Serotype Antigens Research Articles

A Novel, Inexpensive In-House Immunochromatographic Strip Test for Cryptococcosis Based on the Cryptococcal Glucuronoxylomannan Specific Monoclonal Antibody 18B7.

The aim of this study was to develop a novel lateral flow immunochromatoghaphic strip test (ICT) for detecting cryptococcal polysaccharide capsular antigens using only a single specific monoclonal antibody, mAb 18B7. The mAb 18B7 is a well characterized antibody that specifically binds repeating epitopes displayed on the cryptococcal polysaccharide glucuronoxylomannan (GXM). We validated the immunoreactivities of mAb 18B7 against capsular antigens of different cryptococcal serotypes. The mAb 18B7 ICT was constructed as a sandwich ICT strip and the antibody serving in the mobile phase (colloidal gold conjugated mAb 18B7) to bind one of the GXM epitopes while the stationary phase antibody (immobilized mAb18B7 on test line) binding to other remaining unoccupied epitopes to generate a positive visual readout. The lower limit of detection of capsular antigens for each of the Cryptococcus serotypes tested was 0.63 ng/mL. No cross-reaction was found against a panel of antigens isolated from cultures of other pathogenic fungal, except the crude antigen of Trichosporon sp. with the lower limit of detection of 500 ng/mL (~800 times higher than that for cryptococcal GXM). The performance of the mAb 18B7 ICT strip was studied using cerebrospinal fluid (CSF) and serum and compared to commercial diagnostic kits (latex agglutination CALAS and CrAg IMMY). The sensitivity, specificity and accuracy of the mAb18B7 ICT with CSF from patients with confirmed cryptococcal meningitis were 92.86%, 100% and 96.23%, respectively. No false positives were observed with samples from non-cryptococcosis patients. With serum samples, the mAb 18B7 ICT gave a sensitivity, specificity and accuracy of 96.15%, 97.78% and 96.91%, respectively. Our results show that the mAb 18B7 based ICT was reliable, reproducible, and cost-effective as a point-of-care immunodiagnostic test for cryptococcosis. The mAb 18B7 ICT may be particularly useful in countries where commercial kits are not available or affordable.

Structural Studies of the Lipopolysaccharide of Aeromonas veronii bv. sobria Strain K133 Which Represents New Provisional Serogroup PGO1 Prevailing among Mesophilic Aeromonads on Polish Fish Farms

In the present work, we performed immunochemical studies of LPS, especially the O-specific polysaccharide (O-PS) of Aeromonas veronii bv. sobria strain K133, which was isolated from the kidney of carp (Cyprinus carpio L.) during an outbreak of motile aeromonad infection/motile aeromonad septicemia (MAI/MAS) on a Polish fish farm. The structural characterization of the O-PS, which was obtained by mild acid degradation of the LPS, was performed with chemical methods, MALDI-TOF mass spectrometry, and 1H and 13C NMR spectroscopy. It was revealed that the O-PS has a unique composition of a linear tetrasaccharide repeating unit and contains a rarely occurring sugar 2,4-diamino-2,4,6-trideoxy-D-glucose (bacillosamine), which may determine the specificity of the serogroup. Western blotting and ELISA confirmed that A. veronii bv. sobria strain K133 belongs to the new serogroup PGO1, which is one of the most commonly represented immunotypes among carp and trout isolates of Aeromonas sp. in Polish aquacultures. Considering the increase in the MAI/MAS incidences and their impact on freshwater species, also with economic importance, and in the absence of an effective immunoprophylaxis, studies of the Aeromonas O-antigens are relevant in the light of epidemiological data and monitoring emergent pathogens representing unknown antigenic variants and serotypes.

Serologic monitoring of herds with and without bacterin vaccination for Actinobacillus pleuropneumoniae

