Enhancing production of the malaria asexual blood-stage vaccine candidate PfRipr5 in insect cells by modulating expression vector and culture temperature

Despite the recent approval of the first malaria vaccine RTS,S/AS01, its efficacy in children and infants is still modest. Therefore, continued development of new, improved malaria vaccines, including asexual blood-stage vaccines such as the one herein targeted, is essential to reach desired levels of protection against disease and mortality. In this study, the insect cell-baculovirus expression vector system (IC-BEVS) was used to produce a malaria asexual blood-stage vaccine candidate based on PfRipr5 antigen and compared to traditional mammalian (HEK293) cell system. PfRipr5 could be expressed to higher levels in IC-BEVS, with higher protein purity and reactivity to a conformational anti-PfRipr monoclonal antibody than its mammalian counterpart. The performance of IC-BEVS was further improved by modulating the expression vector sequence and culture temperature. The addition to the expression vector of (i) one alanine (A) amino acid residue adjacent to the signal peptide cleavage site, and (ii) a glycine-serine linker (GGSGG) between the PfRipr5 sequence and the purification tag, resulted in up to 2.2-fold increase in the expression of secreted PfRipr5. In addition, lowering temperature from standard 27 °C to 22 °C at the time of infection improved PfRipr5 productivity by up to 1.7-fold. Noteworthy, a synergistic effect was attained by combining both optimization strategies, enabling to increase expression of extracellular PfRipr5 by up to 4-fold and process yield post-purification by 5.2-fold, while maintaining same degree of protein purity and reactivity. This work highlights the potential of insect cells to produce the PfRipr5 malaria vaccine candidate and the importance of optimizing the expression vector and culture conditions to boost expression of secreted proteins

Cutoff Values of Serum IgG4 and Histopathological IgG4+ Plasma Cells for Diagnosis of Patients with IgG4-Related Disease

IgG4-related disease is a new disease classification established in Japan in the 21st century. Patients with IgG4-related disease display hyper-IgG4-gammaglobulinemia, massive infiltration of IgG4+ plasma cells into tissue, and good response to glucocorticoids. Since IgG4 overexpression is also observed in other disorders, it is necessary to diagnose IgG4-related disease carefully and correctly. We therefore sought to determine cutoff values for serum IgG4 and IgG4/IgG and for IgG4+/IgG+ plasma cells in tissue diagnostic of IgG4-related disease. Patients and Methods. We retrospectively analyzed serum IgG4 concentrations and IgG4/IgG ratio and IgG4+/IgG+ plasma cell ratio in tissues of 132 patients with IgG4-related disease and 48 patients with other disorders. Result. Serum IgG4 >135  mg/dl demonstrated a sensitivity of 97.0% and a specificity of 79.6% in diagnosing IgG4-related disease, and serum IgG4/IgG ratios >8% had a sensitivity and specificity of 95.5% and 87.5%, respectively. IgG4+cell/IgG+ cell ratio in tissues >40% had a sensitivity and specificity of 94.4% and 85.7%, respectively. However, the number of IgG4+ cells was reduced in severely fibrotic parts of tissues. Conclusion. Although a recent unanimous consensus of all relevant researchers in Japan recently established the diagnostic criteria for IgG4-related disease, findings such as ours indicate that further discussion is needed

Relationship between advanced glycation end products and plaque progression in patients with acute coronary syndrome: the JAPAN-ACS Sub-study.

Background: The Japan Assessment of Pitavastatin and Atorvastatin in Acute Coronary Syndrome (JAPAN-ACS) trial demonstrated that early aggressive statin therapy in patients with ACS significantly reduces plaque volume (PV). Advanced glycation end products (AGEs) and the receptors of AGEs (RAGE) may lead to angiopathy in diabetes mellitus (DM) and may affect on the development of coronary PV. The present sub-study of JAPAN-ACS investigates the association between AGEs and RAGE, and PV.Methods: Intravascular ultrasound (IVUS)-guided percutaneous coronary intervention (PCI) was undertaken, followed by the initiation of statin treatment (either 4 mg/day of pitavastatin or 20 mg/day of atorvastatin), in patients with ACS. In the 208 JAPAN-ACS subjects, PV using IVUS in non-culprit segment > 5 mm proximal or distal to the culprit lesion and, serum levels of AGEs and soluble RAGE (sRAGE) were measured at baseline and 8-12 months after PCI.Results: At baseline, no differences in the levels of either AGEs or sRAGE were found between patients with DM and those without DM. The levels of AGEs decreased significantly with statin therapy from 8.6 ± 2.2 to 8.0 ± 2.1 U/ml (p < 0.001), whereas the levels of sRAGE did not change. There were no significant correlations between changes in PV and the changes in levels of AGEs as well as sRAGE. However, high baseline AGEs levels were significantly associated with plaque progression (odds ratio, 1.21; 95% confidence interval, 1.01 - 1.48; p = 0.044) even after adjusting for DM in multivariate logistic regression models.Conclusions: High baseline AGEs levels were associated with plaque progression in the JAPAN-ACS trial. This relationship was independent of DM. These findings suggest AGEs may be related to long-term glucose control and other oxidative stresses in ACS.Trial registration: NCT00242944. © 2013 Fukushima et al.; licensee BioMed Central Ltd