Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    On the role of vaccine dose and antigenic distance in the transmission dynamics of Highly Pathogenic Avian Influenza (HPAI) H5N1 virus and its selected mutants in vaccinated animals
    Sitaras, Ioannis - \ 2017
    Wageningen University. Promotor(en): M.C.M. Jong, co-promotor(en): B. Peeters. - Wageningen : Wageningen University - ISBN 9789463438063 - 209
    avian influenza viruses - avian influenza - disease transmission - vaccines - vaccination - dosage - antigenic variation - mutants - mutations - immunity - vaccine development - virology - epidemiology - aviaire influenzavirussen - aviaire influenza - ziekteoverdracht - vaccins - vaccinatie - dosering - antigene variatie - mutanten - mutaties - immuniteit - vaccinontwikkeling - virologie - epidemiologie

    Influenza virus infections can cause high morbidity and mortality rates among animals and humans, and result in staggering direct and indirect financial losses amounting to billions of US dollars. Ever since it emerged in 1996 in Guangdong province, People’s Republic of China, one particular highly pathogenic avian influenza (HPAI) H5N1 virus has spread globally, and is responsible for massive losses of poultry, as well as human infections. For these reasons, HPAI H5N1 is considered as one of the viruses possible to cause a future influenza pandemic.

    One of the main reasons why influenza is a recurring problem is its ability to constantly evolve through the selection of mutants that are able to avoid immunity (be it natural or acquired). Due to the accumulation of mutations during genome replication, diverse/variant influenza genome sequences co-exist in a virus pool (quasispecies). These sequences can contain mutations that are able to confer selective advantages to the influenza virus given the opportunity. As a consequence, whenever a situation arises that places the virus under any type of pressure that the dominant virus sequence cannot cope with (i.e. immune pressure, selective receptor binding, etc.), the virus with the genome sequence that allows it to better adapt to that particular pressure becomes selected and takes over.

    Because of the influenza virus’s high rate of mutations, a global surveillance network is in place to monitor changes in circulating strains among humans that would warrant an update of the vaccines used. For human influenza strains, vaccines are updated frequently (every one or two years) and a similar situation holds true for racehorse vaccination. For avian influenza vaccination, however, the situation is different. In most countries, vaccination against avian influenza is not used, and in the countries where vaccines are used (either as routine or emergency measures), they are not updated as frequently as human vaccines are. In addition, in many instances vaccination against avian influenza viruses has met with some spectacular failures, since it failed to produce a level of immunity that would protect against circulating field strains. These vaccination failures have often been attributed to the fact that without constant vaccine updating (as is done for human influenza), the vaccines used are not able to keep up with continuously evolving antigenic variants selected in the field, and thus to protect poultry against them. In addition, since it is known that immune pressure resulting from vaccination can be a driving force in the evolution of influenza viruses and the selection of immune-escape mutants, there is a school of thought that posits that vaccination against avian influenza is not only a very expensive affair (especially if vaccines need to be frequently updated), but can also lead to selection of mutants that are able to avoid vaccination-induced immunity.

    The research reported in this thesis started with addressing the gaps in the knowledge regarding the role of vaccination-induced immunity in the selection of immune-escape mutants of HPAI H5N1, and if there is a way for vaccines to still be able to protect against antigenically-distant variants of the vaccine seed strain, without the need for frequent vaccine updates.

    Our first step in studying influenza virus evolution and selection of immune-escape mutants was to investigate how antigenic pressure may drive the selection of such mutants, and what the effect of the selected mutations on the pathogenicity and transmissibility of the mutants may be. Although there exist a variety of methods to select for influenza virus mutations (i.e. monoclonal antibodies, site-directed mutagenesis, reverse genetics, etc.), none of them is representative of selection as it happens in a vaccinated animal. In Chapter 2, we discuss in detail a laboratory-based system we have developed, in which immune-escape mutants are selected using homologous polyclonal chicken sera, similar to how they are selected in the field due to vaccination- induced immune pressure. We find that selection takes place early on, and additional mutations are selected when immune pressure is increased. Antigenic distances between the selected mutants and their parent strains are also increased throughout the selection process, but not in a linear fashion. Our selection system proved to be robust and replicable, and to be representative of selection in the field, since the mutations we selected for are also found in naturally-selected field isolates, and the antigenic distances between our selected mutants and their parent strains are similar to antigenic distances between vaccine strains and field isolates.

    We continued our research by addressing the roles played by vaccine dose (and resulting immunity) and antigenic distance between vaccine and challenge strains, in the transmission of HPAI H5N1 viruses, by employing transmission experiments using vaccinated chickens (Chapter 3). To our surprise, we found that the effect of antigenic distances between vaccine and challenge strains on transmission is very small compared to the effect of vaccine dose. We then quantified, for the first time, the minimum level of immunity and minimum percentage of the vaccinated population exhibiting said immunity, in order for vaccines to be able to protect against transmission even of strains that are antigenically distant to the vaccine seed strain. Transmission of such strains in well-vaccinated populations would allow for a scenario where vaccination- induced immunity may drive the selection of immune-escape mutants. Our results show that in order for vaccines to prevent transmission of antigenically distant strains (such as the ones resulting from selection due to immune pressure), the threshold level of immunity against these strains should be ≥23 haemagglutination inhibition units (HIU), in at least 86.5% of the vaccinated population. This level of immunity can be estimated by knowing the antigenic distance between the vaccine and challenge (field) strain, and the HI titre against the vaccine strain, which would then allow the approximate level of immunity against the field strain to be deduced. For example, assuming the HI titre against a vaccine strain is 210 HIU, and the distance with the challenge (field) strain is 24 HIU, according to our results the vaccine should be able to protect against the challenge strain, because the difference in HI titres should be around 26 HIU (i.e. above 23 HIU). These results, taken together with our previous work on selection of mutants, where we showed that the antigenic distances between our mutants and their parent strains are representative of distances found in the field, point to the fact that it is unlikely that vaccination-induced immunity can lead to selection of mutants able to escape it, given that a threshold level of immunity in a minimum percentage of the vaccinated population is achieved. As a consequence, we believe that constant vaccine updating may not be necessary for avian influenza viruses, as long as a threshold level of immunity is maintained. This makes vaccination a more attractive control measure, both from a health perspective and a financial one, than just applying biosecurity measures.

