1,720,997 research outputs found
Plant production and immunogenic characterisation of Human papillomavirus chimaeric vaccines
No full textIncludes abstract.Includes bibliographical references (leaves 153-175).Cervical cancer is primarily caused by infection with Human papillomavirus (HPV) and is a global concern, particularly in developing countries which contain ~80% of the cervical cancer burden. Current HPV L1 major capsid protein virus-like particle (VLP)-based vaccines are effective in the type-specific prevention of infection and associated disease. However, the high cost of the vaccines has limited their widespread application, and cytological screening programmes are still required to detect malignant lesions associated with the non-vaccine types, particularly in HIV-infected populations
Production and immunogenicity of chimaeric human papillomavirus-like particle vaccines
No full textIncludes bibliographical references (leaves 129-146).Human papillomavirus (HPV) infection, specifically with oncogenic types, has been implicated in effectively all cervical cancer cases. Cervical cancer is a global health burden, especially in the developing world. Up to 18 types of HPV are considered oncogenic, of which HPV -16 and -18 cause 70% of cervical cancer cases worldwide. Two vaccines are available on the market:
Gardasil(R), targeted against HPV -16, -18; -6 and -11, and Cervarix(TM), against -16 and -18. Both vaccines are based on the L1 capsid proteins of the types they are targeted to and are efficient, pro- phylactic, typespecific vaccines. However, two problems remain: they do not protect against nonvaccine types, that may cause a significant proportion of cancers specifically in African and HIV- positive populations, and they cannot be used to treat existing infections. We designed eight different chimaeric vaccines
Developing country applications of molecular farming: case studies in South Africa and Argentina.
Get PDFMolecular farming is a technology that is very well suited to being applied in developing countries, given the reasonably high level of expertise in recombinant plant development in many centers. In addition, there is an urgent need for products such as inexpensive vaccines and therapeutics for livestock and for some human diseases – and especially those that do not occur or are rare in developed regions. South Africa and Argentina have been at the fore in this area among developing nations, as researchers have been able to use plants to produce experimental therapeutics such as nanoantibodies against rotavirus and vaccines against a wide variety of diseases, including Rabbit haemorrhagic disease virus, Foot and mouth disease virus, Bovine viral diarrhoea virus, bovine rotaviruses, Newcastle disease virus, rabbit and human papillomaviruses, Bluetongue virus, and Beak and feather disease virus of psittacines. A combination of fortuitous scientific expertise in both places, coupled with association with veterinary and human disease research centers, has enabled the growth of research groups that have managed to compete successfully with others in Europe and the USA and elsewhere, to advance this field. This review will cover relevant work from both South Africa and Argentina, as well as a discussion about the perspectives in this field for developing nations.Fil: Rybicki, Edward P.. University of Cape Town; Estados UnidosFil: Hitzeroth, Inga I.. University of Cape Town; Estados UnidosFil: Meyers, Ann. University of Cape Town; Estados UnidosFil: Dus Santos, María José. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Wigdorovitz, Andrés. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin
Development of a plant-made immunoassay for the detection of Porcine circovirus infections in South African swine herds
Get PDFPorcine circovirus type 2 (PCV-2) is considered the major cause of porcine circovirusassociated diseases and is one of the major pathogens in swine producing countries. PCV-2 is a non-enveloped virus with a single stranded circular DNA genome of about 1.8 kb. This encodes the single capsid protein (CP) which is highly immunogenic, as well as a replication-associated protein. Recombinantly expressed CP can selfassemble into virus-like particles (VLPs) that are structurally and immunogenically very similar to native virions. Current commercially available diagnostic kits are VLPbased and are effective at detecting PCV-2 antibodies in sera. However, these diagnostic assays are expensive, therefore limiting their use in developing countries. Plant-based transient expression systems have recently been investigated to express PCV-2 CP for a cheaper diagnostic reagent. The aim of this study was to develop an inexpensive lateral flow device to be able to test for PCV infection in pig herds. Production of PCV-2 CP in Nicotiana benthamiana via transient Agrobacterium-mediated expression was