Objective: Investigate diagnostic serology for Actinobacillus pleuropneumoniae (APP) infections in naturally infected and vaccinated pigs. Materials and methods: The APP status of 12 farms (A-L) was established by lung cultures and isolate serotyping. Screening enzyme-linked immunosorbent assay (ELISA) detected antibodies to ApxIV antigen or multiple APP serotypes. Serotype-specific ELISAs were conducted for serotypes 5 and 7. Seven groups of farm F pigs (serotype 7) were moved to farm K (serotype 5). Autogenous vaccines (V1/V2) prepared from APP serotype 5 cultures from farm K and a commercial, killed APP vaccine (V3) containing serotypes 1, 7, and 15 were used to vaccinate pigs in each group twice or thrice at 3-week intervals, commencing at 10 weeks of age. Blood samples were analyzed with ELISAs specific for serotype 5 and ApxI and ApxII toxins. Serum titers were compared using an analysis of variance. Results: Serotypes 5, 7, 12, or 15 were present in lung cultures. The ApxIV screening ELISA and mix-serotype ELISA regularly detected serotypes 5, 7, and 15. Serotype 12 infections were detected in the mix-serotype ELISA, but not in the ApxIV assays. The serotype 5 or 7 specific ELISA regularly detected herd infections with the relevant serotype. Serotype 5 titers of pigs vaccinated with V1/V2 thrice were higher than those dosed twice with the equivalent volume (P < .05). Pigs receiving V3 showed no serotype 5 antibody response. The ApxI and II titers in V1/V2-vaccinated pigs were higher than controls. Implications: Screening and serotype-specific ELISAs verified APP status. Repeated serotype-specific autogenous APP vaccine doses provided a strong antibody response.

Sarcoidosis development during ulcerative colitis remission in a patient with a susceptible human leukocyte antigen serotype.

The combination of sarcoidosis and ulcerative colitis (UC) is very rare, and its pathogenesis remains unknown. Hereditary factors as well as environmental factors have been speculated, including an association with the human leucocyte antigen (HLA) genotype. A 62-year-old Japanese woman with UC presented with complaint of a cough. Abnormal shadows were evident on the chest X-ray during mesalazine therapy. Multiple indolent subcutaneous nodules were also detected. Transbronchial lung and skin biopsies showed non-caseous epithelioid granulomas, which were pathologically compatible with sarcoidosis. After steroid therapy, she became asymptomatic and the abnormal shadows and subcutaneous nodules disappeared. HLA serological typing revealed that she harbored the sarcoidosis-related HLA-DR14 allele, as well as UC-related HLA-B52 and HLA-DR15 alleles. This case suggests that a susceptible HLA genotype may influence the onset of the combination of sarcoidosis and UC.

On-line separation and quantification of virus antigens of different serotypes in multivalent vaccines by capillary zone electrophoresis: A case study for quality control of foot-and-mouth disease virus vaccines

Accurate quantification of effective antigens of different serotypes is crucial for quality control of multivalent vaccines but challenging. A simple and rapid capillary zone electrophoresis (CZE) method was developed for on-line separation and quantification of foot-and-mouth disease virus (FMDV) antigens in monovalent and bivalent FMDV vaccines. The FMDV peak identity in CZE was demonstrated by the study of FMDV dissociation combined with high performance size exclusion chromatography (HPSEC) analysis. After optimizing CZE conditions including UV detecting wavelength, injection volume, and separation voltage, both serotype A and O FMDV showed good reproducibility (RSD <5%) and linear responses (R2=0.999) between the peak area and FMDV content in the concentration range of 15-400 μg/mL. The two serotypes of FMDV with similar size had different migration time in CZE according to their different zeta potential, which allows them to be separated and quantified, with accuracy of <10% relative error. CZE was then successfully applied for antigen quantification of commercial O monovalent and A/O bivalent FMDV vaccines. Compared with HPSEC, CZE was not only able to quantify each serotype of FMDV, but also free from interference of nucleic acids impurities. In summary, the CZE can be a simple, rapid, and reliable tool for quality control of monovalent and bivalent FMDV vaccines. The CZE method can also be further extended to the quality control of other multivalent virus and virus like particle vaccines.

Development of monospecific polyclonal antibodies against hypervirulent Klebsiella pneumoniae