    To examine the effect the mutations in the haemagglutinin protein of our selected mutants may have in their transmission among chickens vaccinated with the parent strain, we used reverse genetics techniques to insert the HA gene of our most antigenically distant mutant into the parent strain backbone (Chapter 4). We vaccinated animals with a sub-optimal dose of vaccine, and we concluded that the mutations we selected for did not allow the mutant to avoid even low levels of immunity, such as the ones resulting from a sub-optimal vaccine dose (which resembles a poor field vaccination scenario). At the same time, the HA mutations we selected for did not appear to have a negative effect either on the pathogenicity of the mutant, or its ability to transmit to unvaccinated animals, since both parameters were comparable to the parent strain.

    Finally, we studied the role inter-animal variation in immunity – as measured by HI titres – has in the accuracy of antigenic cartography calculations (Chapter 5). We found that using sera from more than one animal significantly increased the accuracy of antigenic distance calculations, since it takes into account individual differences in immune responses to vaccination, an inevitable phenomenon documented in both humans and animals. In addition, we increased the accuracy of antigenic maps by avoiding the use of dimension-reducing algorithms as is currently done. By not reducing the dimensionality of virus positioning in space, our maps retain the original geometry between strains or sera, leading to more accurate positioning (Chapters 2 and 5). We hope that improving the accuracy of antigenic cartography can lead to a more precise surveillance of influenza evolution and better informed decisions regarding the need to update vaccines.

    Taken collectively, our results can improve field vaccination outcomes, since they provide guidelines on how to increase vaccination efficiency in stopping transmission of even antigenically-distant strains. In addition, our method for selecting for immune- escape mutants can be a valuable addition to research on influenza virus evolution. Moreover, policy making decisions regarding vaccination against any type of influenza can also benefit from our improvement on antigenic cartography accuracy, saving unnecessary costs in vaccine updating, and reducing morbidity and mortality of both animals and humans.

    Next-generation salmonid alphavirus vaccine development
    Hikke, M.C. - \ 2016
    Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Gorben Pijlman. - Wageningen : Wageningen University - ISBN 9789462577404 - 159
    alphavirus - atlantic salmon - rainbow trout - vaccine development - immunity - virology - fish culture - aquaculture - biotechnology - alfavirus - europese zalm - regenboogforel - vaccinontwikkeling - immuniteit - virologie - visteelt - aquacultuur - biotechnologie

    ABSTRACT

    Aquaculture is essential to meet the current and future demands for seafood to feed the world population. Atlantic salmon and rainbow trout are two of the most cultured aquaculture species. A pathogen that threatens these species is salmonid alphavirus (SAV). A current inactivated virus vaccine against SAV provides cross-protection against all SAV subtypes in salmonids and reduces mortality amongst infected fish. However, protection is not 100% and due to virus growth at low temperature, the vaccine production process is time consuming. In addition, the vaccine needs to be injected into the fish, which is a cumbersome process. The work described in this thesis aimed to increase the general knowledge of SAV and to assess current vaccine technologies, and to use this knowledge in designing next-generation vaccines for salmonid aquaculture.

    An alternative cell line to support SAV proliferation was identified, however, the virus production time could not yet outcompete the current SAV production system. Making use of the baculovirus insect cell expression system, multiple enveloped virus-like particle (eVLP), and core-like particle (CLP) prototype vaccines were produced in insect cells at high temperature. An in vivo vaccination study showed, however, that these vaccines could not readily protect Atlantic salmon against SAV. The low temperature-dependent replication of SAV was attributed to the glycoprotein E2, and it was found that E2 only correctly travelled to the cell surface at low temperature, and in the presence of glycoprotein E1. The biological impact of this finding was confirmed in the development and in vivo testing of a DNA-launched replicon vaccine. The effective DNA-launched replicon vaccine was extended by delivery of the capsid protein in trans. It was hypothesized that viral replicon particles (VRP) were formed in vivo, which would cause an additional single round of infection and might further elevate the immune response in comparison to the replicon vaccine. A second animal trial indicated that the inclusion of capsid did not yet improve vaccine efficacy. This trial however did show that a DNA vaccine transiently expressing the SAV structural proteins provided superior protection over both replicon vaccines (with and without capsid).

    In this thesis, some virus characteristics, such as the cause of temperature-dependency of SAV replication, of an unique aquatic virus were further explored. The production and in vivo testing of multiple next-generation vaccines defined the prerequisites for induction of a potent immune response in Atlantic salmon. A prototype DNA-launched replicon vaccine has shown potential for further development. The research described in this thesis contributes to the development of next-generation vaccines in the challenging area of fish vaccinology.

    Activation and evasion of the type I Interferon response by infectious bronchitis virus : roles of the accessory proteins
    Kint, J. - \ 2015
    Wageningen University. Promotor(en): Geert Wiegertjes; Huub Savelkoul, co-promotor(en): Maria Forlenza. - Wageningen : Wageningen University - ISBN 9789462573376 - 138
    interferon - coronavirus - infectieus bronchitisvirus - coronaviridae - immuniteitsreactie - kippen - kippenziekten - pluimveeziekten - vaccinontwikkeling - kwantitatieve methoden - eiwit - virale inmenging - interferon - coronavirus - infectious bronchitis virus - coronaviridae - immune response - fowls - fowl diseases - poultry diseases - vaccine development - quantitative methods - protein - viral interference

    SUMMARY

    Viruses are intracellular parasites that exploit the machinery of the host cell to replicate. To defend themselves against invading viruses, animal cells have evolved an anti-viral mechanism, known as the type I interferon response. Through natural selection viruses have in turn evolved mechanisms to counteract or evade the type I IFN response. Coronaviruses are a large group of positive-stranded RNA viruses that cause a range of human and veterinary diseases. Infectious bronchitis virus (IBV) is a member of the genus Gammacoronavirus and it is the causative agent of a highly contagious respiratory disease of poultry. To date, only few studies have investigated the interaction between IBV and the type I IFN response.

    In this thesis, we describe for the first time the activation of the type I interferon response (IFN response) by the Gammacoronavirus IBV, and the repressive role of accessory proteins therein. In Chapter 1 I provide a general introduction into coronaviruses in general and the Gammacoronavirus IBV in particular. I also introduce the IFN response, and highlight differences between the mammalian and chicken IFN response. Finally, I review current knowledge on the roles of coronavirus accessory proteins in counteraction of the IFN response. In Chapter 2 we describe our studies which demonstrated that activation of the IFN response by IBV is dependent on the intracellular double-stranded RNA sensor MDA5. We show that detection of IBV-infection by MDA5 is delayed with respect to the peak of viral replication, and demonstrate that this delay is not due to inhibition of dsRNA detection by IBV. Using mutant viruses that cannot express accessory proteins (null viruses), we found that accessory proteins 3a and 3b of IBV mediate transcription and translation of Ifnβ mRNA.

