Browsing by Author "Hitzeroth, Inga I"
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- ItemOpen AccessDesign and production of a candidate universal influenza A vaccine in Nicotiana benthamiana plants(2017) De Figueiredo Pinto Gomes Pera, Francisco; Hitzeroth, Inga I; Rybicki, Edward PThe influenza A virus is responsible for 250,000 to 500,000 deaths every year worldwide and millions more could die in the event of a serious pandemic. Vaccines against influenza have existed for long, but until today they have been limited by extensive production times and reduced cross-protection between different strains of the virus. This leads to a recurrent need to update the vaccine composition every year, which is both costly and inadequate to fight pandemics. An innovative approach that could improve the vaccine efficacy has been recently developed based on the selection of conserved influenza epitopes with potential to induce broader immune responses. The 23-amino acid extracellular domain of the M2 protein (M2e) is highly conserved among different influenza A strains and thus it seems like an ideal candidate for a universal influenza vaccine. However, due to its small size, it is a poor immunogen when used on its own. The aim of this project was to produce M2e-presenting virus-like particles (VLPs) in Nicotiana benthamiana plants via Agrobacterium-mediated transient expression. Plants are increasingly being examined as alternative recombinant protein expression systems due to their safety, scalability and rapid production times. Moreover, numerous studies suggest the use of recombinant virus-like particles (VLPs) to increase the immunogenicity of antigens. Therefore, to obtain VLPs presenting the M2e epitope, I genetically engineered several different M2e-HA fusion proteins by replacing the hemagglutinin (HA) globular head and main epitope with five tandem repeats of M2e epitope sequences (5xM2e) from human, swine, and avian origin influenza A viruses. To increase the chances of obtaining VLPs, M2e-HA fusions either contained the HA stalk domain (5xM2e-HAstalk) or simply the transmembrane region (5xM2e-HAtrans). Furthermore, the tetramerizing leucine zipper derived from the General Control Protein (GCN4) was also included in some of the constructs to promote particle formation. In total, six different M2e-HA fusions were created: 5xM2e-GCN4-HAstalk, 5xM2e-GCN4-HAtrans, 5xM2e-HAstalk, 5xM2e-HAtrans, 1xM2e-HAstalk and 1xM2e-HAtrans. The expression of these proteins was optimized in plants by testing different conditions and using three different expression vectors. Overall, I was able to show expression after only 3 days post-infiltration for most of the M2e-HA v fusion proteins utilizing the pEAQ-HT and pRIC 3.0 expression vectors whereas expression levels with pTRAc were low or non-detectable. Once the expression of the M2e-HA fusions was optimized, the two proteins with the highest potential to form VLPs were selected for further characterization (5xM2e-HAstalk and 5xM2eHAtrans). Using transmission electron microscopy to analyse purified proteins, both 5xM2eHAstalk and 5xM2e-HAtrans were shown to assemble into VLPs resembling the shape and size of native HA VLPs. These VLPs could also be observed in the apoplastic fractions of infiltrated leaves. However, due to the low number of particles observed, the successful incorporation of the M2e peptide on the surface of the particles was inconclusive, as shown by M2e-specific immuno-gold labelling experiments. Furthermore, contrarily to previous studies, co-expression of the M2e-HA fusions with the M1 protein resulted in a decrease in recombinant protein accumulation and VLP formation in our plant system. A possible inhibition mechanism by the M1 protein is discussed. In summary, this research provides preliminary data to produce universal influenza vaccines in plants. I report here for the first time that M2e fused to either the stalk or transmembrane domain of the HA protein, can self-assemble into VLPs without any other proteins, in N. benthamiana plants. Future work on the immunogenicity of the VLPs produced in this study is required to confirm their potential as a universal influenza vaccine that can be rapidly produced.