Abstract. Darniati, Setiyaningsih S, Agungpriyono DR, Handharyani E. 2021. Development of monospecific polyclonal antibodies against hypervirulent Klebsiella pneumoniae. Biodiversitas 22: 99-105. The aim of the research was to produce monospecific antibodies against Klebsiella pneumoniae serotype K1 and K2. Klebsiella pneumonia isolates were recovered from pneumonic lungs of Aceh cattle. The bacteria were identified by rpoB (RNA polymerase β subunit) PCR amplification specific for K. pneumoniae. The presence of capsule cluster gene magA and k2A was used to characterize capsular serotype K1 and K2, respectively. Antisera were produced using serial immunization of New Zealand White rabbits with K. pneumoniae serotypes K1 and K2. The presence of antibodies was determined by using immunodiffusion test and purification was performed by immunoaffinity purification method. K. pneumoniae antibodies began to appear on the 14th day after the first injection and progressively intensified after the 1st and 2nd booster. The cross-reactivity between the antigen was eliminated by absorbing the antisera with the opposite antigen and several serotypes of non-K1/K2. After purification, serum protein concentrations were substantially decreased from 58.48 µg/µL and 53.99 µg/µL to 2.38 µg / µL and 3.72 µg / µL for K1 and K2, respectively). The SDS-PAGE analysis showed two bands with molecular weight at 54 kDa and 25 kDa representing the heavy and light chains of immunoglobulin G, respectively. Purified polyclonal antiseras against K. pneumoniae serotypes K1 and K2 showed high affinity and specificity for homologous antigens in immunodiffusion and direct agglutination tests, and immunohistochemical staining.

Specific Inhibition of Tumor Growth by T Cell Receptor–Drug Conjugates Targeting Intracellular Cancer-Testis Antigen NY-ESO-1/LAGE-1

Despite the significant therapeutic advances in T-cell immunotherapy, many malignancies remain unresponsive, which might be because of the negative regulation of T cells by the tumor microenvironment (TME). T cells discriminate tumor cells and normal cells through T-cell receptors (TCRs); therefore, we generated a novel type of TCR-drug conjugates (TDCs) by referring antibody-drug conjugations (ADCs) to overcome the effects of the TME on T cells while preserving the specificity of TCR for tumor recognition. We selected HLA-A2/NY-ESO-1157-165 (peptide NY-ESO-1157-165 in complex with human leukocyte antigen serotype HLA-A*02:01) as the antigen and the antigen-specific TCR (1G4113) as the carrier. By sortase A-mediated ligation, we obtained three NY-TCR-vcMMAEs and further studied their properties, antitumor activity, and toxicity in vitro and in vivo. We found that all the NY-TCR-vcMMAEs had high endocytosis efficiency and specifically killed HLA-A2/NY-ESO-1157-165 positive tumor cells. In xenograft models, one of the TDCs, NY-TCR-2M, was effectively and specifically distributed into tumor tissues and inhibited tumor growth without inducing obvious toxicity. Our results demonstrated that TCRs can be carriers of toxic payloads and that the TDCs thus formed can specifically inhibit tumor growth, neglecting the immune microenvironment.

High-Throughput Process Development for the Chromatographic Purification of Viral Antigens.

Chromatography is a widely used method in the biotechnology industry and functions to separate the desired product from process and product related impurities. There is a multitude of resins available based on different modalities (such as charge, hydrophobicity, and affinity) to provide a spectrum of approaches to meet the separation challenges of the diverse products. The challenge of developing viral antigen purification processes is addressed in this method. A unique feature of this product class is that in order to protect against more than one strain of an antigen, vaccines are often multivalent. This entails multiple production processes for each antigen, all of which will require separate development and validation. Ideally, a universal purification method is sought, but differences in the protein subunits (frequently used as the antigens) make this challenging and often-bespoke purification steps are required. This means process development for the chromatographic stages of these products can be particularly challenging and labour intensive. With the numerous choices available, making critical process decisions that are usually unique to each product, process, and strain, can be costly and time-consuming. To address this, scale down purification at <1.0mL column volume and automation approaches are increasingly applied to increase throughput. In this work, a method is described wherein a Tecan Freedom EVO® automated liquid handler is deployed for the evaluation of different resin chemistries and buffer conditions to find a suitable purification strategy. This method allows for the rapid evaluation of the separation viral antigens where limited information on chromatography behavior is known at the early stages of process development. Here, we demonstrate the methodology firstly by explaining the automated purification script and secondly by applying the script for an efficient purification development for different serotypes of rotavirus antigens.