    The observation that IBV delays the activation of the IFN response, prompted us to investigate the sensitivity of IBV to IFN treatment in Chapter 3. Here we show that IBV is relatively resistant to treatment with type I IFN, as relatively high doses of type I IFN are required to decrease propagation of the virus. Next, we studied which viral protein(s) contribute to resistance of IBV to type I IFN and found that absence of accessory proteins 3a and 3b increased sensitivity of IBV to type I IFN, via a presently unknown mechanism. In addition, we observed that independent of accessory proteins 3a and 3b, IBV blocks signaling of IFN by inhibiting phosphorylation and translocation of the transcription factor STAT1. To explain the delayed kinetics of IFN production observed in Chapter 2, we investigated whether delayed protein production was restricted to IFN, or whether IBV, like Alpha- and Betacoronaviruses, inhibits general translation of host proteins (i.e. induces host shutoff). In Chapter 4 we demonstrate that IBV-induced transcription of Ifnβ mRNA leads to the production of relatively little IFN protein. We discovered that limited production of IFN protein by IBV-infected cells is the result of general inhibition of host translation, confirming that IBV induces a shutoff of host-protein production. This finding indicates that evasion of the innate immune system by Gammacoronaviruses may be more similar to that of Alpha- and Betacoronaviruses than previously thought. Using accessory protein null viruses we discovered that accessory protein 5b of IBV is essential for the inhibition of host-protein synthesis by IBV. In Chapter 5 and Chapter 6 we describe the methods used in this thesis to quantify the number of infectious virus particles of IBV as well as methods used to quantify the activation of the type I IFN response in chicken cells. Although the studies described in this thesis have answered several questions about the interaction of IBV with the type I IFN response of its host, they have also raised new questions to be addressed in future research. In the final Chapter of this thesis (Chapter 7), I discuss a number of remaining questions and future perspectives regarding evasion of the IFN response by IBV. Finally, I explore the possible implications of our findings on the in vivo pathogenicity of IBV and on the rational design of attenuated IBV vaccines.

    In conclusion, the work described in this thesis demonstrates for the first time how IBV evades, activates, and antagonises the IFN response. Also, this thesis comprises the first study that describes a function for the accessory proteins of IBV and shows that these poorly understood proteins play an important role in antagonism of the type I IFN response.

    PAT for PER.C6® perfusion cultivation
    Mercier, S.M. - \ 2014
    Wageningen University. Promotor(en): Rene Wijffels, co-promotor(en): Mathieu Streefland. - Wageningen : Wageningen University - ISBN 9789462571396 - 155
    celcultuur vaccins - vaccinontwikkeling - procesontwerp - procesbewaking - procesoptimalisatie - bioreactoren - celkweek - cell culture vaccines - vaccine development - process design - process control - process optimization - bioreactors - cell culture
    IPV v2.0 : upgrading the established inactivated polio vaccine production process
    Thomassen, Y.E. - \ 2014
    Wageningen University. Promotor(en): Rene Wijffels, co-promotor(en): W.A.M. Bakker; L.A. van der Pol. - Wageningen : Wageningen University - ISBN 9789461738561 - 231
    poliomyelitis - geïnactiveerde vaccins - vaccinontwikkeling - bioreactoren - poliomyelitis - inactivated vaccines - vaccine development - bioreactors

    The first vaccine against poliovirus (PV), the causative agent of poliomyelitis, was developed in the 1950s by Jonas Salk. The vaccine (IPV) consists of an injected dose of purified and inactivated wild-type PVs (all three serotypes). Soon after this discovery, at the Rijks Instituut voor de Volksgezondheid (RIV) in Bilthoven, an industrial-scale production process for IPV was developed based on micro-carrier technology and primary monkey kidney cells. In the 1970s, the manufacturing of IPV was scaled up to 300-L by the development of well-controlled bioreactors for cell culture, the so-called “Bilthoven Units” (originally used for bacterial fermentations). In 2004, the Vero cell line was introduced to replace the then used tertiary monkey kidney cells followed by a scale-up from 700 to 1,500-L (from two 350-L to two 750-L bioreactors). IPV manufacturing has been part of the regular vaccine manufacturing activities in Bilthoven ever since the establishment of the IPV production process.

    With polio eradication on our doorstep, the World Health Organization (WHO) is pursuing a new IPV based on non-wild-type strains to increase the biosafety of vaccine manufacturing. In addition, and due to the pending cessation of oral polio vaccines (OPV), the global demand for affordable IPV is increasing. To accommodate these questions two research programs were started at the Netherlands Vaccine Institute (now Institute for Translational Vaccinology). One concerned optimization of the current conventional IPV production process, the other the manufacturing of an affordable sIPV, an IPV based on the attenuated Sabin PV strains normally used for OPV production. The technology of the sIPV production process, developed in the latter project, is also aimed to be transferred to developing countries manufacturers (Chapter 2).

    From the substantial history in polio vaccine production in Bilthoven, a valuable dataset has been generated. Data from over 50 batches at two different production scales (700-L and 1,500-L) were analyzed using multivariate data analysis (MVDA). This statistical method is stimulated by the ICH (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use) to improve scientific understanding of production processes for troubleshooting and improved process control. The initial explorative analysis, performed on single unit operations, indicated consistent manufacturing. Known outliers (e.g., rejected batches) were identified using principal component analysis (PCA). The source of operational variation was pinpointed to variation of input such as cell- and virus culture media. Other relevant process parameters were in control and, using this manufacturing data, could not be correlated to product quality attributes. The gained knowledge of the IPV production process, not only from the MVDA, but also from digitalizing the available historical data, has proven to be useful for troubleshooting, understanding limitations of available data and seeing the opportunity for improvements (Chapter 3).

    One of the gaps in the data was located in the product quantification during processing. The available assay used for determining the D-antigen concentration in in-process samples had high variability. A so-called fast ELISA was developed and qualified for analysis of polio D-antigen. The original 20h-protocol was optimized by minimizing the total incubation time to 1h, and by replacing the signal reagent 3,3',5,5'-tetramethylbenzidine by a chemiluminogenic signal reagent with a theoretical low intrinsic background and high dynamic range. This fast D-antigen ELISA was suitable for measurement of D-antigen concentrations in the different matrixes present during the different unit-operations in the production process (Chapter 4).

