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Infectious diseases, along with cancers, are among the main causes of death among humans worldwide. The production of therapeutic proteins for treating diseases at large scale for millions of individuals is one of the essential needs of mankind. Recent progress in the area of recombinant DNA technologies has paved the way to producing recombinant proteins that can be used as therapeutics, vaccines, and diagnostic reagents. Recombinant proteins for these applications are mainly produced using prokaryotic and eukaryotic expression host systems such as mammalian cells, bacteria, yeast, insect cells, and transgenic plants at laboratory scale as well as in large-scale settings. The development of efficient bioprocessing strategies is crucial for industrial production of recombinant proteins of therapeutic and prophylactic importance. Recently, advances have been made in the various areas of bioprocessing and are being utilized to develop effective processes for producing recombinant proteins. These include the use of high-throughput devices for effective bioprocess optimization and of disposable systems, continuous upstream processing, continuous chromatography, integrated continuous bioprocessing, Quality by Design, and process analytical technologies to achieve quality product with higher yield. This review summarizes recent developments in the bioprocessing of recombinant proteins, including in various expression systems, bioprocess development, and the upstream and downstream processing of recombinant proteins.

Recombinant proteins are gaining enormous importance these days due to their wide application as biopharmaceutical products and proven safety record. Various recombinant proteins of therapeutic and prophylactic importance have been successfully produced in microbial and higher expression host systems. Since there is no specific antiviral therapy available against dengue, the prevention by vaccination is the mainstay in reducing the disease burden. Therefore, efficacious vaccines are needed to control the spread of dengue worldwide. Dengue is an emerging viral disease caused by any of dengue virus 1-4 serotypes that affects the human population around the globe. Dengue virus is a single stranded RNA virus encoding three structural proteins (capsid protein, pre-membrane protein, and envelope protein) and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). As the only licensed dengue vaccine (Dengvaxia) is unable to confer balanced protection against all the serotypes, therefore various approaches for development of dengue vaccines including tetravalent live attenuated, inactivated, plasmid DNA, virus-vectored, virus-like particles, and recombinant subunit vaccines are being explored. These candidates are at different stages of vaccine development and have their own merits and demerits. The promising subunit vaccines are mainly based on envelope or its domain and non-structural proteins of dengue virus. These proteins have been produced in different hosts and are being investigated for development of a successful dengue vaccine. Novel immunogens have been designed employing various strategies like protein engineering and fusion of antigen with various immunostimulatory motif to work as self-adjuvant. Moreover, recombinant proteins can be formulated with novel adjuvants to enhance the immunogenicity and thus conferring better protection to the vaccinees. With the advent of newer and safer host systems, these recombinant proteins can be produced in a cost effective manner at large scale for vaccine studies. In this review, we summarize recent developments in recombinant protein based dengue vaccines that could lead to a good number of efficacious vaccine candidates for future human use and ultimately alternative dengue vaccine candidates.

Technological advancements in the field of chemical threat have made it possible to create extremely dangerous chemical warfare agents (CWA). Hence, the effective protection of personnel is very important in a chemical warfare scenario amidst the current climate of terrorism awareness. In particular, body protection plays a substantial role in the chemical defence considering the urgency of situation in the nuclear, biological and chemical environment. Activated carbon spheres (ACS) based permeable chemical protective clothing (coverall) was developed for protection against CWA. The adsorbent material i.e, ACS used in this protective clothing provided higher adsorption capacity (1029 mg/g in terms of iodine) and low thermal burden (34 °C WBGT index) compared to earlier indigenously developed NBC suit. This article focuses on the extensive evaluation of chemical protective clothing against sulfur mustard (HD), a CWA. The results revealed that the developed protective clothing provided more than 24 h protection against HD. This chemical protective suit is light weight (< 2.75 kg for XL size). It also has higher air permeability (> 30 cm3/s/cm2) as well as less water vapour resistance (< 9.6 m2Pa/W). With continued innovations in materials and attention to key challenges it is expected that advanced, multifunction chemical protective suit will play a pivotal role in the CWA protection scenario.

