Production of a high purity, C-tagged hepatitis B surface antigen fusion protein VLP vaccine for malaria expressed in Pichia pastoris under cGMP conditions.

Virus-like particles (VLPs) induce strong humoral and cellular responses and have formed the basis of some currently licensed vaccines. Here, we present the method used for the production of R21, a VLP-based anti-sporozoite malaria vaccine, under current Clinical Good Manufacturing Practice regulations (cGMP). Previous preclinical studies in BALB/c mice showed that R21 produced almost complete protection against sporozoite challenge with transgenic Plasmodium berghei parasites. Here, we have modified the preclinical production process to enable the production of sufficient quantities of highly pure, clinical-grade material for use in human clinical trials. The R21 construct was re-engineered to include a C-tag to allow affinity-based separation from the major contaminant alcohol oxidase 1 (AOX 1, ~74 kDa). To our knowledge, this is the first use of C-tag technology to purify a VLP vaccine candidate for use in human clinical trials. The R21 vaccine has shown high-level efficacy in an African Phase IIb trial, and multiple clinical trials are underway to assess the safety and efficacy of the vaccine. Our findings support the future use of C-tag platform technologies to enable cGMP-compliant biomanufacturing of high purity yeast-expressed VLP-based vaccines for early phase clinical trials when clinical grade material is required in smaller quantities in a quick time frame.

The Human Adenovirus Type 5 L4 Promoter Is Negatively Regulated by TFII-I and L4-33K.

UNLABELLED: The late phase of adenovirus gene expression is controlled by proteins made in the intermediate phase, including L4 proteins of 22,000- and 33,000-Da apparent molecular mass (L4-22K and -33K proteins) that are expressed initially from the L4 promoter (L4P). The L4P is activated by a combination of viral proteins and cellular p53 and is ultimately inhibited again by its own products. Here, we have examined the L4P of human adenovirus type 5 in detail and have defined its transcription start site, which our data suggest is positioned by a weak TATA box. Rather than contributing positively to promoter activity, a putative initiator element at the transcription start site acts as a target for negative regulation imposed on the L4P by cellular TFII-I. We show that this TFII-I inhibition is relieved by one of the previously defined viral activators of the L4P, the E4 Orf3 protein, which alters the pool of TFII-I in the cell. We also explore further the negative regulation of the L4P by its products and show that the L4-33K protein is more significant in this process than L4-22K. It is the combined actions of positive and negative factors that lead to the transient activation of the L4P at the onset of the late phase of adenovirus gene expression. IMPORTANCE: The adenovirus replication cycle proceeds through multiple phases of gene expression in which a key step is the activation of late-phase gene expression to produce proteins from which progeny particles can be formed. Working with human adenovirus type 5, we showed previously that two proteins expressed from the L4 region of the viral genome perform essential roles in moving the infection on into the late phase; these two proteins are produced by the action of a dedicated promoter, the L4P, and without them the infection does not proceed successfully to progeny generation. In this new work, we delineate further aspects of L4P activity and regulation. Understanding how the L4P works, and how it contributes to activation of the late phase of infection, is important to our understanding of natural infections by the virus, in which late gene expression can fail to occur, allowing the virus to persist.

Preventing spontaneous genetic rearrangements in the transgene cassettes of adenovirus vectors.