    To accommodate research to improve and optimize the current cIPV production process an up-to-date lab-scale version encompassing the legacy inactivated polio vaccine production process was set-up based on the knowledge obtained during the MVDA of historical data. This lab-scale version was designed to be representative of the large scale, meaning a scale-down model, to allow experiments for process optimization that can be readily applied to manufacturing scale. Initially the separate unit operations were scaled-down at setpoint. Subsequently, the unit operations were applied successively in a comparative manner to large-scale manufacturing. This allows the assessment of the effects of changes in one unit operation to the consecutive units at small-scale. The developed scale-down model for cell and virus culture (2.3-L) presents a feasible model with its production scale counterpart (750-L) when operated at setpoint. Also, the scale-down models for the DSP unit operations clarification, concentration, size exclusion chromatography, ion exchange chromatography, and inactivation are in agreement with the manufacturing scale. The small-scale units can be used separately, as well as sequentially, to study variations and critical product quality attributes in the production process. Finally, it has been shown that the scale-down unit operations can be used consecutively to prepare trivalent vaccine at lab-scale with comparable characteristics to the product produced at manufacturing scale (Chapter 5).

    The upcoming of disposables in GMP manufacturing triggered the study on alternatives for the clarification unit (Chapter 5) and alternative Wave-type, bioreactors (Chapter 6). This type of bioreactors makes use of sterilized disposable bags, which could be beneficial in a GMP environment, for example to reduce the cleaning validation burden, or to facilitate change-over to another product using the same equipment. Wave-type bioreactors make use of vertical (standard rocking motion-type) or both vertical and horizontal displacement (CELL-tainer®(CELLution Biotech BV)) for mixing instead of an impeller, which is used in stirred tank reactors. Using the design of experiments (DoE) approach, models for the mixing times in both the CELL-tainer®and the BIOSTAT®CultiBag RM (Sartorius Stedim Biotech) bioreactor (standard rocking motion-type) were developed. The conditions for cultivation of Vero cells in the CELL-tainer®bioreactor were chosen based on comparable mixing times. Vero cells growing adherent to Cytodex 1 microcarriers were cultivated in the CELL-tainer®and in the BIOSTAT®CultiBag RM. Vero cell growth in both bioreactors was comparable with respect to the growth characteristics and main metabolite production and consumption rates. Additionally, poliovirus production in both bioreactors was shown to be similar.

    In view of WHOs pursuit towards an IPV manufacturing process with increased biosafety, the development of sIPV was taken up. Prior to large scale production of clinical lots, an initial proof of principle study was done (Chapter 2). Starting from the conventional IPV (cIPV) production process, minimal adaptations, such as lower virus cultivation temperature, were implemented. Also, the selected disposable filter unit (Chapter 5) was implemented. To quickly prepare sIPV clinical lots and show proof-of-principle of sIPV in human, no further process optimization and/or modernization was done. sIPV was produced at industrial scale followed by formulation of both plain and aluminium adjuvanted sIPV (Chapter 7). The final products met the quality criteria, were immunogenic in rats, showed no toxicity in rabbits and could be released for testing in the clinic. While an immunogenic product, both in animals as in humans was prepared, the product yield was extremely low and further process development will be needed to obtain an affordable sIPV.

    Especially the yield of Sabin PV type 2 after ion exchange chromatography was low. To determine if this effect could be due to a difference in the isoelectric point (pI) of the poliovirus a method for pImeasurement of live virus was developed (Chapter 8). A method for analyzing biological hazardous components (biological safety level 2) was set up for the capillary isoelectric focusing-whole column imaging detection (CIEF-WCID) analyzer. This method is based on closed circuits. Subsequently, the pI’s of complete intact polioviruses were determined. The polioviruses that were analyzed are the commonly used viruses for the production of IPV - Mahoney (type 1), MEF-1 (type 2), and Saukett (type 3) - as well as for OPV - Sabin types 1, 2, and 3. The determined pI's were 6.2 for Mahoney, 6.7 for MEF-1, and 5.8 for Saukett. The pI's of Sabin types 1, 2, and 3 viruses were 7.4, 7.2, and 6.3, respectively. With a pIof 7.2, Sabin PV type 2 is prone to self-aggregation at the pH used during chromatography (pH 7.0). Self-aggregation was thus suggested to be the main cause of low product yield and prevention of this self-aggregation was suggested to be the main focus point for process optimization.

    Besides optimization of the downstream processing, optimization of the upstream processing, i.e. increased virus yields after cell and virus culture was studied (Chapter 9). Vero cells were grown adherent to microcarriers (Cytodex 1; 3 g L-1) using animal component free media in stirred-tank type bioreactors. Different strategies for media refreshment, daily media replacement (semi-batch), continuous media replacement (perfusion) and recirculation of media, were compared with batch cultivation. Cell densities increased using a feed strategy from 1 × 106cells mL-1during batch cultivation to 1.8, 2.7 and 5.0 × 106cells mL-1during semi-batch, perfusion and recirculation, respectively. The effects of these different cell culture strategies on subsequent poliovirus production were investigated. Increased cell densities allowed up to 3 times higher D-antigen levels when compared with that obtained from batch-wise Vero cell culture. However, the cell specific D-antigen production was lower when cells were infected at higher cell densities. This cell density effect is in good agreement with observations for different cell lines and virus types. From the evaluated alternative culture methods, application of a semi-batch mode of operations allowed the highest cell specific D-antigen production. The increased product yields that can easily be reached using these higher cell density cultivation methods, showed the possibility for better use of bioreactor capacity for the manufacturing of polio vaccines to ultimately reduce vaccine cost per dose. Further, the use of animal-component-free cell- and virus culture media shows opportunities for modernization of human viral vaccine manufacturing.

    To assess the affordability of sIPV the manufacturing costs were determined (Chapter 10). The sIPV manufacturing costs, when produced as described in Chapter 7, indicate the requirement for process optimizations. However, the manufacturing costs can be reduced at least a factor 2 when implementing the upstream processing optimization, ACF media and a semi-batch process, as described in Chapter 9. Assuming improvements in downstream processing will result in a process with yields comparable to the cIPV process costs can be further lowered. In addition, a scale-up from two 350-L bioreactors to two 1,000-L bioreactors would nearly halve the manufacturing costs resulting in a more than cost competitive sIPV. These costs analysis showed that an affordable sIPV is feasible.