The spread of dengue fever depends mainly on the availability of favourable breeding sites for its mosquito vectors around human dwellings. To investigate if the various factors influencing breeding habitats can be mapped from space, dengue indices, such as the container index, the house index and the Breteau index, were calculated from Ministry of Public health data collected three times annually in Phitsanulok, Thailand between 2009 and 2011. The most influential factors were found to be temperature, humidity, rainfall, population density, elevation and land cover. Models were worked out using parameters mostly derived from freely available satellite images and fuzzy logic software with parameter synchronisation and a predication algorithm based on data mining and the Decision Tree method. The models developed were found to be sufficiently flexible to accommodate additional parameters and sampling data that might improve prediction of favourable breeding hotspots. The algorithm applied can not only be used for the prediction of near real-time scenarios with respect to dengue, but can also be applied for monitoring other diseases influenced by environmental and climatic factors. The multi-criteria model presented is a cost-effective way of identifying outbreak hotspots and early warning systems lend themselves for development based on this strategy. The proposed approach demonstrates the successful utilisation of remotely sensed images to map mosquito breeding habitats.

Activated carbon fabric or fiber (ACF) is a novel carbonaceous material with exceptionally high adsorption rate and larger adsorption capacity, that has emerged as a rising star in the field of adsorbents. ACF has many advantages over other commercial porous storage materials such as granular activated carbon and powdered activated carbon in terms of adsorption capacity, well defined microporous structure, stability, flexibility and ease of lamination to various substrates. In the last few years, activated carbon fabrics have gained greater choice of interest for use as an adsorbent material in several fields including nuclear, biological and chemical (NBC) protection suit. Viscose rayon, acetate, polyacrylonitrile, pitch, and phenolic based materials are mainly used as precursors for preparation of ACF. ACF or fibres are generally prepared by process comprising stabilisation, carbonisation and activation of precursors. Reviews recent advances and developments in the field of ACF and their utility as an adsorbent material in various fields including NBC scenario. ACF with unmatchable pore structure and surface characteristics at present, with continued innovations and attention to its key challenges, it is expected that ACF will play a pivotal role in diverse environmental, defence, and civil applications.

Chikungunya virus has emerged as one of the most important global arboviral threats over the last decade. Inspite of large scale morbidity, with long lasting polyarthralgia, so far no licensed vaccine or antiviral is available. CHIKV nsP2 protease is crucial for processing of viral nonstructural polypeptide precursor to release enzymes required for viral replication, thus making it a promising drug target. In this study, high cell density cultivation (HCDC) of Escherichia coli in batch process was carried out to produce rCHIKV nsP2pro in a cost-effective manner. The purified nsP2pro and fluorogenic peptide substrate have been adapted for fluorescence resonance energy transfer (FRET) based high throughput screening (HTS) assay with Z’ value and CV of 0.67 ± 0.054 and <10% respectively. We used this cell free HTS system to screen panel of metal ions and its conjugate which revealed zinc acetate as a potential candidate, which was further found to inhibit CHIKV in Vero cells. Scale-up process has not been previously reported for any of the arboviral nonstructural enzymes. The successful scale-up method for viral protease together with a HTS assay could lead to the development of industrial level large-scale screening platform for identification of protease inhibitors against emerging and re-emerging viruses.

Chikungunya, a mosquito-borne viral disease caused by Chikungunya virus (CHIKV), has drawn substantial attention after its reemergence causing massive outbreaks in tropical regions of Asia and Africa. The recombinant envelope 2 (rE2) protein of CHIKV is a potential diagnostic as well as vaccine candidate. Development of cost-effective cultivation media and appropriate culture conditions are generally favorable for large-scale production of recombinant proteins in Escherichia coli. The effects of medium composition and cultivation conditions on the production of recombinant Chikungunya virus E2 (rCHIKV E2) protein were investigated in shake flask culture as well as batch cultivation of Escherichia coli. Further, the fed-batch process was also carried out for high cell density cultivation of E. coli expressing rE2 protein. Expression of rCHIKV E2 protein in E. coli was induced with 1 mM isopropyl-beta-thiogalactoside (IPTG) at ~23 g dry cell weight (DCW) per liter of culture and yielded an insoluble protein aggregating to form inclusion bodies. The final DCW after fed-batch cultivation was ~35 g/l. The inclusion bodies were isolated, solubilized in 8 M urea and purified through affinity chromatography to give a final product yield of ~190 mg/l. The reactivity of purified E2 protein was confirmed by Western blotting and enzyme-linked immunosorbent assay. These results show that rE2 protein of CHIKV may be used as a diagnostic reagent or for further prophylactic studies. This approach of producing rE2 protein in E. coli with high yield may also offer a promising method for production of other viral recombinant proteins.