optimised by comparing two expression vectors, pEAQ-HT and pCBP2, and VLPs were also expressed in Escherichia coli. VLPs produced in plants and in E. coli were used to set up a lateral flow device. In addition, various purification methods of VLPs such as ion exchange chromatography (IEC) and sucrose gradient ultracentrifugation were explored to obtain pure VLPs free of bacterial contamination. The VLPs were successfully expressed in N. benthamiana with both pEAQ-HT and pCBP2, and VLPs were subsequently purified on discontinuous sucrose gradients by ultracentrifugation. The assembly of the CP was assessed by transmission electron microscopy, which showed the presence of assembled VLPs. To further purify the VLPs IEC was used, and fully assembled VLPs which were free of contamination were prepared. Purified VLPs expressed in plants and E. coli were successfully used as coating antigen in lateral flow devices, which were able to detect PCV-2 CP antibodies in CP-immunised rabbit sera. E. coli-made VLPs showed higher affinity to PCV-2 antibodies compared to plant-made VLPs. In conclusion, this study has successfully demonstrated the potential to use a plantbased transient expression system to produce affordable diagnostic reagent, especially for developing countries. This is the first study that expressed PCV-2 VLPs using a pCBP-2 expression vector and used PCV-2 VLPs as a coating reagent in the development of a lateral flow test as a proof of concept
Development of a potential challenge model and plant-produced vaccine candidate for beak and feather disease virus
No full textPsittacine beak and feather disease (PBFD), the most prevalent viral disease affecting psittacines, is caused by beak and feather disease virus (BFDV). An outbreak of the disease has been reported in wild endangered Cape parrots (Poicephalus robustus), which is endemic to South Africa. No treatment or vaccine is commercially available. In this study, an investigation into the outbreak was undertaken. BFDV diversity was assessed and viral load and clinical signs correlated. A plant-produced BFDV subunit vaccine was produced in parallel with a corresponding challenge model. Cape parrots were assessed and 53 blood samples collected. Viral load was determined using quantitative real-time PCR (qPCR), and 22 BFDV full-length genome sequences acquired to infer phylogenetic relatedness. The capsid gene (cp) was optimised for transient Agrobacterium-mediated expression in whole-plant Nicotiana benthamiana (N. benthamiana). Virus-like particles (VLPs) were purified and analysed using transmission electron microscopy. Virions from a Palm cockatoo (Probosciger aterrimus) were purified and a BFDV dsDNA molecular clone was synthesised and replication assessed in 293TT mammalian cells and N. benthamiana using rolling circle replication and qPCR. Two distinct BFDV phylogenetic clusters were reported for Cape parrots, and a direct correlation was seen between viral load in the blood and clinical signs in PBFD-afflicted birds. The CP was successfully expressed in N. benthamiana, and increased through optimisation of Agrobacterium infiltration density and the inclusion of the NSs silencing suppressor. The CP formed VLPs, which were shown to be morphologically similar to infectious virions. The dsDNA molecular clone was shown to replicate autonomously in mammalian 293TT cells, and in plants with the assistance of the Bean yellow dwarf virus replication associated protein (Rep). BFDV genetic diversity in Cape parrots highlights the importance of ensuring new strains are not inadvertently introduced into the wild. This is the first systematic investigation of virus diversity in Cape parrots and assessment of BFDV viral load in a wild psittacine population. The CP was successfully produced in planta and presence of VLPs suggests the possibility of developing pseudovirions. This is the first reported replication of BFDV in tissue culture, and will greatly expand the scope of available research
Plant production of Gonococcal peptide vaccine, candidate peptide display with HPVs
No full textHuman Papillomavirus (HPV) and Neisseria gonorrhoeae (Ng) are prominent pathogens responsible for a significant proportion of the global burden attributed to sexually transmitted infections. While vaccines targeting HPV have seen success, an effective vaccine against gonorrhea remains elusive, as antimicrobial resistance continues to be a growing threat. This study explores the innovative approach of plant-based production for a dual vaccine against HPV16 and gonorrhea, utilizing N. benthamiana as the expression host. The vaccine design involves the incorporation of a gonorrhea peptide, mimicking the 2C7 epitope of the surface molecule LOS on gonorrhea, into the surface DE loop of HPV16 virus-like particles (VLPs). These chimeric VLPs as well as HPV16 VLPs were expressed and successfully purified