A high-throughput approach to vaccine bioprocess development

Vaccination is currently the predominant tool in the prevention of infectious disease. Each year, an estimated 2-3 million lives are saved worldwide and infant mortality has been significantly reduced. Despite substantial recent advances, vaccine manufacturing can still be laborious owing to difficulties in development, lengthy clinical trials, and stringent regulations. In light of the SARS-CoV-2 (Covid-19) pandemic, the need for a development platform which can rapidly screen potential candidates and/or a vaccine scaffold capable of adaptability to new disease targets has never been more apparent. To meet this need, the breadth of vaccine types under exploration has rapidly expanded. DNA and RNA vaccines offer the opportunity for rapid manufacture but can be poorly immunogenic, whilst subunit vaccines can require complex processing. Virus-like particles (VLPs) have the potential to address these two factors. Tandem Core VLPs, expressed in the methylotrophic yeast Pichia pastoris, are an exciting alternative to current manufacturing methods. They have excellent potential, both as standalone vaccines for the virus from which they are derived, or as scaffolds for the display of foreign antigens. The hepatitis B core antigen (HBC) can spontaneously self-assemble, forming icosahedral particles that are inherently immunogenic. Tandem Core HBC VLPs have been genetically modified in the major insertion region (MIR) enabling surface display of up two epitopes of interest when assembled. For HBC VLPs to be considered a viable vaccine candidate, their bioprocessing must be optimized. Currently, there are various issues to address including problems with formation, solubility and immunogenicity, which are often clone dependent. In this work, Tandem Core VLPs, consisting of genetically linked HBC monomers carrying different epitopes in the MIR will be used to develop a high-throughput platform and explore the impact of different inserts on VLP production and processing. Influenza will be used as a model pathogen owing to its persistence as a public health threat. The aim of this work is to develop a vaccine platform, defined by Adalja et al., (2019) as “a technology in which the underlying, nearly identical mechanism, device, delivery vector or cell line was employed for [design of] multiple target vaccines”. The equipment and methods developed in this work were considered to enable: (1) thorough investigation of three HBC VLP candidates in an attempt to identify a universal bioprocess, irrespective of surface displayed epitopes; (2) formation of a small-scale high throughput platform which could be implemented for rapid screening of new disease targets or to allow fine-tuning of processes for epitope-dependent optimisation. After initial studies using an ‘empty’ HBC VLP (HBC-K1,K1), the ambr®250 modular was used to investigate upstream bioprocessing of three influenza specific candidates (HBC -HA2,3M2E, -LAH3,K1 and -3M2E,K1), exploring different fermentation induction strategies and to identify epitope related differences. Following this, the most readily soluble candidate (HBC-LAH3,K1) was selected for further upstream optimisation combining ambr® 250 experimentation with statistical Design of Experiments (DoE). An improved process was identified enabling an increase in VLP titre, a 34% increase in biomass compared to the initial condition, and a 6% decrease in process time compared to methanol induction. This process was then applied to the production of the alternative VLP constructs. The improved feeding regime resulted in higher biomass and soluble HBC yield for all three VLPs. Subsequent downstream process studies on the primary recovery of VLP candidates was then necessary to account for the reduced volumes associated with miniaturised fermentation studies, and to bridge the gap between upstream processing and purification. Building on previous work, a high-throughput, small scale cell disruption method was investigated using Adaptive focused acoustics®. A 96-well plate workflow was demonstrated, enabling suitable VLP release and recovery with a ~99.7% reduction in sample volume, in comparison to high pressure homogenisation (HPH). Finally, chromatography screening was undertaken using high-throughput PreDictor® plates to rapidly identify separation conditions for the various vaccine candidates. Studies were conducted to investigate suitable resins and binding/elution conditions and to determine the influence of the physicochemical properties of the displayed epitopes on separation performance. Multiple resins were identified as being suitable for VLP purification, and results were useful to manipulate chromatographic separation (5mL column scale) conditions for the VLPs to achieve improved product yield and purity profiles. Overall, this research suggests that a high-throughput vaccine development platform can be realised through the integration of numerous small-scale single-use equipment, techniques and methodologies. Namely, the use of the ambr®250 bioreactors, AFA® cell disruption in 96-well plates and 96-well PreDictor™ resin plates. Combined with statistical DoE, this platform can be used to rapidly optimise production and purification conditions for novel vaccine technologies such as HBC Tandem Core VLPs. The improved bioprocessing of these constructs paves the way for future vaccine candidates which exploit HBC as a vaccine scaffold. These findings have implications for reducing the time taken to develop vaccine manufacturing processes and prepare for disease outbreaks based on ‘Pathogen X’