- ItemOpen AccessHuman papillomavirus (HPV) type 16 E7 protein bodies cause tumour regression in mice(2014-05-24) Whitehead, Mark; Öhlschläger, Peter; Almajhdi, Fahad N; Alloza, Leonor; Marzábal, Pablo; Meyers, Ann E; Hitzeroth, Inga I; Rybicki, Edward PAbstract Background Human papillomaviruses (HPV) are the causative agents of cervical cancer in women, which results in over 250 000 deaths per year. Presently there are two prophylactic vaccines on the market, protecting against the two most common high-risk HPV types 16 and 18. These vaccines remain very expensive and are not generally affordable in developing countries where they are needed most. Additionally, there remains a need to treat women that are already infected with HPV, and who have high-grade lesions or cervical cancer. Methods In this paper, we characterize the immunogenicity of a therapeutic vaccine that targets the E7 protein of the most prevalent high-risk HPV - type 16 – the gene which has previously been shown to be effective in DNA vaccine trials in mice. The synthetic shuffled HPV-16 E7 (16E7SH) has lost its transforming properties but retains all naturally-occurring CTL epitopes. This was genetically fused to Zera®, a self-assembly domain of the maize γ-zein able to induce the accumulation of recombinant proteins into protein bodies (PBs), within the endoplasmic reticulum in a number of expression systems. Results High-level expression of the HPV 16E7SH protein fused to Zera® in plants was achieved, and the protein bodies could be easily and cost-effectively purified. Immune responses comparable to the 16E7SH DNA vaccine were demonstrated in the murine model, with the protein vaccine successfully inducing a specific humoral as well as cell mediated immune response, and mediating tumour regression. Conclusions The fusion of 16E7SH to the Zera® peptide was found to enhance the immune responses, presumably by means of a more efficient antigen presentation via the protein bodies. Interestingly, simply mixing the free PBs and 16E7SH also enhanced immune responses, indicating an adjuvant activity for the Zera® PBs.
- ItemOpen AccessImmunogenic assessment of plant-produced Human papillomavirus type 16 chimaeric L1:L2 virus-like particles and the production of an encapsidated therapeutic DNA vaccine candidate(2017) Chabeda, Eva Aleyo; Hitzeroth, Inga I; Rybicki, Edward PCervical cancer caused by infection with Human papillomavirus (HPV) is the 4th most common cancer in women globally, and results in an estimated 266 000 deaths every year. Current vaccines are based on the immunodominant L1 major capsid protein, which assembles into virus-like particles (VLPs) that are highly effective in type-specific prevention of cervical infection. However, these vaccines are produced in expensive cell culture systems, are type-specific and do not induce the regression of established infections. The cervical cancer burden (~80%) is mainly in developing countries due to limited healthcare resources, therefore there is a need for more broadly protective and affordable vaccines. Plants provide an alternative platform to produce cheaper vaccines, given their scalability, rapid production and low risk of contamination. The L2 minor capsid protein has sequence regions that are highly conserved across several HPV types, and HPV-16 L2 peptides 108-120, 65-81, 56-81 and 17-36 have been shown to elicit cross-neutralising antibodies. To increase the immunogenicity of L2, second-generation L1:L2 chimaeric VLP (cVLP) vaccines have been investigated. In this study, the 4 L2 peptides above were used to generate plant-produced HPV-16-derived L1:L2 chimaeras. The L2 epitopes were substituted into the DE loop of HPV-16 L1 at position 131 (SAC) or between the helix 4 and β-J structural region at position 431 (SAE). All chimaeras were transiently expressed in Nicotiana benthamiana via Agrobacterium-mediated transfer. Optimisation of expression was conducted by comparing protein expression levels over several days using 4 plant expression vectors, with the highest yields obtained by targeting protein to the chloroplast or with the use of a self-replicating vector. The chloroplast targeted SAC chimaeras predominantly assembled into higher order structures (T=1 VLPs and T=7 VLPs), whereas SAE chimaeras assembled into capsomeres or formed aggregates, indicating that the length, sequence and substitution position of L2 epitopes affects VLP assembly. All SAC chimaeras in addition to SAE 65-81 (smaller epitope not previously tested in chimaeras) were used in vaccination studies in mice, and their immunogenic potential analysed in pseudovirion-based neutralisation assays (PBNAs). Of the 7 heterologous HPVs tested, cross-neutralisation was observed with HPV-11, -18 and -58. Only the anti-SAE 65-81 serum showed neutralisation of homologous HPV-16, suggesting that antibodies detected from all candidate vaccines were mostly non-neutralising, and that the position of the L2 epitope display is critical to maintaining L1-specific neutralising epitopes. Lastly, to address the lack of therapeutic efficacy of current vaccines, I aimed to develop a novel E7 DNA vaccine delivered by plant-made pseudovirions (PsVs). A geminivirus-derived self-replicating plasmid encoding a shuffled E7 (E7SH) sequence that has no transformation ability but contains natural cytotoxic T-lymphocyte epitopes, was constructed using Goldenbraid technology and co-expressed in plants with HPV-16 or HPV-35 L1- and L2-encoding expression vectors. The pseudogenome was successfully encapsidated into plant-made PsVs. These PsVs were capable of infecting mammalian cells and encapsidated replicons expressed E7SH showing the promise of this candidate vaccine as a future combination prophylactic and therapeutic vaccine.