First-generation, E1/E3-deleted adenoviral vectors with diverse transgenes are produced routinely in laboratories worldwide for development of novel prophylactics and therapies for a variety of applications, including candidate vaccines against important infectious diseases, such as HIV/AIDS, tuberculosis, and malaria. Here, we show, for two different transgenes (both encoding malarial antigens) inserted at the E1 locus, that rare viruses containing a transgene-inactivating mutation exhibit a selective growth advantage during propagation in E1-complementing HEK293 cells, such that they rapidly become the major or sole species in the viral population. For one of these transgenes, we demonstrate that viral yield and cytopathic effect are enhanced by repression of transgene expression in the producer cell line, using the tetracycline repressor system. In addition to these transgene-inactivating mutations, one of which occurred during propagation of the pre-viral genomic clone in bacteria, and the other after viral reconstitution in HEK293 cells, we describe two other types of mutation, a small deletion and a gross rearranging duplication, in one of the transgenes studied. These were of uncertain origin, and the effects on transgene expression and viral growth were not fully characterized. We demonstrate that, together with minor protocol modifications, repression of transgene expression in HEK293 cells during viral propagation enables production of a genetically stable chimpanzee adenovirus vector expressing a malarial antigen which had previously been impossible to derive. These results have important implications for basic and pre-clinical studies using adenoviral vectors and for derivation of adenoviral vector products destined for large-scale amplification during biomanufacture.

Adenovirus late-phase infection is controlled by a novel L4 promoter.

During human adenovirus 5 infection, a temporal cascade of gene expression leads ultimately to the production of large amounts of the proteins needed to construct progeny virions. However, the mechanism for the activation of the major late gene that encodes these viral structural proteins has not been well understood. We show here that two key positive regulators of the major late gene, L4-22K and L4-33K, previously thought to be expressed under the control of the major late promoter itself, initially are expressed from a novel promoter that is embedded within the major late gene and dedicated to their expression. This L4 promoter is required for late gene expression and is activated by a combination of viral protein activators produced during the infection, including E1A, E4 Orf3, and the intermediate-phase protein IVa2, and also by viral genome replication. This new understanding redraws the long-established view of how adenoviral gene expression patterns are controlled and offers new ways to manipulate that gene expression cascade for adenovirus vector applications.

Generation of cell lines to complement adenovirus vectors using recombination-mediated cassette exchange.

BACKGROUND: Adenovirus serotype 5 (Ad5) has many favourable characteristics for development as a gene therapy vector. However, the utility of current Ad5 vectors is limited by transient transgene expression, toxicity and immunogenicity. The most promising form of vector is the high capacity type, which is deleted for all viral genes. However, these vectors can only be produced to relatively low titres and with the aid of helper virus. Therefore a continuing challenge is the generation of more effective Ad5 vectors that can still be grown to high titres. Our approach is to generate complementing cell lines to support the growth of Ad5 vectors with novel late gene deficiencies. RESULTS: We have used LoxP/Cre recombination mediated cassette exchange (RMCE) to generate cell lines expressing Ad5 proteins encoded by the L4 region of the genome, the products of which play a pivotal role in the expression of Ad5 structural proteins. A panel of LoxP parent 293 cell lines was generated, each containing a GFP expression cassette under the control of a tetracycline-regulated promoter inserted at a random genome location; the cassette also contained a LoxP site between the promoter and GFP sequence. Clones displayed a variety of patterns of regulation, stability and level of GFP expression. Clone A1 was identified as a suitable parent for creation of inducible cell lines because of the tight inducibility and stability of its GFP expression. Using LoxP-targeted, Cre recombinase-mediated insertion of an L4 cassette to displace GFP from the regulated promoter in this parent clone, cell line A1-L4 was generated. This cell line expressed L4 100K, 22K and 33K proteins at levels sufficient to complement L4-33K mutant and L4-deleted viruses. CONCLUSIONS: RMCE provides a method for rapid generation of Ad5 complementing cell lines from a pre-selected parental cell line, chosen for its desirable transgene expression characteristics. Parent cell lines can be selected for high or low gene expression, and for tight regulation, allowing viral protein expression to mirror that found during infection. Cell lines derived from a single parent will allow the growth of different vectors to be assessed without the complication of varying complementing protein expression.

Adenovirus serotype 5 L4-22K and L4-33K proteins have distinct functions in regulating late gene expression.