A high-throughput single-use platform for vaccine bioprocess development

Vaccination is the predominant tool in the prevention of infectious disease. Considering the SARS-CoV-2 (Covid-19) pandemic, the need for a development platform, capable of rapid candidate screening and/or a vaccine scaffold capable of adaptability to new disease targets, has never been more apparent. VLPs, expressed in the methylotrophic yeast Pichia pastoris, offer an exciting alternative to current manufacturing methods due to their potential as scaffolds for foreign antigen display. The hepatitis B core antigen (HBC) can spontaneously self-assemble, forming icosahedral particles that are inherently immunogenic. Tandem Core HBC (TC-HBC) VLPs have been genetically modified in the major insertion region (MIR) enabling display of up to two epitopes of interest when assembled. For TC-HBC VLPs to be considered a viable vaccine candidate, their bioprocessing must be optimized. Currently, there are various issues to address, including problems with formation, solubility, and immunogenicity. Please click Additional Files below to see the full abstract

The Role of Sonority in Jargonaphasia.

The Sonority Sequencing Principle suggests that the relative sonority rank among sounds can explain intrasyllabic and transsyllabic phoneme ordering patterns in normal speakers. The values of segments can be ranked from most to least sonority as follows: Vowels, Glides, Liquids, Nasals and Obstruents. The unmarked order of segments within an initial demisyllable is O-N-L-G from the periphery toward the peak, and G-L-N-O in a final demisyllable from the peak toward the periphery. The sonority slope of an initial demisyllable will be steep to maximize the perceptual saliency of syllable onset. In a final demisyllable, especially if embedded, the slope will be flatter to maintain sonority contrast between abutting syllables. This study answered the following questions: (1) What syllable shape and sonority profile patterns (in four types of demisyllables) are present in the neologistic and legitimate English utterances of three fluent aphasics, (2) Are these patterns similar to those observed in the well-formed utterances of normal speakers, (3) Does the sonority principle facilitate neologism analysis, (4) Can sonority be incorporated further into models of sentence production and, (5) What implications for sonority theory and for theories of neology are suggested by results. Data from three neologistic jargonaphasics were audio-recorded during expressive language tasks. Neologisms were phonemically transcribed by three independent listeners. A demisyllable data base for target-related neologisms, abstruse neologisms, and English words was compiled for each subject. Summary frequency distributions for demisyllable shapes and sonority profiles were obtained and tested for each word type. Results from extensive demisyllable analyses suggested the following: (1) demisyllable shapes for neologisms and English words were most often of the form CV or CVC, (2) intrasyllabic and transsyllabic sonority profiles in neologisms and English words were most frequently of the preferred patterns, although some exceptions were noted, (3) demisyllable shapes and sequence preferences were similar to patterns found in legitimate English words and, (4) sonority may constrain the operation of mechanisms that create neologisms, whether viewed from a serial or parallel model of language production. Results suggest that sonority may partially govern construction of normal and impaired phonological forms

Virus-like particles: A flexible platform for universal influenza vaccine development