- ItemOpen AccessRecombinant expression of beak and feather disease virus capsid protein and assembly of virus-like particles in Nicotiana benthamiana(BioMed Central, 2017-09-11) Regnard, Guy L; Rybicki, Edward P; Hitzeroth, Inga IBackground: Beak and feather disease virus (BFDV) is an important disease causing agent affecting psittacines. BFDV is highly infectious and can present as acute, chronic or subclinical disease. The virus causes immunodeficiency and is often associated with secondary infections. No commercial vaccine is available and yields of recombinant BFDV capsid protein (CP) expressed in insect cells and bacteria are yet to be seen as commercially viable, although both systems produced BFDV CP that could successfully assemble into virus-like particles (VLPs). Plants as expression systems are increasingly becoming favourable for the production of region-specific and niche market products. The aim of this study was to investigate the formation and potential for purification of BFDV VLPs in Nicotiana benthamiana. Methods: The BFDV CP was transiently expressed in N. benthamiana using an Agrobacterium-mediated system and plant expression vectors that included a bean yellow dwarf virus (BeYDV)-based replicating DNA vector. Plant-produced BFDV CP was detected using immunoblotting. VLPs were purified using sucrose cushion and CsCl density gradient centrifugation and visualised using transmission electron microscopy. Results: In this study we demonstrate that the BFDV CP can be successfully expressed in N. benthamiana, albeit at relatively low yield. Using a purification strategy based on centrifugation we demonstrated that the expressed CP can self-assemble into VLPs that can be detected using electron microscopy. These plant-produced BFDV VLPs resemble those produced in established recombinant expression systems and infectious virions. It is possible that the VLPs are spontaneously incorporating amplicon DNA produced from the replicating BeYDV plant vector. Conclusions: This is the first report of plant-made full-length BFDV CP assembling into VLPs. The putative pseudovirions could be used to further the efficacy of vaccines against BFDV.
- ItemOpen AccessSafety and immunogenicity of plant-produced African horse sickness virus-like particles in horses(BioMed Central, 2018-10-11) Dennis, Susan J; O’Kennedy, Martha M; Rutkowska, Daria; Tsekoa, Tsepo; Lourens, Carina W; Hitzeroth, Inga I; Meyers, Ann E; Rybicki, Edward PAbstract African horse sickness (AHS) is caused by multiple serotypes of the dsRNA AHSV and is a major scourge of domestic equids in Africa. While there are well established commercial live attenuated vaccines produced in South Africa, risks associated with these have encouraged attempts to develop new and safer recombinant vaccines. Previously, we reported on the immunogenicity of a plant-produced AHS serotype 5 virus-like particle (VLP) vaccine, which stimulated high titres of AHS serotype 5-specific neutralizing antibodies in guinea pigs. Here, we report a similar response to the vaccine in horses. This is the first report demonstrating the safety and immunogenicity of plant-produced AHS VLPs in horses.
- ItemRestrictedA unique isolate of beak and feather disease virus isolated from budgerigars (Melopsittacus undulatus) in South Africa(Springer Verlag, 2010) Varsani, Arvind; de Villiers, Gillian K; Regnard, Guy L; Bragg, Robert R; Kondiah, Kulsum; Hitzeroth, Inga I; Rybicki, Edward PBeak and feather disease virus (BFDV), the causative agent of psittacine beak and feather disease (PBFD) infects psittaciformes worldwide. We provide an annotated sequence record of three full-length unique genomes of BFDV isolates from budgerigars (Melopsittacus undulatus) from a breeding farm in South Africa. The isolates share[99% nucleotide sequence identity with each other and *96% nucleotide sequence identity to two recent isolates (Melopsittacus undulatus) from Thailand but only between 91.6 and 86.6% identity with all other full-length BFDV sequences. Maximum-likelihood analysis and recombination analysis suggest that the South African budgerigar BFDV isolates are unique to budgerigars, are non-recombinant in origin, and represent a new genotype of BFDV.