from plants and demonstrated self-assembly into VLPs. The choice of N. benthamiana as the expression system is informed by its suitability for efficient and cost-effective recombinant protein production. Immunological evaluations were conducted in mice to assess the immune response elicited by the dual vaccine. The mice displayed robust antibody responses against both HPV and gonorrhea, indicating the insertion of the peptide does not disrupt the binding of antibodies to HPV16 and highlights the potential of the vaccine candidate to induce a dual protective immunity. To assess the protective efficacy of the vaccine candidate, a challenge model was developed and optimized, involving the use of HPV16 PsVs containing luciferase (FLuc). The challenge model was refined through a comparison of secreted and non-secreted reporter proteins, followed by comparison of plant and mammalian expression systems. This showed thatHPV16 PsVs containing non-secreted FLuc produced in mammalian cell culture were optimal for the challenge. Mice vaccinated with the plant-produced dual vaccine demonstrated protection against HPV infection upon challenge. In summary the chimeric and HPV16 VLP vaccine candidates were expressed in an Agrobacterium-mediated transient system in plants. The immunogenicity of the vaccine candidates was accessed in a mouse model and a further challenge model. The results demonstrated the potential of using VLPs in the display of foreign epitopes in the fight against gonorrhea and HPV. The findings contribute valuable insights into the development of vaccines against sexually transmitted infections, paving the way for innovative strategies in the field of VLP display molecules and plant molecular pharming
Towards the development of plant-made PsVs as potential delivery vehicles for therapeutic HPV vaccines
Get PDFInfection with human papillomavirus (HPV) is the leading cause of cervical cancer, the fourth most common cancer in women globally. Cervical cancer results in an estimated 604,000 new cases and 342,000 deaths each year, with the majority of these cases reported in sub-Saharan Africa. HPV-16 is a high-risk oncogenic subtype and, along with HPV-18, is associated with >70% of all cervical cancers. While current vaccines can prevent infection with high-risk HPVs, they cannot induce regression of persistent infections, thus the development of vaccines that function therapeutically is required. DNA vaccines are ideal candidates for therapeutic treatment; however, naked DNA vaccines are associated with ineffective presentation to antigen presenting cells (APCs). This limitation can be overcome by using pseudovirions (PsVs) as vaccine delivery vehicles. HPV PsVs are highly immunogenic synthetic viral particles consisting of L1 and L2 capsid proteins, which self-assemble to package pseudogenome DNA. Pre-existing immunity to high-risk HPVs through natural infection and vaccination, however, preclude their use as delivery vehicles for DNA vaccines. The development of non-human papillomaviruses (PVs) PsVs for gene delivery is a novel alternative. PV PsVs are conventionally produced in mammalian cells. However, plants have demonstrated potential as an alternative platform for rapid PsV production due to their scalability and cost-efficiency. Therefore, the aim of this study was to investigate plant-based production of bovine papillomavirus 1 (BPV-1) PsVs encapsidating a HPV-16 therapeutic DNA vaccine candidate, and to assess their infectivity in mammalian cells. Initially, strategies to optimise Agrobacterium-mediated transient expression of BPV-1 L1 and L2 capsid proteins in Nicotiana benthamiana were explored. Approaches such as an increased acetosyringone concentration for recombinant Agrobacterium induction, a heat-shock treatment of post-infiltrated plants, and an extended in planta maturation were investigated. A pseudogenome encoding secreted embryonic alkaline phosphatase (SEAP) was co-infiltrated with BPV-1 L1- and L2-encoding expression vectors. L1 protein expression and particle assembly were confirmed with western blotting and transmission electron microscopy (TEM) respectively. However, no SEAP expression was observed following infection of HEK293TT cells with the plant-made particles and none of the strategies investigated were conclusively found to increase BPV-1 PsV yield. Several geminivirus-derived self-replicating reporter plasmids encoding a HPV-16 shuffled E7 (E7SH) sequence were constructed using In-Fusion cloning. These constructs were co-expressed in N. benthamiana with expression vectors encoding BPV-1 or HPV-35 L1 and L2 proteins. HPV-35 was chosen as it has been shown to encapsidate a Zera®E7SH DNA vaccine in plants for delivery to mammalian cells in vitro. Following purification, rolling circle amplification (RCA) analysis showed successfully encapsidation of the E7SH-based pseudogenomes within