Adenoviruses express up to 20 distinct mRNAs from five major late transcription unit (MLTU) regions, L1 to L5, by differential splicing and polyadenylation of the primary transcript. MLTU expression is regulated at transcriptional and posttranscriptional levels. The L4-33K protein acts as a splicing factor to upregulate several MLTU splice acceptor sites as the late phase progresses. The L4 region also expresses a 22K protein whose sequence is related to the sequence of L4-33K. L4-22K is shown here also to have an important role in regulating the pattern of MLTU gene expression. An adenovirus genome containing a stop codon in the L4-22K open reading frame expressed low levels of both structural and nonstructural late proteins compared to the wild-type (wt) adenovirus genome; a decrease in intermediate proteins, IVa2 and IX, was also observed. However, early protein synthesis and replication were unaffected by the absence of L4-22K. Intermediate and late protein expression was restored to wt levels by L4-22K expressed in trans but not by L4-33K. Increased MLTU promoter activity, resulting from stabilization of the transcriptional activator IVa2 by L4-22K, made a small contribution to this restoration of late gene expression. However, the principal effect of L4-22K was on the processing of MLTU RNA into specific cytoplasmic mRNA. L4-22K selectively increased expression of penton mRNA and protein, whereas splicing to create penton mRNA is known not to be increased by L4-33K. These results indicate that L4-22K plays a key role in the early-late switch in MLTU expression, additional to and distinct from the role of L4-33K.

Simian adenovirus vector production for early-phase clinical trials: A simple method applicable to multiple serotypes and using entirely disposable product-contact components.

A variety of Good Manufacturing Practice (GMP) compliant processes have been reported for production of non-replicating adenovirus vectors, but important challenges remain. Most clinical development of adenovirus vectors now uses simian adenoviruses or rare human serotypes, whereas reported manufacturing processes mainly use serotypes such as AdHu5 which are of questionable relevance for clinical vaccine development. Many clinically relevant vaccine transgenes interfere with adenovirus replication, whereas most reported process development uses selected antigens or even model transgenes such as fluorescent proteins which cause little such interference. Processes are typically developed for a single adenovirus serotype - transgene combination, requiring extensive further optimization for each new vaccine. There is a need for rapid production platforms for small GMP batches of non-replicating adenovirus vectors for early-phase vaccine trials, particularly in preparation for response to emerging pathogen outbreaks. Such platforms must be robust to variation in the transgene, and ideally also capable of producing adenoviruses of more than one serotype. It is also highly desirable for such processes to be readily implemented in new facilities using commercially available single-use materials, avoiding the need for development of bespoke tools or cleaning validation, and for them to be readily scalable for later-stage studies. Here we report the development of such a process, using single-use stirred-tank bioreactors, a transgene-repressing HEK293 cell - promoter combination, and fully single-use filtration and ion exchange components. We demonstrate applicability of the process to candidate vaccines against rabies, malaria and Rift Valley fever, each based on a different adenovirus serotype. We compare performance of a range of commercially available ion exchange media, including what we believe to be the first published use of a novel media for adenovirus purification (NatriFlo® HD-Q, Merck). We demonstrate the need for minimal process individualization for each vaccine, and that the product fulfils regulatory quality expectations. Cell-specific yields are at the upper end of those previously reported in the literature, and volumetric yields are in the range 1 × 1013 - 5 × 1013 purified virus particles per litre of culture, such that a 2-4 L process is comfortably adequate to produce vaccine for early-phase trials. The process is readily transferable to any GMP facility with the capability for mammalian cell culture and aseptic filling of sterile products.

Survey of the perceived professional, educational and personal needs of physiotherapists in primary care and community settings.

