster injections which were given without an adjuvant. The Quil-A® was used as a control adjuvant. The ELISA was performed to measure the antibodies against pneumococcal type 14 polysaccharide (Pn14PS). Results The level of total antibodies against Pn14PS antigen was no different between the mouse groups with or without adjuvant codelivery. Codelivery of the PCV with chitosan nanoparticles as well as the Quil-A adjuvant elicited IgG1, IgG2a, IgG2b, and IgG3 antibodies. Meanwhile, codelivery of liposome nanoparticles elicited mainly IgG1 antibodies against the Pn14PS. Conclusions The chitosan and liposome nanoparticles as adjuvant codelivery were successfully synthesized. These nanoparticles have different shapes in particle formation, liposome nanoparticle with their unilamellar shape and chitosan nanoparticles in large shape due to the aggregation of small-size particles. Codelivery of chitosan nanoparticles has more effect on the IgG subclass antibody production than that of liposome nanoparticles in a mouse model.