    Chikungunya virus-like particle vaccine
    Metz, S.W.H. - \ 2013
    Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Gorben Pijlman. - S.L. : s.n. - ISBN 9789461735652 - 139
    chikungunyavirus - virusachtige deeltjes - virusziekten - aedes albopictus - vectoren, ziekten - vaccins - vaccinontwikkeling - genexpressie - baculovirus - insecten - celcultuur vaccins - chikungunya virus - virus-like particles - viral diseases - aedes albopictus - disease vectors - vaccines - vaccine development - gene expression - baculovirus - insects - cell culture vaccines

    Chikungunya virus (CHIKV) is an arthropod-borne alphavirus (family Togaviridae) and is the causative agent of chikungunya fever. This disease is characterised by the sudden onset of high fever and long-lasting arthritic disease. First identified in Tanzania in 1952, CHIKV has re-emerged in the last decade causing large outbreaks throughout Africa, Asia and Southern Europe. Increased CHIKV spread is mainly caused by its adaptation to a new mosquito vector, the Asian tiger mosquito Ae. albopictus, which is able to colonize more temperate regions. Currently, there are no antiviral treatments or commercial vaccines available, to prevent CHIKV infections. However, increased vector spread and clinical manifestations in humans, have triggered vaccine development. A broad range of vaccine strategies have been proposed and described, including inactivated virus formulations, live-attenuated virus, chimeric virus vaccines, DNA vaccines, adenoviral vectored vaccines, subunit protein vaccines and virus-like particle (VLP) formulations. However, these vaccination strategies have specific limitations in manufacturing, immunogenicity, safety, recombination and large scale production. Many, if not all safety problems do not apply for subunit or VLP based vaccines, except for the recombinant origin of the vaccine.

    Recently, a CHIKV VLP-based vaccine was developed and provided protection in both mice and non-human primates. Even though this VLP approach is a safe, efficient and promising alternative to other vaccine strategies, large scale DNA plasmid transfection into mammalian cells and VLP yield of transfected cells remains challenging in terms of industrial production. These problems are alleviated by using the recombinant baculovirus-insect cell expression system.

    In this thesis, recombinant baculoviruses were constructed to produce CHIKV glycoprotein E1 and E2 subunits and VLPs.For the production of CHIKV-E1 and E2 subunits, both protein genes were cloned downstream the polyhedrin gene (polh) promoter of in an Autographica californica multiple nucleopolyhedrovirus backbone, together with their authentic signal peptides 6K(E1) and E3(E2). Deletion of the C-terminal transmembrane domain, generated secreted versions of E1 (E1ΔTM or sE1) and E2 (E2ΔTM or sE2). A substantial amount of recombinant protein was glycosylated and processed by furin. The secreted CHIKV subunits were purified from the medium and were able to induce neutralizing antibodies in rabbits. For the production of the VLPs, the complete structural polyprotein (capsid, E3, E2, 6K, E1) was cloned downstream the AcMNPV polh promoter. E3E2 precursor processing and glycosylation appeared to be more efficient when E3E2 were expressed as part of the whole structural polyprotein cassette, compared to the individually expressed E3E2. The VLPs were isolated from the medium fraction and were morphologically similar to wild type CHIKV. A similar strategy was used to produce VLPs from another alphavirus, the salmonid alphavirus (SAV). Here, however, the normal baculovirus expression temperature of 27°C appeared to be detrimental for SAV-E3E2 furin cleavage and SAV-VLP production. E2-glycoprotein processing was shown to be temperature dependent and a tailored temperature-shift regime was designed in which Sf9-cells were infected with a recombinant baculovirus expressing the SAV structural proteins, and incubated at 27°C for 24 h, followed by a processing phase of 72 h at 15°C. Using this temperature regime, SAV-VLPs were produced that were morphologically indistinguishable from wild type SAV and underscores the flexibility of the baculovirus-insect cell expression system.

    The immunogenicity of purified CHIKV-sE1 and -sE2 subunits and purified CHIKV-VLPs were then tested in a lethal vaccination-challenge mouse model, in IFN α/β, -γ receptor null AG129 mice. The innate immune system of these mice was made dysfunctional. This vaccine-challenge study clearly showed that VLPs provided superior protection, compared to their subunit counterparts. The subunits provided only partial protection and induced low neutralizing antibody titres. Immunization with the VLPs fully protected mice against lethal challenge and induced significant higher neutralizing antibody titress. Even though neutralizing antibody titres were lower after subunit immunization, this study showed that a minor neutralizing antibody response is sufficient to protect mice from lethal CHIKV challenge. Next, the CHIKV VLPs were tested for their ability to induce complete protection in an adult wild-type immune-competent mouse model, in which mice develop arthritic disease after CHIKV infection. The VLPs were able to induce full protection after a single immunization of 1 µg VLPs, without the use of adjuvants. In addition, IgG isotyping revealed a balanced IgG1-IgG2c response, suggesting a role for both humoral and cellular immunity in the protection against CHIKV infection. Mice served as a proxy for primates and vaccination trials in primates are next on the agenda.

    This thesis is a typical example of the opportunities for the recombinant baculovirus-insect cell expression system in viral vaccine development, especially in vaccine development for other arboviruses. Although the CHIKV-VLPs produced in insect cells are amenable for large-scale production, the production process and downstream processing need to be carefully designed and optimized before CHIKV VLPs can be produced on an industrial scale. However, the data presented in this thesis show that CHIKV-VLPs produced in insect cells using recombinant baculoviruses represents as a new, safe, non-replicating and effective vaccine candidate against CHIKV infections.

    Chikungunya virus (CHIKV) is an arthropod-borne alphavirus (family Togaviridae) and is the causative agent of chikungunya fever. This disease is characterised by the sudden onset of high fever and long-lasting arthritic disease. First identified in Tanzania in 1952, CHIKV has re-emerged in the last decade causing large outbreaks throughout Africa, Asia and Southern Europe. Increased CHIKV spread is mainly caused by its adaptation to a new mosquito vector, the Asian tiger mosquito Ae. albopictus, which is able to colonize more temperate regions. Currently, there are no antiviral treatments or commercial vaccines available, to prevent CHIKV infections. However, increased vector spread and clinical manifestations in humans, have triggered vaccine development. A broad range of vaccine strategies have been proposed and described, including inactivated virus formulations, live-attenuated virus, chimeric virus vaccines, DNA vaccines, adenoviral vectored vaccines, subunit protein vaccines and virus-like particle (VLP) formulations. However, these vaccination strategies have specific limitations in manufacturing, immunogenicity, safety, recombination and large scale production. Many, if not all safety problems do not apply for subunit or VLP based vaccines, except for the recombinant origin of the vaccine.

    Recently, a CHIKV VLP-based vaccine was developed and provided protection in both mice and non-human primates. Even though this VLP approach is a safe, efficient and promising alternative to other vaccine strategies, large scale DNA plasmid transfection into mammalian cells and VLP yield of transfected cells remains challenging in terms of industrial production. These problems are alleviated by using the recombinant baculovirus-insect cell expression system.