The emphasis of UK Government policy on primary-care-based services has led to more physiotherapists working in the community. The aims of the present study were to identify the perceived professional, educational and personal needs of community physiotherapists, and to determine good practice in meeting these needs. A survey of physiotherapists working in 15 National Health Service community trusts in the West Midlands was carried out in September 2000. The survey questionnaire was developed through focus groups and mailed to a random sample of 200 community physiotherapists. The response rate was 67%, and the median age group of the respondents was 21-30 years. The participants worked mainly in 'urban but not inner city' areas, most commonly in domiciliary (31%, n = 38) and general practitioner surgery/health centre (26%, n = 32) locations. Fifty-one per cent (n = 66) of respondents had no specific learning objectives for continuing professional development (CPD); those with such objectives were more positive as to their helpfulness than those without them (Mann-Whitney U-test z = 2.519, P = 0.012). Fifty-three per cent (n = 68) also often/very often found it problematic getting cover for their caseloads so that they could take part in CPD activities. Access to library resources and use of computers were problems, as were confidence in appraising literature and opportunities to discuss research evidence with colleagues. Fifty-nine per cent (n = 77) of respondents indicated that they often/very often felt stressed by the size of their caseloads. Colleague support included mentorship, peer review, journal clubs, clinical interest groups and multidisciplinary in-service training; respondents with experience of these resources expressed more positive attitudes to them than those without (Mann-Whitney U-test z = 2.871, P < 0.0005 for each). Forty-two per cent (n = 54) indicated that there were problems with safety issues. This study has identified needs that will have an impact on the ability of community physiotherapists to meet the demands of clinical governance. National Health Service management at all levels has a responsibility to facilitate the education, training and support of community physiotherapists.

Role of apoptosis and cytokines in influenza virus morbidity.

Influenza virus is a major human pathogen that causes epidemics and pandemics with increased morbidity and, especially in the elderly and those with pre-existing medical conditions, increased mortality. Influenza is characterised by respiratory symptoms and constitutional symptoms. Whilst knowledge of the mechanisms underlying host and tissue specificity has advanced considerably of late we still know relatively little about other aspects of influenza virus virulence. In this review, we will explore what is known about the role of apoptosis in respiratory epithelial cell damage and the role of cytokines in inflammation and constitutional symptoms with particular emphasis on the link between apoptosis, inflammation, fever and cytokine production.

Laboratory-Scale Production of Replication-Deficient Adenovirus Vectored Vaccines.

Replication-deficient adenoviruses are potent vaccine development platforms used extensively for human and animal candidate vaccines, largely due to their very good safety and immunogenicity profile. In this chapter we describe a method that can be used in any laboratory for the scalable production of replication-deficient adenovirus vector vaccines to GLP for preclinical studies in animal models, including definitive experimental studies in large target animal species for veterinary applications. We use human adenovirus serotype 5 (HAdV5) as an example, but the method can be easily adapted for use with other adenovirus serotypes from different species of origin.

Simian adenoviruses as vaccine vectors.

Replication incompetent human adenovirus serotype 5 (HAdV-C5) has been extensively used as a delivery vehicle for gene therapy proteins and infectious disease antigens. These vectors infect replicating and nonreplicating cells, have a broad tissue tropism, elicit high immune responses and are easily purified to high titers. However, the utility of HAdV-C5 vectors as potential vaccines is limited due to pre-existing immunity within the human population that significantly reduces the immunogenicity of HAdV-C5 vaccines. In recent years, adenovirus vaccine development has focused on simian-derived adenoviral vectors, which have the desirable vector characteristics of HAdV-C5 but with negligible seroprevalence in the human population. Here, we discuss recent advances in simian adenovirus vaccine vector development and evaluate current research specifically focusing on clinical trial data.

Enhancing cellular immunogenicity of MVA-vectored vaccines by utilizing the F11L endogenous promoter.