Human influenza remains a global public health threat, namely due to its evolutionary adaptability, which hinders effective prevention. Vaccination is currently the predominant tool in the prevention of infectious disease. However, current production methods for influenza vaccines are not only logistically inadequate in the face of a pandemic, but also rely on targeting two surface proteins on the influenza virus, which are prone to antigenic drift. As a consequence, a new vaccine needs to be developed for each new seasonal epidemic. Additionally, the vaccine strain needs to be selected around eight months prior to administration and can often be mismatched leaving the population unprotected. A ‘universal’ vaccine, effective irrespective of the surface proteins, would be desirable to offer cross-protectivity across strains. Tandem core virus-like particles (VLPs), expressed in methylotrophic yeast Pichia pastoris, are an exciting alternative to current manufacturing methods. VLPs, due to their inherent safety profile and advances in genetic engineering, have excellent potential both as standalone vaccines for the virus from which they are derived, or as platforms for the display of foreign antigens. The hepatitis B core antigen (HBcAg) is able to spontaneously self-assemble, forming icosahedral particles that are inherently immunogenic. Moreover, the HBcAg is capable of carrying antigen inserts in the major insertion region (MIR) which are displayed on the particle surface. In order for VLPs to be considered a viable alternative, their bioprocessing must be optimized. Currently, various issues are at play including problems with formation, solubility and immunogenicity, often clone dependent. In this work, two genetically linked HBcAg monomers, carrying different inserts in the MIR, were used to study the effects on fermentation efficiencies using two different induction strategies. Rationalizing an induction strategy would enable the development of an efficient process to produce and purify VLPs. Results indicate that increased biomass is not always synonymous with increased protein expression. Moreover, protein expression and solubility appear to be linked with the complexity of the inserts displayed on the VLP surface. The aim of this work is to improve the bioprocessing of VLPs in a microbial expression system, using tandem core technology. This proposed method is cheap and rapidly scalable, reduces the cost per dose and eliminates the long production timelines associated with current manufacturing. The very nature of VLPs and the comparable ease of production would enable this to be promoted as a platform process, for a myriad of disease targets

Ethnic minority livelihoods contesting state visions of 'ideal farmers' in Vietnam's northern borderlands

Since the 1990s, several Vietnamese state policies have focused on whole-heartedly integrating upland ethnic minority farmers into the market economy. These policies revolve around interventions related to natural resource use, agricultural intensification, and cash-cropping, in a quest to produce 'ideal farmers.' Simultaneously, the growing frequency of extreme weather extremes has been impacting upland livelihoods in important ways. Consequently, farmers must now navigate an increasingly complex socio-political and natural environment when making livelihood decisions. This study focuses on a mountainous district in the Sino-Vietnamese borderlands. Through in-depth qualitative fieldwork with ethnic minority semi-subsistence farmers and local officials, we delve into the ways in which farmers respond to the unpredictable interplay of state interventions and extreme weather events. Rooted in contemporary political ecology debates, we adopt a multi-scalar approach while drawing on actor-oriented livelihood conceptualizations. Our findings show that the Vietnamese state has failed to convince upland farmers to fully commit to state-endorsed cash-cropping schemes. Yet, farmers do not necessarily reject such opportunities outright. Rather, they navigate and rework state-supported opportunities, all while remaining acutely attuned to local physical environment limits, important social networks, and cultural norms and expectations

From flask to large scale high cell density production of ω-transaminase using auto- induction media

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Caseworker-Perceived Caregiver Substance Abuse and Child Protective Services Outcomes

The authors used data from the National Survey of Child and Adolescent Well-Being to examine associations of child protective services (CPS) caseworkers' perceptions of caregiver substance abuse with their perceptions of the severity of risk and harm a child experienced as a result of alleged maltreatment, as well as with whether a family experienced a range of CPS outcomes. The outcomes included whether the family received services from CPS, was substantiated for maltreatment, experienced child removal, and was subject to a termination of parental rights (TPR) petition. The authors also compared the magnitude of the association between caseworker-perceived caregiver substance abuse and each outcome to that of the association between other maltreatment-related risk factors and each outcome. Findings suggest that, all else equal, caseworker-perceived caregiver substance abuse is associated with increased caseworker perceptions that children have experienced severe risk and harm and also with an increased probability of each of the CPS outcomes except TPR. Moreover, these associations are equal in magnitude or larger than those between the other risk factors and the outcomes. These findings imply that CPS decisions are heavily influenced by caseworker perceptions of caregiver substance abuse, regardless of the presence of other risk factors for child maltreatment