    In this thesis, recombinant baculoviruses were constructed to produce CHIKV glycoprotein E1 and E2 subunits and VLPs.For the production of CHIKV-E1 and E2 subunits, both protein genes were cloned downstream the polyhedrin gene (polh) promoter of in an Autographica californica multiple nucleopolyhedrovirus backbone, together with their authentic signal peptides 6K(E1) and E3(E2). Deletion of the C-terminal transmembrane domain, generated secreted versions of E1 (E1ΔTM or sE1) and E2 (E2ΔTM or sE2). A substantial amount of recombinant protein was glycosylated and processed by furin. The secreted CHIKV subunits were purified from the medium and were able to induce neutralizing antibodies in rabbits. For the production of the VLPs, the complete structural polyprotein (capsid, E3, E2, 6K, E1) was cloned downstream the AcMNPV polh promoter. E3E2 precursor processing and glycosylation appeared to be more efficient when E3E2 were expressed as part of the whole structural polyprotein cassette, compared to the individually expressed E3E2. The VLPs were isolated from the medium fraction and were morphologically similar to wild type CHIKV. A similar strategy was used to produce VLPs from another alphavirus, the salmonid alphavirus (SAV). Here, however, the normal baculovirus expression temperature of 27°C appeared to be detrimental for SAV-E3E2 furin cleavage and SAV-VLP production. E2-glycoprotein processing was shown to be temperature dependent and a tailored temperature-shift regime was designed in which Sf9-cells were infected with a recombinant baculovirus expressing the SAV structural proteins, and incubated at 27°C for 24 h, followed by a processing phase of 72 h at 15°C. Using this temperature regime, SAV-VLPs were produced that were morphologically indistinguishable from wild type SAV and underscores the flexibility of the baculovirus-insect cell expression system.

    The immunogenicity of purified CHIKV-sE1 and -sE2 subunits and purified CHIKV-VLPs were then tested in a lethal vaccination-challenge mouse model, in IFN α/β, -γ receptor null AG129 mice. The innate immune system of these mice was made dysfunctional. This vaccine-challenge study clearly showed that VLPs provided superior protection, compared to their subunit counterparts. The subunits provided only partial protection and induced low neutralizing antibody titres. Immunization with the VLPs fully protected mice against lethal challenge and induced significant higher neutralizing antibody titress. Even though neutralizing antibody titres were lower after subunit immunization, this study showed that a minor neutralizing antibody response is sufficient to protect mice from lethal CHIKV challenge. Next, the CHIKV VLPs were tested for their ability to induce complete protection in an adult wild-type immune-competent mouse model, in which mice develop arthritic disease after CHIKV infection. The VLPs were able to induce full protection after a single immunization of 1 µg VLPs, without the use of adjuvants. In addition, IgG isotyping revealed a balanced IgG1-IgG2c response, suggesting a role for both humoral and cellular immunity in the protection against CHIKV infection. Mice served as a proxy for primates and vaccination trials in primates are next on the agenda.

    This thesis is a typical example of the opportunities for the recombinant baculovirus-insect cell expression system in viral vaccine development, especially in vaccine development for other arboviruses. Although the CHIKV-VLPs produced in insect cells are amenable for large-scale production, the production process and downstream processing need to be carefully designed and optimized before CHIKV VLPs can be produced on an industrial scale. However, the data presented in this thesis show that CHIKV-VLPs produced in insect cells using recombinant baculoviruses represents as a new, safe, non-replicating and effective vaccine candidate against CHIKV infections.

    Next-generation outer membrane vesicle vaccines from concept to clinical trials
    Waterbeemd, B. van de - \ 2013
    Wageningen University. Promotor(en): Rene Wijffels, co-promotor(en): Michel Eppink. - [S.l.] : s.n. - ISBN 9789461734983
    vaccins - vaccinontwikkeling - neisseria meningitidis - vaccines - vaccine development - neisseria meningitidis

    Only vaccines containing outer membrane vesicles (OMV) have successfully stopped Neisseria meningitidis serogroup B epidemics. The OMV vaccines, however, provide limited coverage and are difficult to produce. This is caused by an obligatory detergent treatment, which removes lipopolysaccharide (LPS), a toxic OMV component. This thesis explored an alternative approach, based on OMV with attenuated lpxL1-LPS and a detergent-free process. The alternative approach is referred to as ‘next-generation OMV’ and provided vaccines with improved immunological and biochemical properties. In addition, quantitative proteomics demonstrated a preferred protein composition. This provided justification for further development towards clinical trials. After optimization of specific process steps, an improved pilot-scale production process was developed. The quality of OMV from this optimized process was stable and within pre-set specifications for nine consecutive batches. Studies in mice and rabbits suggested that next-generation OMV are immunogenic and safe for parenteral use in humans. Therefore these vaccines are now ready for clinical evaluation. Several groups are developing broadly protective OMV vaccines against N. meningitidis serogroup B, but also against other serogroups and other pathogens. OMV therefore have the potential to become a versatile technology platform for prophylactic and therapeutic vaccines. Such a platform requires a reliable production process to generate substantial quantities of high quality product. The process described in this thesis is well-suited for this purpose. The results encourage technology transfer to a commercial partner, with the goal to translate nextgeneration OMV technology into actual vaccines and improve global public health.

    Development of an influenza virus vaccine using the baculovirus-insect cell expression system : implications for pandemic preparedness
    Cox, M.M.J. - \ 2009
    Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Monique van Oers. - [S.l. : S.n. - ISBN 9789085854791 - 135
    vaccinontwikkeling - vaccins - influenza - baculovirus - insecten - hemagglutininen - celcultuur vaccins - recombinant vaccins - vaccine development - vaccines - influenza - baculovirus - insects - haemagglutinins - cell culture vaccines - recombinant vaccines
    Key word

    Influenza, rHA, vaccine, baculovirus, insect cells, production, pandemic preparedness

    Influenza (or flu) is a highly contagious, acute viral respiratory disease that occurs seasonally in most parts of the world and is caused by influenza viruses. Influenza vaccination is an effective way to reduce the complications and the mortality rate following influenza infections. The currently available influenza vaccines are manufactured in embryonated chicken eggs, a 40-year old production technology. The research in this thesis was aimed at the design, validation and development of a production process for a recombinant hemagglutinin (rHA) influenza vaccine for the prevention of seasonal influenza. The viral surface protein HA is the key antigen in the host response to influenza virus since neutralizing antibodies directed against HA can mitigate or prevent infection. The baculovirus-insect cell system was selected for the synthesis of rHA molecules. The designed process was used to manufacture candidate trivalent rHA vaccines, which were tested in four clinical studies in a total of more than 3000 human subjects age 18 - 92 to support licensure of FluBlok under the “Accelerated Approval” procedure in the United States (U.S.). These studies demonstrated that the purified rHA protein was well tolerated and resulted in a strong and long lasting immune response. In addition, the novel vaccine provided cross protection against drifted influenza viruses. In response to the emergence of the new H1N1 A/California /04/2009 influenza strain, the outlined design was used to produce a rHA vaccine candidate and merely 6 weeks later, the first batches of vaccine were ready for human clinical testing. There are two especially important advantages to the use of this technology from a public health perspective: First, the insect cell-baculovirus system has demonstrated the potential to facilitate safe and expeditious responses to health care emergencies such as the one currently posed by the novel H1N1 virus pandemic and secondly, the rHA vaccine does not contain ovalbumin or other antigenic proteins that are present in eggs and may therefore be administered to people who are egg-allergic.