the plant-made PsVs. However, TEM showed that PsV yields were low and no E7 expression was observed following infection of HEK293TT cells with the plant-made PsVs. These results indicated that encapsidation efficiency of PsVs produced in plants is low. BPV1 and HPV-35 PsVs were also produced in HEK293TT cells and comparative analyses with plant-made particles revealed that, while the pseudogenomes were successfully encapsidated in the PsVs produced in both systems, only the HEK293TT-made PsVs effectively delivered their packaged DNA into mammalian cells. These findings demonstrate the ability of PsVs to self-assemble and encapsidate pseudogenome DNA in planta while also revealing potential limitations of plant-based PsV production. For plant-made PsVs to reach their potential in gene delivery, further optimisation and characterisation of plant-based PV PsV expression is required
Enhancement of plant expression vectors using replication and silencing suppressor elements
Get PDFMolecular farming is gaining traction as a cost-effective platform to produce recombinant proteins. Further improvements can be made, however, to increase overall yield especially for difficult to express proteins. In this study virus-derived silencing suppressors and replication elements were used with the aim of increasing expression and yield of enhanced green fluorescent protein (EGFP) and the Zika PrME polyprotein in Nicotiana benthamiana. A comparison of four viral silencing suppressor proteins was performed: these were tomato spotted wilt virus non-structural protein, NSs, tomato aspermy virus (TAV) 2b, tomato bushy stunt virus P19 and begomovirus alphasatellite Rep. Differences in EGFP expression in N. benthamiana due to the silencing suppression were determined using immunoblotting and fluorescence of EGFP. In addition, replication elements from three viruses (bean yellow dwarf virus [BeYDV], beak and feather disease virus [BFDV] and begomovirus alphasatellite) were assembled into novel plant expression vectors using GoldenBraid (GB) cloning technology and assessed using EGFP. Finally, the two approaches were combined in an attempt to express the Zika PrME polyprotein, which was assessed using immunoblotting. EGFP expression was found to be greatest in the presence of the TAV 2b protein and no difference in fluorescence intensity between the original BeYDV replicating plant expression vector and that constructed using GB could be detected; however, the GB assembly of the BFDV and alphasatellite plant expression vectors was unsuccessful. The TAV 2b combined with the BeYDV replicating elements were used for the expression of Zika PrME. The gene was successfully cloned into the replicating BeYDV vector and a vector that does not replicate (negative control). The PrME was not detected using anti-His tag immunoblotting despite optimisation for Agrobacterium infiltration density, harvest day post infiltration, signal peptides and buffers during extraction. In this study I demonstrated the following: that the TAV 2b protein out-performed all other silencing suppressors; that the GB cloning technology can be successfully applied in the development of novel plant expression vectors, although further optimisation is required for these and for Zika PrME expression. Further work in characterising the effect of silencing suppression on recombinant protein expression can be assessed using RT-qPCR to measure the effect on mRNA levels. In summary, these improvements in plant recombinant protein expression can be readily applied to large scale production of novel therapeutics and vaccines
The Immunogenicity of Plant-produced Human Papillomavirus (HPV) Virus-like particles (VLPs) in Mice
Get PDFCervical cancer is caused by infection with high-risk Human papillomaviruses (HPVs). It is ranked fourth among the top cancers in women worldwide, with ~87% of the global cervical cancer cases reported in developing countries. The HPV L1 capsid protein can self-assemble into virus-like particles (VLPs) that are structurally like native virions, which is the foundation on which commercially available vaccines have been developed. There are 3 commercially available HPV vaccines that are effective at preventing HPV infections, but are expensive, therefore limiting their use in the poorer developing countries where they are most needed. Thus, there is a need for more cost-effective HPV vaccines for use in these countries. Over the years, the use of plants to produce vaccines has begun to be more favourably looked upon as a costeffective alternative to conventionally used expression systems. The aim of this study was to evaluate the plant-based transient expression system as a tool to produce potentially cost-effective HPV L1 VLP-based vaccines, particularly for developing countries. Firstly, the L1 proteins of the 8 most common high-risk Human papillomavirus types