Serotype distribution of Streptococcus pneumonia in children with invasive disease in Turkey: 2015-2018

ABSTRACT Objectives: To determine the serotype distribution of pneumococcus causing invasive pneumococcal disease (meningitidis, bacteremia and empyema) in children in Turkey, and to observe potential changes in this distribution in time to guide effective vaccine strategies. Methods: We surveyed S. pneumoniae with conventional bacteriological techniques and with real-time polymerase chain reaction (RT-PCR) in samples of cerebrospinal fluid (CSF), blood and pleural fluid. S. pneumoniae strains were isolated from 33 different hospitals in Turkey, which are giving health services to approximately 60% of the Turkish population. Results: A total of 167 cases were diagnosed with invasive pneumococcal disease between 2015 and 2018. We diagnosed 52 (31.1%) patients with meningitis, 104 (62.2%) patients with bacteremia, and 11 (6.6%) patients with empyema. Thirty-three percent of them were less than 2 years old and 56% less than 5 years old. Overall PCV13 serotypes accounted for 56.2% (94/167). The most common serotypes were 19 F (11.9%), 1 (10.7%) and 3 (10.1%). Conclusions: Besides the increasing frequency of non-vaccine serotypes, vaccine serotypes continue to be a problem for Turkey despite routine and high-rate vaccination with PCV13 and significant reduction reported for the incidence of IPD in young children. Since new candidate pneumococcal conjugate vaccines with more serotype antigens are being developed, continuing IPD surveillance is a significant source of information for decision-making processes on pneumococcal vaccination.

Development and serology based efficacy assessment of a trivalent foot-and-mouth disease vaccine

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals throughout the world. The endemicity of this disease in Bangladesh has been causing high economic loss and an impediment to the full potential surge of livestock industries. In Bangladesh, vaccination using imported or locally produced FMD vaccines is the existing practice of controlling the disease, although vaccine failure cases are very common. Hence, to address the problem, the present study was envisaged to develop an effective FMD vaccine tailored to the circulating indigenous foot-and-mouth disease virus (FMDV) strains. Three local circulating FMDVs O/BAN/TA/Dh-301/2016 (MK088170.1), A/BAN/CH/Sa-304/2016 (MK088171.1) and Asia1/BAN/DH/Sa-318/2018 (MH457186.1) isolates were selected as vaccine strains based on recent epidemiology, genetic and antigenic analyses. These serotype O, A and Asia1 vaccine strains showed strong antigenic relationship (r1 > 0.3) with 100% to 75% of the respective circulating viruses. The candidate viruses were successfully inactivated by 3.0 mM binary ethylenimine within 7–10 h after the onset of inactivation. Extrapolation of inactivation kinetics confirmed < 1 log10 TCID50 in a 10000-liter batch liquid preparation after 24 h inactivation cycle. The inactivated virus particles were significantly (p < 0.05) concentrated and the trivalent vaccine was formulated using 6 µg per dose per serotype antigen payload. The trivalent vaccine was administered in divided doses in different groups of cattle. All doses of the vaccine elicited significantly (p < 0.05) higher levels of antibodies as early as 14-day post-vaccination (dpv) and peak antibody titers were achieved in 28 dpv. The ‘full dose’ (6.0 µg per dose per serotype) vaccine elicited antibody titers expected to confer protection in 100% cattle of the respective group and maintained such level of antibodies beyond 180 dpv. Thus, the trivalent FMD vaccine prepared with 6.0 µg antigen per dose per serotype of the selected candidate viruses will confer protection against circulating FMDVs of Bangladesh and its neighboring countries.

Development and evaluation of a new recombinant protein vaccine (YidR) against Klebsiella pneumoniae infection

Vaccination is a promising approach to prevent Klebsiella infection; however, the high heterogeneity of strains is a limiting factor. The best antigenic target for an anti-Klebsiella vaccine should be expressed by all or most of strains. We previously found YidR protein to be highly conserved among K. pneumoniae strains independently of antigen serotype. Therefore, in the present study, we developed a recombinant YidR protein vaccine and evaluated its protective efficacy against lethal challenge with K. pneumoniae in a mouse model. The yidR gene was cloned in Escherichia coli for recombinant expression. The lethal dose (LD100) of K. pneumoniae was determined and lethal challenge was carried out after immunization with recombinant purified YidR. After immunization, the concentration of total serum IgG was significantly higher in YidR-immunized mice than in non-immunized mice, indicating strong induction of antibodies. Mice were challenged with LD100 of K. pneumoniae, and significantly lower murine sepsis and higher body weight were observed in YidR-immunized mice compared to unvaccinated controls. Moreover, ∼90% of YidR-immunized mice survived beyond 10 days of observation, whereas none of the control mice survived past 48 h. The protective effect of YidR recombinant protein vaccine was demonstrated and YidR may be a promising vaccine candidate to prevent klebsiellosis.