Families Coping With Financial Loss Following the Deepwater Horizon Oil Spill

Objective: This study examines family strategies for coping and adaptation to social disruption from the 2010 Deepwater Horizon oil spill (DHOS) in south Louisiana. Background: The DHOS is a technological disaster of unprecedented scale and ongoing impact, including the socioeconomic disruption of families. Method: Using data from focus groups, grounded‐theory methods informed a thematic analysis of spill‐related economic loss and coping mechanisms among families in the spill‐affected region. Results: Key findings were as follows: (a) long‐term economic impacts persisted but were nuanced and differed across places; (b) for families living in multistressor environments, concerns about the DHOS spilled over into other aspects of social functioning and became enmeshed with perceptions of other environmental stressors; and (c) economic exposure after the DHOS affected families differently based on social position and community social structure. Conclusion: This study contributes to existing knowledge on technological disaster and family resilience in the face of environmental shocks and stressors, underscoring the utility of the conservation of resources model of stress in this area of research. Implications: This research offers information about family‐level response to oil spill impacts and may be of interest to policymakers and practitioners who work to support resilience in disaster contexts

Effects of the nicotinic agonist varenicline, nicotinic antagonist r-bPiDI, and DAT inhibitor R-modafinil on co-use of ethanol and nicotine in female P rats.

Rationale: Co-users of alcohol and nicotine are the largest group of polysubstance users worldwide. Commonalities in mechanisms of action for ethanol (EtOH) and nicotine proposes the possibility of developing a single pharmacotherapeutic to treat co-use. Objectives: Toward developing a preclinical model of co-use, female alcohol-preferring (P) rats were trained for voluntary EtOH drinking and i.v. nicotine self-administration in three phases: (1) EtOH alone (0 vs. 15%, 2-bottle choice); (2) nicotine alone (0.03 mg/kg/infusion, active vs. inactive lever); and (3) concurrent access to both EtOH and nicotine. Using this model, we examined the effects of (1) varenicline, a nicotinic acetylcholine receptor (nAChR) partial agonist with high affinity for the α4β2 subtype; (2) r-bPiDI, a subtype-selective antagonist at α6β2* nAChRs; and (3) (R)-modafinil, an atypical inhibitor of the dopamine transporter (DAT). Results: In Phases 1 and 2, pharmacologically relevant intake of EtOH and nicotine was achieved. In the concurrent access phase (Phase 3), EtOH consumption decreased while nicotine intake increased relative to Phases 1 and 2. For drug pretreatments, in the EtOH access phase (Phase 1), (R)-modafinil (100 mg/kg) decreased EtOH consumption, with no effect on water consumption. In the concurrent access phase, varenicline (3 mg/kg), r-bPiDI (20 mg/kg), and (R)-modafinil (100 mg/kg) decreased nicotine self-administration, but did not alter EtOH consumption, water consumption, or inactive lever pressing. Conclusions: These results indicate that therapeutics which may be useful for smoking cessation via selective inhibition of α4β2 or α6β2* nAChRs, or DAT inhibition, may not be sufficient to treat EtOH and nicotine co-use

Pseudotyping of VSV with Ebola virus glycoprotein is superior to HIV-1 for the assessment of neutralising antibodies

Ebola virus (EBOV) is an enveloped, single-stranded RNA virus that can cause Ebola virus disease (EVD). It is thought that EVD survivors are protected against subsequent infection with EBOV and that neutralising antibodies to the viral surface glycoprotein (GP) are potential correlates of protection. Serological studies are vital to assess neutralising antibodies targeted to EBOV GP; however, handling of EBOV is limited to containment level 4 laboratories. Pseudotyped viruses can be used as alternatives to live viruses, which require high levels of bio-containment, in serological and viral entry assays. However, neutralisation capacity can differ among pseudotyped virus platforms. We evaluated the suitability of EBOV GP pseudotyped human immunodeficiency virus type 1 (HIV-1) and vesicular stomatitis virus (VSV) to measure the neutralising ability of plasma from EVD survivors, when compared to results from a live EBOV neutralisation assay. The sensitivity, specificity and correlation with live EBOV neutralisation were greater for the VSV-based pseudotyped virus system, which is particularly important when evaluating EBOV vaccine responses and immuno-therapeutics. Therefore, the EBOV GP pseudotyped VSV neutralisation assay reported here could be used to provide a better understanding of the putative correlates of protection against EBOV