in Africa (HPV 16, 18, 31, 33, 35, 45, 52, and 58) and 2 low risk types (HPV 6 and 34) were transiently expressed in Nicotiana benthamiana. The proteins were purified via isopycnic ultracentrifugation using sucrose and Optiprep™ density gradients, and the assembly of VLPs assessed by transmission electron microscopy (TEM). To further assess whether the VLPs are immunogenic, HPV 35, 52 and 58 were selected for mice studies. These were selected in particular, as HPV 35 is the fifth most prevalent type in Africa, and HPV 52 and 58 are among the most frequently reported high-risk types in Sub-Saharan Africa. VLPs representing the 3 HPV types were quantified and prepared for immunization in mice. The commercially available Gardasil® HPV VLP vaccine was used as a positive control. The immunogenicity of the vaccines was evaluated by testing for the presence of anti-L1 antibodies in sera from immunized mice using enzyme-linked immunosorbent assays (ELISAs) and western blots. Sera from immunized mice were also tested for the presence of neutralizing antibodies using pseudovirion based neutralization assays (PBNAs). L1 proteins of all 10 HPV types tested were successfully expressed in N. bethamiana, and TEM analysis showed that expression resulted in the successful formation of fully assembled VLPs (40-60nm) as well as small VLPs and/or capsomeres (25-39nm). The analysis of the immune response showed that type-specific L1-specific antibodies were produced which were able to successfully neutralize homologous pseudovirions (PsVs) in PBNAs. Sera from mice immunized with plant-produced VLPs were further tested against heterologous HPV 6, 16, 18, 31, and 45 PsVs. However, none of the tested heterologous HPVs were neutralized, suggesting that plant-made VLPs induced type-specific neutralizing antibodies only. In conclusion, this study successfully demonstrated the potential for using plant-based transient expression systems to produce affordable and immunogenic HPV vaccines, particularly for developing countries. This is the first study describing the expression of 10 HPV L1 proteins in plants, marking a step towards the development of cheaper HPV vaccines which could be combined to generate an effective multivalent vaccine against HPVs
Setting up a platform for plant-based influenza virus vaccine production in South Africa
Get PDFAbstract Background During a global influenza pandemic, the vaccine requirements of developing countries can surpass their supply capabilities, if these exist at all, compelling them to rely on developed countries for stocks that may not be available in time. There is thus a need for developing countries in general to produce their own pandemic and possibly seasonal influenza vaccines. Here we describe the development of a plant-based platform for producing influenza vaccines locally, in South Africa. Plant-produced influenza vaccine candidates are quicker to develop and potentially cheaper than egg-produced influenza vaccines, and their production can be rapidly upscaled. In this study, we investigated the feasibility of producing a vaccine to the highly pathogenic avian influenza A subtype H5N1 virus, the most generally virulent influenza virus identified to date. Two variants of the haemagglutinin (HA) surface glycoprotein gene were synthesised for optimum expression in plants: these were the full-length HA gene (H5) and a truncated form lacking the transmembrane domain (H5tr). The genes were cloned into a panel of Agrobacterium tumefaciens binary plant expression vectors in order to test HA accumulation in different cell compartments. The constructs were transiently expressed in tobacco by means of agroinfiltration. Stable transgenic tobacco plants were also generated to provide seed for stable storage of the material as a pre-pandemic strategy. Results For both transient and transgenic expression systems the highest accumulation of full-length H5 protein occurred in the apoplastic spaces, while the highest accumulation of H5tr was in the endoplasmic reticulum. The H5 proteins were produced at relatively high concentrations in both systems. Following partial purification, haemagglutination and haemagglutination inhibition tests indicated that the conformation of the plant-produced HA variants was correct and the proteins were functional. The immunisation of chickens and mice with the candidate vaccines elicited HA-specific antibody responses. Conclusions We managed, after synthesis of two versions of a single gene, to produce by transient and transgenic expression in plants, two variants of a highly pathogenic avian influenza virus HA protein which could have vaccine potential. This is a proof of principle of the potential of plant-produced influenza vaccines as a feasible pandemic response strategy for South Africa and other developing countries.</p
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