    From process understanding to process control: application of PAT on the cultivation of Bordetella pertussis for a whole cell vaccine
    Streefland, M. - \ 2009
    Wageningen University. Promotor(en): Hans Tramper, co-promotor(en): Dirk Martens; E.C. Beuvery; L.A. van der Pol. - [S.l. : S.n. - ISBN 9789085853572 - 181
    celcultuur vaccins - vaccinontwikkeling - bordetella pertussis - verwerkingskwaliteit - procesbewaking - cell culture vaccines - vaccine development - bordetella pertussis - processing quality - process control
    De kwaliteit van farmaceutische producten wordt geborgd door een op Good Manufacturing Practice gebaseerd kwaliteits systeem, waarbij kwaliteitscontrole van het eindproduct een belangrijke rol vervult. Recentelijk is hierin verandering gekomen en vragen regelgevers bij overheden steeds meer om een kwaliteitssysteem dat al tijdens het proces operationeel is. Voor chemische farmaca wordt dit al steeds meer door de industrie toegepast, maar voor biologische producten blijkt die nog steeds moeilijk. In dit proefschrift wordt een methode beschreven die on line monitoring en kwaliteitscontrole voor een complex biologisch product (een cellulair vaccin) mogelijk maakt. Door te identificeren welke procesparameters invloed hebben op de kritieke eigenschappen van het product, kan een controle systeem worden ingericht dat deze kritische parameters tijdens het proces monitort en beheerst. Hiermee verschuift de kwaliteitscontrole van achteraf naar tijdens het proces. Dit maakt de productie van vaccins flexibeler, goedkoper en bovenal veiliger voor de patient.
    Towards a Neisseria meningitidis B vaccine : introducing systems biology in process development
    Baart, G.J.E. - \ 2008
    Wageningen University. Promotor(en): Hans Tramper, co-promotor(en): Dirk Martens; E.C. Beuvery. - [S.l.] : S.n. - ISBN 9789085049975 - 276
    neisseria meningitidis - vaccinontwikkeling - wiskundige modellen - celmetabolisme - modelleren - bioproceskunde - neisseria meningitidis - vaccine development - mathematical models - cell metabolism - modeling - bioprocess engineering

    Towards a Neisseria meningitidis B vaccine
    Neisseria meningitidis is a bacterium that is only found in humans and can cause the diseases meningitis or septicaemia, especially in young children. At the Netherlands Vaccine Institute a vaccine against serogroup B meningococci, which causes about 50% of the disease cases worldwide, is currently being developed. The pathogen itself is the basis of the vaccine. To obtain more insight in the metabolism of meningococci, a mathematical model of metabolism was constructed, using its genome sequence as a starting point and the validity of this metabolic model was checked under different experimental conditions. The gathered knowledge was used to design a synthetic medium for growth of serogroup B meningococci and for the development of a cultivation process. In addition, the research has led to the realization of equipment in which bulk production of serogroup B meningococci is possible, which brings a publically available vaccine a step closer.



    Whooping cough vaccines: production of virulent B. pertussis
    Thalen, M. - \ 2008
    Wageningen University. Promotor(en): Hans Tramper, co-promotor(en): Dirk Martens; T.W. Graaf. - S.l. : S.n. - ISBN 9789085049531 - 149
    kinkhoest - bordetella pertussis - vaccins - vaccinontwikkeling - biologische productie - bioreactoren - kweekmedia - pertussis - bordetella pertussis - vaccines - vaccine development - biological production - bioreactors - culture media
    key words: acellular, fed batch, metabolism, pertussis toxin