Modified vaccinia virus Ankara (MVA)-vectored vaccines against malaria, influenza, tuberculosis and recently Ebola virus are in clinical development. Although this vector is safe and immunogenic in humans, efforts remain on-going to enhance immunogenicity through various approaches such as using stronger promoters to boost transgene expression. We previously reported that endogenous MVA promoters such as pB8 and pF11 increased transgene expression and immunogenicity, as compared to the conventional p7.5 promoter. Here, we show that both promoters also rivalled the mH5 promoter in enhancing MVA immunogenicity. We investigated the mechanisms behind this improved immunogenicity and show that it was a result of strong early transgene expression in vivo, rather than in vitro as would normally be assessed. Moreover, keeping the TK gene intact resulted in a modest improvement in immunogenicity. Utilizing pB8 or pF11 as ectopic promoters at the TK locus instead of their natural loci also increased transgene expression and immunogenicity. In addition to a reporter antigen, the pF11 promoter was tested with the expression of two vaccine antigens for which cellular immunogenicity was significantly increased as compared to the p7.5 promoter. Our data support the use of the pF11 and pB8 promoters for improved immunogenicity in future MVA-vectored candidate vaccines.

Correlation between levels of apoptosis, levels of infection and haemagglutinin receptor binding interaction of various subtypes of influenza virus: does the viral neuraminidase have a role in these associations.

Previously, we have shown that an H3N2 influenza virus (clone 7a) induced more apoptosis in MDCK cells than an H1N1 (A/Fiji) influenza virus and that the virion neuraminidase (NA) played a role in the induction of apoptosis. In this study we have examined a further 6 N2 (H3/H2) and 3 N1 (Hsw/H1) viruses and confirmed that the N2 viruses induce more apoptosis in MDCK cells than the N1 viruses. Furthermore, the level of apoptosis, the level of cell infection and the NA activity of the virus preparations paralleled each other for all the viruses. The levels of infection depended upon the degree of interaction of the viral haemagglutinin (HA) with its receptors: while all the viruses utilised NeuAc alpha-2,6 Gal containing receptors, the H3/H2 viruses showed a greater interaction than the Hsw/H1 viruses. Removal of sialic acid from virions by treatment with bacterial NA enhanced infection and apoptosis but the effect was much greater for the A/Fiji virus than for the clone 7a virus. Thus, while the relative interaction of the HAs for their receptors is the major factor influencing infectivity and apoptosis, the viral NA possibly plays an indirect role by removing sialic acid from the HA, thereby increasing its receptor binding.

Influenza A virus-induced apoptosis is a multifactorial process: exploiting reverse genetics to elucidate the role of influenza A virus proteins in virus-induced apoptosis.

Three influenza viruses, A/Puerto Rico/8/34-A/England/939/69 clone 7a (H3N2), A/Fiji/15899/83 (H1N1), and A/Victoria/3/75 (H3N2), induce different levels of apoptosis in vitro at equal moi; Clone 7a > A/Victoria > A/Fiji. Previous studies have shown that several viral proteins from clone 7a and A/Fiji, including PB2, NA, NS1, M1, and M2, induce apoptosis when expressed individually fused to the herpes simplex virus tegument protein, VP22. However, this did not reflect viral protein-protein-RNA interactions known to occur within infected cells. To explore the role of viral proteins in apoptosis under infection conditions, recombinant viruses with single or triple gene exchanges were generated using A/Victoria or clone 7a as the background virus. Inserting the A/Fiji NS or PB2 gene into A/Victoria or clone 7a significantly reduced the level of apoptosis compared to the parent virus while clone 7a PA or NP genes increased apoptosis. Inserting A/Fiji NA or HA or clone 7a NS, M, NA, or HA genes individually into A/Victoria had no significant effect on apoptosis. Surprisingly, inserting the M, NA, and HA genes of A/Fiji together into clone 7a reduced apoptosis, whereas inserting clone 7a M, NA, and HA together into A/Fiji increased apoptosis. These results suggest that no single virus protein induces apoptosis and that the combination of genes required may be strain specific, highlighting the difficulty of predicting the virulence of new strains that arise in nature. No support for the view that apoptosis is essential for high virus yields was obtained as high virus yields were obtained with viruses that induced both high and low levels of apoptosis.