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Enhancing production of the malaria asexual blood-stage vaccine candidate PfRipr5 in insect cells by modulating expression vector and culture temperature
Get PDFDespite 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
Long-term propagation of influenza A virus and its defective interfering particles: Analyzing dynamic competition to select antiviral candidates
Get PDFInfluenza A virus (IAV) is a major threat to human health. A potential antiviral therapy against influenza disease could be the intranasal application of defective interfering particles (DIPs) [1,2]. During intracellular IAV replication, these IAV DIPs are randomly generated. They contain at least one defective interfering (DI) RNA, typically a genome segment with a large internal deletion, rendering them replication-incompetent. During co-infection with infectious standard virus (STV), DIPs impede STV replication via a growth advantage [3] and by stimulation of the innate immune response [4]. In this work, we profiled the propagation competition between a variety of DIPs for selection toward accumulation of highly interfering DIPs in Madin-Darby canine kidney (MDCK) suspension cell culture.
To that end, we propagated IAV and its DIPs over 21 days using a small-scale two-stage cultivation system. Illumina-based next-generation sequencing (NGS) in combination with a lately reported bioinformatic pipeline was utilized to detect and quantify specific deletion junctions within the virus population [5]. During long-term propagation, both the infectious and total virus titers oscillated periodically, a characteristic pattern of DIP and STV interplay [6]. NGS analysis revealed that the highest de novo formation and accumulation of DI vRNAs occurred on the polymerase-encoding segments. Moreover, we identified an accumulation of short DI vRNAs with an optimum fragment length underlining their replication advantage. Deletion junctions were usually situated near both vRNA ends. For efficient DI vRNA propagation, retaining the incorporation signal but not the entire bundling signal was required. Few DI vRNAs propagated to high levels toward the end of cultivation, while the level of others declined. Reverse genetics was utilized to generate purely clonal DIPs derived from DI vRNAs of segment 1 that showed the highest increase in accumulation during cultivation. For this, genetically engineered adherent and suspension MDCK cells complementing the defect in segment 1 were employed [7,2]. During co-infection with STV, these DIPs displayed a higher interfering efficacy relative to DIPs derived from less competitive DI vRNAs.
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Overviews of vaccine technology for neglected diseases in developing Latin America country - Peru
Get PDFImmunization has been the greatest strategy to prevent sickness and death associated with infectious diseases [1]. So, vaccine technology has evolved to produce more immunogenic vaccines and to avoid the exposure to disease-causing organisms during manufacturing and treatment [2]. Conversely, 2.5 million people are still dying globally each year from vaccine-preventable diseases since the underuse of vaccines and lack vaccines against non-well studied pathogens [2]. Only Cuba, Brazil and Argentina are listed as producers of vaccines against infectious diseases such as yellow fever, leishmaniasis and rotavirus [3]. The roles of mentioned developing countries are crucial because the produced vaccines are economically affordable which increase an increment of vaccine coverage [3]. However, neglected communities are still susceptible to pathogens that are non-well documented since they do not represent a high risk for public health. In Peru, several deaths are associated with the infection of bacteria borne diseases and along to the last decades and preventative treatment is not yet available. It has been developed some research aimed to develop a vaccine against Carrion’s disease in The National Institute of Health from Peru. The employed procedure to produce the vaccine against Carrion’s disease has not described the parameters used for the purification and characterization to obtain a highly pure molecule for animal trial, consequently, the unexpected results could be obtained because the vaccine candidate did not achieve the basic standard for the evaluation [4]. In conclusion, there are few the developing Latin American countries involved in the vaccine technology field, and it is required to integrate effort among global institutions to face diseases that affect neglected communities in developing countries.
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Use of a parallel bioreactor scaledown system for optimisation of a perfusion-based upstream process for adenovirus production
Get PDFThe recent pandemic emphasises the need for vaccine producers to be able to respond rapidly to the need for large quantities for global distribution. Here, we report work to optimise a perfusion-based upstream approach. Perfusion can enhance volumetric productivity of adenoviral vectors, but the complexity of perfusion culture and the lack of suitable scale-down models has hindered work to establish the complex relationships between variables affecting the process.
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Rotavirus VP6 nanotubes show an antigen form-dependent adjuvant activity: Zika virus envelope protein monomer vs Zika virus-like particles
Get PDFThe structural protein of rotavirus (RV) VP6 can self-assemble into tubular polymeric structures under specific conditions of pH and ionic strength when expressed in recombinant systems. Previous studies have shown that RV VP6 nanotubes (VP6NT) have an adjuvant effect on the immunogenicity of norovirus virus-like particles (VLPs) in mice (Blazevic et al., 2011; Malm et al., 2016). The present study focused on the determination of adjuvant activity of VP6NT on the immunogenicity of monomers of the viral envelope (E) protein or of Zika virus (ZikV) VLPs. ZikV infection can cause congenital malformations in fetuses and the Guillain-Barré syndrome in adults, as the most severe consequences. To date, there is no treatment or vaccine available against ZikV. Several vaccine candidates against this virus have been reported and E protein has been selected as the primary antigenic determinant.
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Advancing vaccine development and manufacture in Africa
Get PDFEmerging and known infectious diseases pose a constant threat to the health and prosperity of Africa and its people. Africa, like other regions of the world with a high burden of vaccine preventable diseases, has benefited immensely from vaccines and Africa’s growing population means an increase in the continuing need for vaccines. By 2050, Africa’s population will be 2.5 billion and one in four people in the world will be African.
However, Africa has limited capacity to produce vaccines, yet is the most in need of routine vaccines. Currently less than 1% of vaccines used in Africa is made in Africa while Unicef supplies more than 60% of its global demand for vaccines to Africa.
Dependency on external vaccines supply has rendered Africa vulnerable in epidemics and pandemics, and poorly prepared to respond adequately to emergency situations during disease outbreaks.
Local vaccine development and production in Africa is therefore essential to support Africa’s growing needs and specific disease burdens as well as boost socio-economic development and the development of skills in biotechnology, and positively impact industrial and life science development.
However, building local capacity would require the right level of political and technical support, including a clear, well-funded and coherent regional policymaking and planning approach, allowing development of the necessary ecosystem to establish a viable, competitive and sustainable vaccine manufacturing capability.
An overview of the work of the African Vaccine Manufacturing Initiative (AVMI) and its mission of advancing vaccine development and manufacturing capacity in Africa will be provided. Collaborative efforts with partners, progress to date and the need for an African vaccine manufacturing policy will be discussed.
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CuAAC for inorganic preceramic polymer synthesis
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Diffusional and microstructural profiles in metallic-to-UHTC conversion by carbonization
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Phase evolution in thermally annealed metallic-UHTC composites
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