Long-Term Protection Elicited by a DNA Vaccine Candidate Expressing the prM-E Antigen of Dengue Virus Serotype 3 in Mice

Dengue virus (DENV) is the causative agent of dengue, and its incidence has increased 30-fold in the past five decades. Among the four cocirculating serotypes, DENV3 is associated with an increased number of severe infections and has become widespread. Vaccination is the mainstay of prevention in reducing disease burden. Previously, the protective efficacy of DNA vaccine candidates toward DENV1, 2, and 4 was confirmed in mice. In this study, a DNA vaccine candidate (pVAX1-D3ME) expressing the prM and E proteins of DENV3 was constructed, and then the immunogenicity and protection were assessed in mice to further develop a tetravalent dengue vaccine. Moreover, the cross-reactive immune responses against the other three serotypes were investigated. The results showed that three doses of 50 μg of pVAX1-D3ME were sufficient to induce strong antigen-specific T cell responses and robust and consistent neutralizing antibodies. Additionally, immunization with pVAX1-D3ME offered protective immunity against not only DENV3 but also the other three serotypes, which could be observed even after 12 months. This study shows great promise for the further evaluation of a dengue tetravalent DNA vaccine candidate in large animal models, including non-human primates.

Immunological characterisation and immunoprotective efficacy of functional domain antigens of botulinum neurotoxin serotype A

Botulinum neurotoxins (BoNTs) are highly toxic proteins that mediate their effects by binding to neuronal receptors and block the neutralizing ability of therapeutic antibodies. Vaccination is currently the most effective strategy to prevent botulism. In this study, a series of recombinant functional domain antigens of BoNT/A were prepared and identified, and their immunoprotective efficacies were explored and compared. Our results showed that all antigens produced strong humoral immune responses, although their protective effects against the toxin were different. Only the Hc and HN-L antigens produced strong protective effects and afforded complete immunoprotection. In addition, the combined vaccine groups showed that there was no synergistic effect on immune responses after antigen combination, suggesting that the integrity of the toxin antigen or domain is crucial to the immune effects. Studies of the dose-dependent immunoprotective effects further confirmed that the Hc domain antigen afforded more effective protective potency than the HN-L antigen, equivalent to the immune effect of the full-length toxin (Hc + HN-L combination group). Overall, our results demonstrated that the Hc domain elicited a strong protective immune response and also provided basic data and theoretical support for the development of Hc-based BoNT/A subunit vaccine. Therefore, the receptor binding domain Hc is implicated as a promising target antigen of the BoNT/A recombinant subunit vaccine as an alternative to the toxoid vaccine.

Evaluation of Long-term Antibody Response and Cross-serotype Reaction in Ducks Immunised with Recombinant Riemerella Anatipestifer Outer Membrane Protein A and CpG ODN.

IntroductionRiemerella anatipestifer (RA) infections can lead to high mortality in ducklings. Inactivated vaccines against RA are commercially available, but they fail to provide cross-protection against various serotypes. We have previously demonstrated that a subunit vaccine containing recombinant outer membrane protein A (rOmpA) antigen of serotype 2 formulated with CpG oligodeoxynucleotides (ODN) as the adjuvant was able to stimulate both humoral and cellular immunities.Material and MethodsIn the present study, thirty healthy 7-day-old Pekin ducks were randomly assigned to three equal treatment groups: rOmpA-vaccinated, rOmpA + CpG-vaccinated, and control. Vaccine was injected intramuscularly and a booster dose of the same vaccine was given two weeks after primary immunisation. The long-term antibody response and cross-serotype reaction of this vaccine were evaluated in ducks.ResultsCompared to ducks immunised with rOmpA alone, ducks immunised with rOmpA + CpG ODN had significantly (p < 0.05) increased serum antibody titre from two weeks until nine months after primary immunisation. In addition, expression of cytokines including interferon (IFN)-α, IFN-γ, interleukin (IL)-6, and IL-12 was significantly (p < 0.05) enhanced in PBMC of ducks immunised with rOmpA + CpG ODN two weeks after primary immunisation. Antibodies from ducks immunised with the rOmpA + CpG ODN vaccine could also detect RA serotypes 1 and 6 in Western blot analysis.ConclusionCombination of rOmpA and CpG ODN could be a feasible strategy for developing a subunit RA vaccine with long term and broader-ranging protection.

