Physicochemical and biological characterization of SB2, a biosimilar of Remicade® (infliximab)

A biosimilar is a biological medicinal product that contains a version of the active substance of an already authorized original biological medicinal product. Biosimilarity to the reference product (RP) in terms of quality characteristics, such as physicochemical and biological properties, safety, and efficacy, based on a comprehensive comparability exercise needs to be established. SB2 (Flixabi® and Renflexis®) is a biosimilar to Remicade® (infliximab). The development of SB2 was performed in accordance with relevant guidelines of the International Conference on Harmonisation, the European Medicines Agency, and the United States Food and Drug Administration. To determine whether critical quality attributes meet quality standards, an extensive characterization test was performed with more than 80 lots of EU- and US-sourced RP. The physicochemical characterization study results revealed that SB2 was similar to the RP. Although a few differences in physicochemical attributes were observed, the evidence from the related literature, structure-activity relationship studies, and comparative biological assays showed that these differences were unlikely to be clinically meaningful. The biological characterization results showed that SB2 was similar to the RP in terms of tumor necrosis factor–α (TNF-α) binding and TNF-α neutralization activities as a main mode of action. SB2 was also similar in Fc-related biological activities including antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, neonatal Fc receptor binding, C1q binding, and Fc gamma receptor binding activities. These analytical findings support that SB2 is similar to the RP and also provide confidence of biosimilarity in terms of clinical safety and efficacy.     

Effect of dietary ascorbic acid on growth and non-specific immune responses of tiger puffer, Takifugu rubripes

We report nutritional physiology and non-specific immune responses of ascorbic acid (AA) in puffer fish for the first time. This study aimed to examine the essentiality and requirements of AA in diets for the tiger puffer, Takifugu rubripes based on growth performance, liver AA and bone collagen concentration, and non-specific immune responses. Five casein-gelatin based semi-purified diets were formulated to contain five graded levels of l-ascorbyl-2-monophosphate at 0, 40, 80, 160 and 700mg/kg (designated as AMP0, AMP40, AMP80, AMP160 and AMP700, respectively) and fed to triplicate groups of fish. After 10weeks of feeding trial, growth performances of fish (initial body weight, 35g) fed the AMP0 were significantly lower compared to that of fish fed diets supplemented with AMP. The fish fed the AMP0 diet also exhibited significantly lower hematocrit, condition factor and hepatosomatic index compared to the fish fed diets supplemented with AMP. Phagocytic activity (NBT assay) was significantly lower in fish fed the AMP0 diet than in fish fed the AMP containing diets. Plasma lysozyme activity of fish fed the AMP80 and AMP160 was significantly higher than that of fish fed the AMP0. Dietary supplementation of AMP significantly increased the liver superoxide dismutase in the fish. Myeloperoxidase activity of fish fed the AMP0 was significantly lower compared to that of fish fed the AMP containing diets. Bone collagen level tended to increase numerically and total AA concentration in liver of fish was significantly increased in a dose dependent manner by the supplementation of AMP. Therefore, tiger puffer requires exogenous ascorbic acid and the optimum dietary level could be 29mg AA/kg diet for normal growth and physiology. Dietary AA concentration over 82mg/kg could be required to enhance non-specific immune responses of the fish. However, it does not seem that the fish needs an overdose of dietary AA (>160mg/kg) for better non-specific immune responses.     

Plasmid DNA encoding CCR7 ligands compensate for dysfunctional CD8+ T cell responses by effects on dendritic cells

Lymphotoxin alpha-deficient (LTalpha-/-) mice, which lack lymph nodes and possess a disorganized spleen, develop dysfunctional CD8+ T cells upon HSV infection and readily succumb to herpes encephalitis. Such mice do develop apparently normal peptide-specific CD8+ T cell responses, as measured by MHC class I tetramer staining, but the majority of cells fail to become cytotoxic or express peptide-induced IFN-gamma production. In the present study, we demonstrate that functional defects of CD8+ T cells in LTalpha-/- mice can be largely rectified by the administration of plasmid DNA encoding CCR7 ligands before HSV infection. Treated mutant mice developed increased peptide-specific cytotoxic responses, enhanced numbers of CD8+ T cells capable of producing IFN-gamma, as well as improved resistance to HSV challenge. The corrective effect of chemokine treatment appeared to result from improved dendritic cell-mediated Ag presentation. Thus, a major consequence of the treatment was an increase in splenic dendritic cell number in CCR7 ligand-treated LTalpha-/- mice with such splenocyte populations showing improved APC activity in vitro. Our results document that functional defects of CD8+ T cells can be corrected, and indicate the value of plasmid vector encoding appropriate chemokines to achieve such immunotherapy.     

Induction of CD8 T-cell-specific systemic and mucosal immunity against herpes simplex virus with CpG-peptide complexes

Oligodeoxynucleotides (ODN) containing unmethylated CpG motifs exert powerful adjuvant activity in vivo and in vitro. Administered with antigen they induce a population of antigen-specific CD8+ T cells. In this study we immunized C57BL/6 mice with bioactive CpG ODN combined with an immunodominant epitope derived from herpes simplex virus (HSV) glycoprotein B (amino acids 498 to 505; SSIEFARL) and analyzed the magnitude and durability of the peptide-specific response. The effectiveness of the CD8+ T-cell response as measured by peptide-specific tetramers, peptide-induced intracellular gamma interferon expression, and resistance to systemic and mucosal challenge during the acute and memory phases was compared with the response induced by immunization with recombinant vaccinia virus encoding SSIEFARL as a minigene (VvgB(498-505)). Confirming the reports of others, our results demonstrate that the CpG ODN-peptide approach generates an antigen-specific CD8+ T-cell population, but the frequency of CD8+ T cells is lower than that induced by VvgB(498-505). Nevertheless, the protection level was comparable when mice were systemically and mucosally challenged during the acute phase. However, such responses by both groups waned with time and were functionally less effective. Still, our results indicate that the CpG ODN-peptide immunization system holds promise as a means of selectively inducing a CD8+ T-cell response against HSV.     