    The production of acellular pertussis in comparison with whole cell pertussis
    vaccines demands 5 to 25 times the amount of B. pertussis' virulence factors such
    as pertussis toxin (PT), to produce the same number of vaccine doses. An
    increase in the volumetric productivity by employing fed-batch rather than the
    currently used batch cultivations of B. pertussis could reduce the cost price of
    acellular pertussis vaccines. This study defined the conditions that enable fed
    batch cultivations at high specific PT production. A solution containing lactate and
    glutamate was fed to the cultures at various rates. The feed rate and whether or
    not the fed substrates were completely consumed, significantly influenced cellular
    metabolism. If lactate was detectable in the culture broth while glutamate was not,
    poly-hydroxy-butyrate (PHB) was formed. Any PHB present was metabolized
    when glutamate became detectable again in the culture liquid. At higher lactate
    and glutamate concentrations, free fatty acids were produced. Though toxic, free
    fatty acids were not the reason cultures stopped growing. By choosing appropriate
    conditions, a cell density of 6.5 g.L-1 dry weight was reached, i.e. a 7-fold increase
    compared to batch culture. The metabolic mechanisms behind the formation of
    PHB and fatty acids are discussed, as well as how to further increase the cell
    density. The PT production stopped at 12 mg.L-1, well before growth stopped,
    indicating that regulatory mechanisms of PT production may be involved.
    Neonatal diarrhoea in pigs: alpha- and beta(2)-toxin produced by Clostridium perfringens
    Hendriksen, S.W.M. ; Leengoed, L.A.M.G. van; Roest, H.I.J. ; Nes, A. van - \ 2006
    Tijdschrift voor Diergeneeskunde 131 (2006)24. - ISSN 0040-7453 - p. 910 - 913.
    varkenshouderij - clostridium perfringens - diergeneeskunde - diarree - biggenziekten - toxinen - vaccinatie - vaccinontwikkeling - autovaccins - diagnostische technieken - diagnose - pig farming - clostridium perfringens - veterinary science - diarrhoea - piglet diseases - toxins - vaccination - vaccine development - autogenous vaccines - diagnostic techniques - diagnosis - beta2 toxin - animals - prevalence - piglets - gene
    Since 2001 the Pig Health Unit of Utrecht University has been consulted by various pig farms regarding neonatal diarrhoea. When preventive measures against E. coli-induced diarrhoea had no or limited results, the diarrhoeic piglets were investigated further. The microbiological and pathological findings were indicative of infection with Clostridium perfringens. Toxin typing by polymerase chain reaction led to the detection of genes encoding a-toxin (cpa) and beta2-toxin (cpb2). Surprisingly, alpha- and beta2-toxin-producing C. perfringens was isolated from all tested herds with piglets with neonatal diarrhoea. From our observations, it is likely that many herds in the Netherlands are infected with beta2-toxin-producing C. perfringens strains. As present vaccines lack beta2-toxoid and thus do not provide piglets with protection against beta2-induced diarrhoea.
    Development of a novel subunit vaccine against East Coast fever based on the Theileria parva sporozoite surface protein p67
    Kaba, S.A. - \ 2003
    Wageningen University. Promotor(en): Just Vlak; R.W. Goldbach, co-promotor(en): Monique van Oers. - [S.l.] : S.n. - ISBN 9789058088895 - 120
    theileria parva - rundvee - protozoëninfecties - sporozoïten - oppervlakteantigenen - vaccinontwikkeling - infectieziekten - theileria parva - cattle - protozoal infections - sporozoites - surface antigens - vaccine development - infectious diseases
    Theileriaparva is an intracellular protozoan parasite and the causative agent of a lethal cattle disease, called East Coast fever (ECF). This disease poses a major constraint on improvement of cattle production in Eastern, Central andSouthern Africa, especially for smallholder farmers. The protozoa are transmitted to cattle in the form of sporozoites by the brown-ear tick, Riphicephalus appendiculatus . The sporozoites invade lymphocytes, where they develop into schizonts. In addition, they induce a large-scale uncontrolled proliferation of the lymphocytes, leading to severe clinical symptoms, like weight loss, pyrexia, anaemia, terminal respiratory distress and finally death ensues within two to three weeks, if the animal is not treated. The disease can be cured with antibiotics, and this is the basis for the current method of vaccination, called "Infection and Treatment", where animals are injected with T. parva sporozoites and are, simultaneously, treated with antibiotics over a longer period of time. This vaccination method, however, is far from convenient, since the production of large amounts of sporozoites is very time consuming and a cold environment is needed to keep the sporozoites alive. In addition, the use of a live vaccine has pertinent risks, especially when the instructions for antibiotic treatment are not strictly followed and the immunity engendered is strain-specific. 

    The research described in this thesis was aimed at the possibilities of developing a subunit vaccine against East Coast fever, based on the production of T. parva sporozoite surface major protein p67. This protein is present on the outside of sporozoites and plays a crucial role in the entry of sporozoites into lymphocytes and is the major antigen producing neutralising antibodies. The first objective was to produce large amounts of p67 in a near-authentic conformation. Production of recombinant p67 in bacterial expression systems had failed to produce correctly processed protein and large amounts were needed to achieve a reasonable (70 %) level of protection. The baculovirus-insect cell expression system forms a valuable alternative for the expression of large amounts of near-authentic and immunologically active proteins. Previous attempts, however, to produce p67 in insect cells resulted in low levels of recombinant protein, which had a conformation different from the native p67 protein. Again large quantities were needed to protect cattle against ECF.

    In the research described in thesis several types of novel baculovirus vectors were constructed to produce different regions of p67 in insect cells. In the first set of vectors, various domains of p67 were expressed as separate entities, but this resulted in low levels of expression. For the second set, domains of p67 were fused to the carboxy-terminus of the "green fluorescent protein" (GFP), a visible marker, leading to a considerable increase in yield of recombinant p67. In addition, GFP:p67 fusion polypeptides were recognised by a monoclonal antibody (TpM12), which was raised against native p67 and capable of neutralising sporozoites. On the contrary, only a small portion of full length, non-fused p67 expressed in insect cells was recognised by this antibody. Fusion to GFP, thus, appeared to increase the stability of p67 and to result in a more native configuration of the recombinant protein. In a third set of baculovirus vectors, N and C terminal domains of p67 were fused to the baculovirus envelope protein GP64. This resulted in the display of recombinant p67 on the outside of insect cells as well as on the surface of budded baculovirus particles. The TpM12 epitope was also conserved when p67 was fused to GP64.

    P67 could also be expressed as a secreted soluble protein. The rationale behind this experiment was to ultimately facilitate the purification of the recombinant protein. This was achieved by removal of a putative transmembrane domain and fusion of p67 to a specific signal peptide derived from honeybee melittin. Deletion of the viral genes, chitinase and v-cathepsine from the baculovirus genome enhanced the integrity and increased the stability of this secreted p67 protein. Unfortunately, the secreted form was no longer recognised by TpM12, and hence, had a conformation different from p67 in sporozoites. Therefore, the secreted p67 was not tested in further immunological studies.

    In order to select the best recombinant p67 products for extensive vaccine trials, the various fusion proteins combining domains of p67 with GFP or GP64 were tested in mice for their immunogenicity and, especially, the ability to induce neutralising antibodies. In mice, the p67 molecule, lacking both its signal peptide and transmembrane region, and fused to GFP (GFP:p67ΔSS) gave the best humoral immune response, followed by the p67 C-terminal domain coupled to GP64 (GP64:p67C). These two immunogens were tested in cattle, in combination with a water-in-oil or a saponin-based adjuvant. Also in cattle, a high level of sero-conversion was obtained using a total of 100 µg recombinant p67 for immunisation divided over two needle injections. Moreover, the antisera raised in mice and cattle neutralised the infectivity of T. parva sporozoites in an in-vitro assay. Subsequently, in Kenya Boran cattle were vaccinated with GFP:p67DSS or with GP64-p67C. After a primary immunisation followed by a single booster, T. parva stabilated sporozoites were injected to test whether the vaccines protected the animals from ECF. Eighty five percent of the animals was protected from the lethal disease (ECF) using a much lower dose of recombinant protein than was used in the earlier studies.

    The research described in this thesis exploited the versatility of the baculovirus-insect cell expression system and showed that an ECF subunit vaccine based on recombinant p67, in a better conformation and formulated in an optimal adjuvant, can be used effectively in a vaccination program. Both of the proteins tested are good candidates for the development of a commercial ECF subunit vaccine and may contribute substantially to improvement in cattle productivity and poverty alleviation in sub-SaharanAfrica

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