Analyzing and Reanalyzing the Genome: Findings from the MedSeq Project

Although genome sequencing is increasingly available in clinical and research settings, many questions remain about the interpretation of sequencing data. In the MedSeq Project, we explored how much effort is required to evaluate and report on more than 4,500 genes reportedly associated with monogenic conditions, as well as pharmacogenomic (PGx) markers, blood antigen serotyping, and polygenic risk scores in 100 individuals (50 with cardiomyopathy and 50 healthy) randomized to the sequencing arm. We defined the quality thresholds for determining the need for Sanger confirmation. Finally, we examined the effort needed and new findings revealed by reanalyzing each genome (6-23months after initial analysis; mean 13months). Monogenic disease risk and carrier status were reported in 21% and 94% of participants, respectively. Only two participants had no monogenic disease risk or carrier status identified. For the PGx results (18 genotypes in six genes for five drugs), the identified diplotypes prompted recommendation for non-standard dosing of at least one of the analyzed drugs in 95% of participants. For blood antigen studies, we found that 31% of participants had a rare blood antigen genotype. In the cardiomyopathy cohort, an explanation for disease was identified in 48% of individuals. Over the course of the study, 14 variants were reclassified and, upon reanalysis, 18 new variants met criteria for reporting. These findings highlight the quantity of medically relevant findings from a broad analysis of genomic sequencing data as well as the need for periodic reinterpretation and reanalysis of data for both diagnostic indications and secondary findings.

Japanese encephalitis virus prM-E antigen immunization conferred protection against challenge by four different serotypes of Dengue viruses in mice.

Dengue virus (DENV) and Japanese encephalitis virus (JEV) are closely related mosquito-borne flaviviruses. Together, they caused most arthropod-borne diseases in the world. Previously, we had demonstrated that both live attenuated and inactivated JE vaccines elicited cross-protection against DENV infection, and a DNA vaccine candidate expressing JEV prM-E protein (named pCAG-JME) could provide effective protection against JEV infection in mice. In this study, we examined whether the same pCAG-JME could elicit cross-protection against DENV infection. Our results showed that pCAG-JME indeed induced cross-reactive antibodies and cross-protection against four different serotypes of DENV in mice. Interestingly, pCAG-JME-immunized mice also generated both Th1 and Th2 responses when stimulated by all four different serotypes of DENV antigens. Moreover, cross-primed CD8+ T cell response was also detected following the stimulation of DENV proteins using intracellular cytokine staining. In addition, sera from pCAG-JME-immunized mice significantly reduced the mortality caused by DENV2 infection in severe combination immunodeficiency mouse. These results suggest that both JE and DENV cross-reactive antibodies and cross-primed T cells might play important roles in the cross-protection. The findings of this study also indicate a possibility of developing novel multivalent genetically engineered vaccines against both JEV and DENV.

The Dengue ED3 Dot Assay, a Novel Serological Test for the Detection of Denguevirus Type-Specific Antibodies and Its Application in a Retrospective Seroprevalence Study

There are four distinct antigenic serotypes of dengue viruses (DENV-1-4). Sequential infections with different serotypes lead to cross-reactive but also serotype-specific neutralizing antibody responses. Neutralization assays are considered as gold standard for serotype-specific antibody detection. However, for retrospective seroprevalence studies, access to large serum quantities is limited making neutralization assays well-nigh impossible. Therefore, a serological test, wasting only 10 µL serum, was developed using fusion proteins of maltose binding protein and E protein domain 3 (MBP-ED3) as antigens. Twelve MBP-ED3 antigens for DENV-1-4, three MBP-ED3 antigens for WNV, JEV, and TBEV, and MBP were dotted onto a single nitrocellulose strip. ED3 dot assay results were compared to virus neutralization and ED3 ELISA test results, showing a >90% accordance for DENV-1 and a 100% accordance for DENV-2, making the test specifically useful for DENV-1/-2 serotype-specific antibody detection. Since 2010, DENV-1 has replaced DENV-2 as the dominant serotype in Cambodia. In a retrospective cohort analysis, sera collected during the DENV-1/-2 endemic period showed a shift to DENV-2-specific antibody responses in 2012 paralleled by the decline of DENV-2 infections. Altogether, the ED3 dot assay is a serum-, time- and money-saving diagnostic tool for serotype-specific antibody detection, especially when serum samples are limited.

