Successful selection of an infection‐protective anti‐Staphylococcus aureus monoclonal antibody and its protective activity in murine infection models

Recent clinical trials to develop anti‐methicillin‐resistant Staphylococcus aureus (MRSA) therapeutic antibodies have met unsuccessful sequels. To develop more effective antibodies against MRSA infection, a panel of mAbs against S. aureus cell wall was generated and then screened for the most protective mAb in mouse infection models. Twenty‐two anti‐S. aureus IgG mAbs were obtained from mice that had been immunized with alkali‐processed, deacetylated cell walls of S. aureus. One of these mAbs, ZBIA5H, exhibited life‐saving effects in mouse models of sepsis caused by community‐acquired MRSA strain MW2 and vancomycin‐resistant S. aureus strain VRS1. It also had a curative effect in a MW2‐caused pneumonia model. Curiously, the target of ZBIA5H was considered to be a conformational epitope of either the 1,4‐β‐linkage between N‐acetylmuramic acid and N‐acetyl‐D‐glucosamine or the peptidoglycan per se. Reactivity of ZBIA5H to S. aureus whole cells or purified peptidoglycan was weaker than that of most of the other mAbs generated in this study. However, the latter mAbs did not have the protective activities against S. aureus that ZBIA5H did. These data indicate that the epitopes that trigger production of high‐yield and/or high‐affinity antibodies may not be the most suitable epitopes for developing anti‐infective antibodies. ZBIA5H or its humanized form may find a future clinical application, and its target epitope may be used for the production of vaccines against S. aureus infection.     

Molecular cloning of HCV and clinical application

Abstract: Fifty-five clones encoding epitopes of HCV were isolated from Japanese patients. Their amino acid homology (AAH) to the sequence of prototype (HCV-1) ranged from 47% to 94%. These sequences cover 60% of the HCV genome lacking M/E and NS2 regions suggesting a very low or lacking immunogenecity for these regions. Two test kits for detection of anti-HCV antibody were developed using a combination of a synthetic peptide (AR142) containing the epitope of N14 (QRKTKRSTNRR) having a homology to the core of HCV of | fr | sol 8/11AA and a non-fusion recombinant protein Y19 starting from amino acid number (AAN) 1380 to 1507 in the NS3 region showing a AAH to the HCV-1 of 90%, and a combination of a mixture of three synthetic peptides of S29 AAN of 1–30, 38–65 and 47–74 of the core and a non-fused recombinant protein S4 AAN of 1287–1506 having a 93% AAH of the NS3 region. They showed almost the same order of sensitivity and specificity of the second-generation kits when tested with serum from blood donors and patients with non-A, non-B hepatitis. It should also be stressed that in all of the complete responders of a recombinant α-interferon therapy, the antibody levels against AR142 gradually decreased during and after the treatment. In 1992, studies performed for 125 patients with hepatocellular carcinoma in our clinic shows that of these 16 patients might developed from either chronic non-B, non-C liver diseases or chronic liver diseases caused by mutant(s) of HCV as their serum were negative for HBsAg and second-generation of anti-HCV.     

Oxidative folding of synthetic polypeptides S‐protected as tert‐butylthio derivatives

A new method for oxidative folding of synthetic polypeptides assembled by stepwise solid phase synthesis is introduced. Folding is obtained in excellent yields by reacting S‐tert‐butylthiolated polypeptides with a 100‐fold molar excess of cysteine at 37 °C in a slightly alkaline buffer containing chaotropic salts, and in the presence of air‐oxygen. This novel protocol has been applied to the folding of S‐tert‐butylthiolated human thymus and activation‐regulated chemokine (hu‐TARC) derivatives as well as to larger segments of Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins. Folded P. falciparum polypeptides have been used as substrates of endoproteinase Glu‐C (Glu‐C) and endoproteinase Asp‐N (Asp‐N) in an attempt to identify their disulfide connectivities. Particular practical advantages of the present method are (i) easy purification and storage of the S‐protected peptide derivatives, (ii) elimination of the risk of cysteine alkylation during the acidolytic cleavage deprotection and resin cleavage steps, (iii) possibility to precisely evaluate the extent of folding and disulfide bond formation by mass spectrometry, and (iv) facile recovery of the final folded product. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Immunotherapeutic approaches in Hepatocellular carcinoma: Building blocks of hope in near future