Development of a test strip reader for a lateral flow membrane-based immunochromatographic assay

A low-cost, simple strip reader system using a linear movement mechanism of CD-ROM deck has been developed to characterize a lateral flow membrane-based immunochromatographic assay. The test strip reader was assembled by a CD-ROM deck and home-made optical head especially designed for immunoassays. The optical head for detecting reflected light from the test strip surface consists of green light-emitting diode, large area silicon photodiode, and anodized aluminum mounting block providing a slit structure for cutting light from the LED. The stepping motor of the deck was operated in the full step mode, whose distance of each reading point is about 0.15 mm. The performance of the strip reader was tested by analysis of HBV (hepatitis B virus) antigen test kit. This strip reader can be useful for inexpensive, disposable, and membrane-based assays that provide visual evidence of the presence of an analyte in a liquid sample.     

DNA chip replication for a personalized DNA chip

We report the replication technology of DNA chip using by sequence specific localization of nucleic acids via hybridization and electric transfer of the nucleic acids onto a new substrate without losing their array information. The denatured DNA fragments are first spotted and UV-cross-linked on a nylon membrane. The membrane is then immersed and hybridized in a DNA mixture solution that contains all complementary sequences of the nucleic acids to be hybridized with the DNA fragments on the membrane. The hybridized DNA fragments are transferred to another membrane at the denatured condition. After separating two membranes, the transferred membrane contains a complementary array of DNA fragments. This method can be used for the replication of the same copy of DNA chip repeatedly and moreover could be applied for a personalized DNA chip fabrication, where specific information of each spot of DNA chip is originated from the genetic information of a personal sample.     

Systemic and mucosal immunity induced by oral somatic transgene vaccination against glycoprotein B of pseudorabies virus using live attenuated Salmonella typhimurium

Glycoprotein B mediates the absorption and penetration of the pseudorabies virus in the form of an immunodominant Ag, and represents a major target for the development of new vaccines. This study evaluated the efficiency of live attenuated Salmonella typhimurium SL7207 for the oral delivery of DNA vaccine encoding the pseudorabies virus glycoprotein B (pCI-PrVgB) in vivo, leading to the generation of both systemic and mucosal immunity against the pseudorabies virus Ag. An oral transgene vaccination of pCI-PrVgB using a Salmonella carrier produced a broad spectrum of immunity at both the systemic and mucosal sites, whereas the intramuscular administration of a naked DNA vaccine elicited no mucosal immunoglobulin (Ig)A response. Interestingly, the Salmonella-mediated oral transgene vaccination of the pseudorabies virus glycoprotein B biased the immune responses to the Th2-type, as determined by the IgG2a/IgG1 ratio and the cytokine production profile. However, oral vaccination mediated by Salmonella harbouring pCI-PrVgB showed inferior protection to systemic immunization against virulent pseudorabies virus infection. The expression of transgene delivered by Salmonella bacteria in antigen-presenting cells of both the systemic and mucosal-associated lymphoid tissues was further demonstrated. These results highlight the potential use of live attenuated S. typhimurium for an oral transgene pseudorabies virus glycoprotein B vaccination to induce broad immune responses.     

Two genetic lineages of sea slaters, Ligia (Crustacea: Isopoda) in South Korea: a population genetic approach

In this study, the species composition and population genetic properties of the sea slater, Ligia, in South Korea were investigated using mitochondrial and nuclear gene sequences. Two groups of sea slaters, genetically isolated from each other, a Western Group (WG) and an Eastern Group (EG) were identified. These groups exhibited considerable genetic divergence from Ligia exotica, previously recorded as a species inhabiting this country. These results indicate that there may be two species of Ligia in South Korea, but there is a small probability that both groups are L. exotica. A comparison of their genetic properties indicates that WG has a higher effective population size than EG, and that EG may have experienced a recent expansion, implying that it has a shorter history in South Korea than WG. These findings suggest that the South Korean sea slater populations may have been established as a result of several colonization events that can be traced on a continental scale by phylogeographic studies of sea slaters.     

Multiple alternating immunizations with DNA vaccine and replication incompetent adenovirus expressing gB of pseudorabies virus protect animals against lethal virus challenge

The prime-boost vaccination with DNA vaccine and recombinant viral vector has emerged as an effective prophylactic strategy to control infectious diseases. Here, we compared the protective immunities induced by multiple alternating immunizations with DNA vaccine (pCIgB) and replication-incompetent adenovirus (Ad-gB) expressing glycoprotein gB of pseudorabies virus (PrV). The platform of pCIgB-prime and Ad-gB-boost induced the most effective immune responses and provided protection against virulent PrV infection. However, priming with pCIgB prior to vaccinating animals by the DNA vaccine-prime and Ad-boost protocol provided neither effective immune responses nor protection against PrV. Similarly, boosting with Ad-gB following immunization with DNA vaccine-prime and Ad-boost showed no significant responses. Moreover, whereas the administration of Ad-gB for primary immunization induced Th2-type-biased immunity, priming with pCIgB induced Th1-type-biased immunity, as judged by the production of PrV-specific IgG isotypes and cytokine IFN-gamma. These results indicate that the order and injection frequency of vaccine vehicles used for heterologous prime-boost vaccination affect the magnitude and nature of the immunity. Therefore, our demonstration implies that the prime-boost protocol should be carefully considered and selected to induce the desired immune responses.