Japanese encephalitis virus (JEV) is the most important cause of encephalitis in most Asian regions. JEV envelope domain III (JEV EDIII) protein is involved in binding to host receptors, and it contains specific epitopes that elicit virus-neutralizing antibodies. A highly immunogenic, recombinant JEV EDIII protein was expressed in Escherichia coli. In order to take this vaccine candidate for further studies, recombinant JEV EDIII protein was produced employing a pilot-scale fermentation process. Recombinant JEV EDIII protein expressed as inclusion bodies (IBs) was solubilized in 8 M urea and renatured by on-column refolding protocol in the presence of glycerol. A three-step purification process comprising of affinity chromatography, ion-exchange chromatography (IEX) based on salt, and IEX based on pH was developed. About ~124 mg of highly purified and biologically active EDIII protein was obtained from 100 g of biomass. Biological function of the purified EDIII protein was confirmed by their ability to generate EDIII-specific antibodies in mice that could neutralize the virus. These findings suggest that recombinant JEV EDIII protein in combination with compatible adjuvant is highly immunogenic and elicit high-titer neutralizing antibodies. Thus, recombinant JEV EDIII protein produced at large scale can be a potential vaccine candidate.

Japanese encephalitis (JE) is one of the leading causes of acute encephalopathy affecting children and adolescents in the tropics. Optimization of media was carried out for enhanced production of recombinant JE virus envelope domain III (EDIII) protein in Escherichia coli. Furthermore, batch and fed-batch cultivation process in E. coli was also developed in optimized medium. Expression of this protein in E. coli was induced with 1 mM isopropyl-β-thiogalactoside and yielded an insoluble protein aggregating to form inclusion bodies. The inclusion bodies were solubilized in 8 M urea, and the protein was purified under denaturing conditions using Ni-NTA affinity chromatography. After fed-batch cultivation, the recombinant E. coli resulted in cell dry weight and purified protein about 36.45 g l⁻¹ and 720 mg l⁻¹ of culture, respectively. The purity of the recombinant JE virus EDIII protein was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, and reactivity of this protein was determined by Western blotting and ELISA with JE virus-infected human serum samples. These results establish the application of this protein to be used for the diagnosis of JE virus infection or for further studies in vaccine development. This process may also be suitable for the high-yield production of other recombinant viral proteins.

Summary Japanese encephalitis is a major public health problem in South‐East Asia and Western Pacific countries. The recombinant nonstructural 1 (rNS1) protein of Japanese encephalitis virus is a potential diagnostic as well as vaccine candidate. Developments of cost‐effective and simple culture media as well as appropriate culture conditions are generally favourable for large‐scale production of recombinant proteins. The effects of medium composition and cultivation conditions on the production of rNS1 protein were investigated in shake flask culture as well as batch cultivation of Escherichia coli. Further, the fed‐batch process was also carried out for high cell density cultivation (HCDC) of E. coli expressing rNS1 protein. Isopropyl‐β‐d‐thiogalactopyranoside (IPTG) was used to induce the expression of rNS1 protein at ∼ 13 g dry cell weight per litre of culture. The final dry cell weight after fed‐batch cultivation was ∼ 17 g l−1. The Inclusion bodies were isolated and purified through affinity chromatography to give a final product yield of ∼ 142 mg l−1. The reactivity of purified protein was confirmed by Western blotting and Enzyme linked immunosorbent assay. These results show that rNS1 protein may be used as a diagnostic reagent or for further prophylactic studies. This approach of producing rNS1 protein in E. coli with high yield may also offer promising method for production of other viral recombinant proteins.