Hepatocellular carcinoma (HCC) is the most common type of primary hepatic cancer and is among the major causes of mortality due to cancer. Due to the lack of efficient conventional therapeutic options for this cancer, particularly in advanced cases, novel treatments including immunotherapy have been considered. However, despite the encouraging clinical outcomes after implementing these innovative approaches, such as oncolytic viruses (OVs), adoptive cell therapies (ACT), immune checkpoint blockades (ICBs), and cancer vaccines, several factors have restricted their therapeutic effect. The main concern is the existence of an immunosuppressive tumor microenvironment (TME). Combination of different ICBs or ICBs plus tyrosine kinase inhibitors have shown promising results in overcoming these limiting factors to some extent. Combination of programmed cell death ligand-1 (PD-L1) antibody Atezolizumab and vascular endothelial growth factor (VEGF) antibody Bevacizumab has become the standard of care in the first-line therapy for untestable HCC, approved by regulatory agencies. This paper highlighted a wide overview of the direct and indirect immunotherapeutic strategies proposed for the treatment of HCC patients and the common challenges that have hindered their further clinical applications.     

Economic simulation of batch and continuous aqueous two-phase purification for viral products

Vaccine manufacturing strategies that lower capital and production costs could improve vaccine access by reducing the cost per dose and encouraging localized manufacturing. Continuous processing is increasingly utilized to drive lower costs in biological manufacturing by requiring fewer capital and operating resources. Aqueous two-phase systems (ATPS) are a liquid–liquid extraction technique that enables continuous processing for viral vectors. To date, no economic comparison between viral vector purifications using traditional methods and ATPS has been published. In this work, economic simulations of traditional chromatography-based virus purification were compared to ATPS-based virus purification for the same product output in both batch and continuous modes. First, the modeling strategy was validated by re-creating a viral subunit manufacturing economic simulation. Then, ATPS capital and operating costs were compared to that of a traditional chromatography purification at multiple scales. At all scales, ATPS purification required less than 10% of the capital expenditure compared to chromatography-based purification. At an 11 kg per year production scale, the ATPS production costs were 50% less than purification with chromatography. Other chromatography configurations were explored, and may provide a production cost benefit to ATPS, but the purity and recovery were not experimentally verified. Batch and continuous ATPS were similar in capital and production costs. However, manual price adjustments suggest that continuous ATPS plant-building costs could be less than half that of batch ATPS at the 11 kg per year production scale. These simulations show the significant reduction in manufacturing costs that ATPS-based purification could deliver to the vaccine industry.     

A DNA vaccine against chimeric AFP enhanced by HSP70 suppresses growth of hepatocellular carcinoma

Alpha-Fetoprotein (AFP) is produced principally in fetal liver, gastrointestinal tract and the yolk sac which is temporarily present during embryonic development. AFP is overexpressed in the majority of Hepatocellular Carcinoma (HCC) and thus offers an attractive target for immunotherapy against this neoplasm. Here, we report that anti-HCC effects were achieved in a therapeutic setting with a DNA vaccine encoding mouse AFP and co-expressing Heat Shock Protein 70 (HSP70) gene. We also demonstrated that this vaccine elicited a marked and highly effective AFP specific CTL response against AFP-positive target cells. This vaccine also induced the prolongation of life span in mice bearing the tumor and the eradication of HCC. It is anticipated that vaccine strategies such as this may contribute to the effective future treatment of Hepatocellular Carcinoma. Ying-hua Lan and Yong-guo Li contributed equally to this work.