Insulin autoimmune syndrome possibly caused by coenzyme Q10.

A 52-year-old woman was transported for reduced consciousness. Her blood glucose was only 19 mg/dL, but her blood immunoreactive insulin and insulin antibody levels were high at 250 μU/mL and 50 U/mL, respectively. She had no history of insulin treatment, but she had been taking coenzyme Q10 supplements for three months. Her human leukocyte antigen serotype was DR4. After stopping coenzyme Q10, her hypoglycemia disappeared and immunoreactive insulin and insulin antibody levels normalized. Based on the above, she was diagnosed with insulin autoimmune syndrome caused by coenzyme Q10. It is necessary to be aware of the onset of insulin autoimmune syndrome due to coenzyme Q10. Its pathogenesis requires clarification.

Improving vaccines against Streptococcus pneumoniae using synthetic glycans

Streptococcus pneumoniae remains a deadly disease in small children and the elderly even though conjugate and polysaccharide vaccines based on isolated capsular polysaccharides (CPS) are successful. The most common serotypes that cause infection are used in vaccines around the world, but differences in geographic and demographic serotype distribution compromises protection by leading vaccines. The medicinal chemistry approach to glycoconjugate vaccine development has helped to improve the stability and immunogenicity of synthetic vaccine candidates for several serotypes leading to the induction of higher levels of specific protective antibodies. Here, we show that marketed CPS-based glycoconjugate vaccines can be improved by adding synthetic glycoconjugates representing serotypes that are not covered by existing vaccines. Combination (coformulation) of synthetic glycoconjugates with the licensed vaccines Prevnar13 (13-valent) and Synflorix (10-valent) yields improved 15- and 13-valent conjugate vaccines, respectively, in rabbits. A pentavalent semisynthetic glycoconjugate vaccine containing five serotype antigens (sPCV5) elicits antibodies with strong in vitro opsonophagocytic activity. This study illustrates that synthetic oligosaccharides can be used in coformulation with both isolated polysaccharide glycoconjugates to expand protection from existing vaccines and each other to produce precisely defined multivalent conjugated vaccines.

RNA ISOLATION OF DENGUE VIRUS TYPE 1 WITH DIFFERENT PRECIPITATION SOLVENTS: DIMETHYL SULFOXIDE, ACETONE, AND ETHANOL 70%

Dengue Hemorrhagic Fever (DHF) is caused by dengue viruses that belong to Flaviviridae. The disease is known to be caused by 4 types of dengue viruses, namely DENV-1, DENV-2, DENV-3, and DENV-4 associated with antigenic. Dengue virus is a virus RNA that causes illness with clinical manifestations of Dengue Fever, Dengue Hemorrhagic Fever and Dengue Shock Syndrome. The aim of research was to determine the effectiveness of dimethyl sulfoxide, acetone, and ethanol 70% as precipitation solvent in the process of RNA isolation. The method used was Reverse Transcription - Polymerase Chain Reaction (RT-PCR) and Polymerase Chain Reaction (PCR) with specific primers for dengue virus type 1 (DENV-1). RNA isolation can be done easily using an RNA Isolation Kit. Use of RNA Isolation Kit results in a purer RNA isolate from contaminants and from RNA degradation. In generally the isolation is using cold ethanol / alcohol with concentration 90-95%. Ethanol / Alcohol does not dissolve RNA and light density of alcohol lighter than water makes RNA rise and hover on the surface. In RNA isolation solvent precipitation that used are acetone, ethanol 70%, and DMSO. In qualitative RNA measurements using agarose gel electrophoresis and was examined under the UV light-illuminator and quantitative RNA measurements using Nanodrop spectrophotometry with absorbance ratio at 260/280 and 260/230 showed a good result indicated by the appearance of the band on electrophoresis results in PCR. While the measurement quantitatively is showed that there was still protein contamination but the results are quite good because it does not much different from the ratio set in the reference. Acetone, ethanol 70%, and DMSO can be used as a substitute of 96% ethanol in the process of RNA isolation in DENV-1 virus and can also be applied to other dengue virus because the structure of the 4th antigen serotype is very similar one with the other and no effect.