Use of baculovirus expression vectors: development of diagnostic reagents, vaccines and morphological counterparts of bluetongue virus

The productivity and flexibility of insect baculovirus expression vectors and the ability of the baculovirus genome to incorporate (and express) large amounts of foreign DNA allows this system to be used for both single and multiple gene expression. Using the system, bluetongue virus (BTV) genes have been expressed to develop diagnostic reagents and vaccines as well as to understand the basic structures of the virions. BTV which causes disease in ruminants in many parts of the world, consists of 10 double-stranded RNA segments enclosed by double capsids that are composed by 7 structural proteins. Since each protein is encoded by a single RNA species, DNA clones of all 10 RNA species were synthesized and individually expressed in baculovirus vectors at high levels. This has yielded proteins that have been shown to be excellent diagnostic and vaccine reagents. In addition, multiple expression vectors have been used to synthesize morphological structures (viral and subviral) representing BTV.     

A suite of parallel vectors for baculovirus expression

The expression of proteins using recombinant baculoviruses is a mature and widely used technology. However, some aspects of the technology continue to detract from high throughput use and the basis of the final observed expression level is poorly understood. Here, we describe the design and use of a set of vectors developed around a unified cloning strategy that allow parallel expression of target proteins in the baculovirus system as N-terminal or C-terminal fusions. Using several protein kinases as tests we found that amino-terminal fusion to maltose binding protein rescued expression of the poorly expressed human kinase Cot but had only a marginal effect on expression of a well-expressed kinase IKK-2. In addition, MBP fusion proteins were found to be secreted from the expressing cell. Use of a carboxyl-terminal GFP tagging vector showed that fluorescence measurement paralleled expression level and was a convenient readout in the context of insect cell expression, an observation that was further supported with additional non-kinase targets. The expression of the target proteins using the same vectors in vitro showed that differences in expression level were wholly dependent on the environment of the expressing cell and an investigation of the time course of expression showed it could affect substantially the observed expression level for poorly but not well-expressed proteins. Our vector suite approach shows that rapid expression survey can be achieved within the baculovirus system and in addition, goes some way to identifying the underlying basis of the expression level obtained.     

Development of baculovirus triple and quadruple expression vectors: co-expression of three or four bluetongue virus proteins and the synthesis of bluetongue virus-like particles in insect cells.

Baculovirus multiple gene transfer vectors pAcAB3 and pAcAB4 have been developed to facilitate the insertion of three or four foreign genes respectively into the Autographa californica nuclear polyhedrosis virus (AcNPV) genome by a single co-transfection experiment. The pAcAB3 vector contains a polyhedrin promoter and two p10 promoters on either side of the polyhedrin promoter but in opposite orientations. The pAcAB4 vector has an additional polyhedrin promoter in opposite orientation to the first copy that is in juxtaposition to the first p10 promoter. Each of these derived vectors (pAcAB3, pAcAB4) have been used for the simultaneous expression of three or four bluetongue virus (BTV) genes respectively. When Spodoptera frugiperda cells were infected with the recombinant virus (AcBT-3/2/7/5) expressing the four major structural genes of BTV, double-capsid, virus-like particles consisting of VP2, VP3, VP5 and VP7 of BTV were assembled.     

Laboratory rearing of immature Culicoides peregrinus Kieffer,a potential vector of bluetongue virus

Culicoides peregrinus (Kieffer) (Diptera: Ceratopogonidae) was reared from egg to adult using four different combinations of food and substrate (T1: absorbent cotton, mud broth with 2% yeast; T2: 1% agar, mud broth with 2% yeast; T3: absorbent cotton, nutrient broth; T4: absorbent cotton, 2% yeast). Field‐collected engorged females exhibited mean fecundity of 82.45 ± 4.00. The highest rate of emergence and largest adults were obtained in T1, and the lowest rate of emergence was observed in T4. Two‐way analyses of variance with post hoc Tukey tests showed significant differences in age at pupation, pupal weight and wing length among the various treatments, although the sex ratio was 1 : 1 in all food/substrate combinations. The successful rearing of immature C. peregrinus is an encouraging step towards the establishment of a laboratory colony of this prevalent species associated with livestock.     

Expression of human immunodeficiency virus genes using baculovirus expression system

The structural protein genes of HIV-1 and HIV-2 have been expressed inSpodoptera frugiperda (SF) cells using baculovirus expression system. The noncoding flanking sequences of HIV structural genes were removed and a putative ribosome binding site was placed in front of the open reading frame of each gene by using crossover linker mutagenesis. The coding sequences of thegag, pol, env, andvif proteins were inserted intoAutographa californica nuclear polyhedrosis virus (AcNPV) so that HIV genes were under the control of the AcNPV polyhedrin promoter. All recombinant AcNPV-infected SF cells express high levels of HIV structural proteins. Detailed strategies of recombinant AcNPV construction for high level protein expression are presented.     

Spatial distribution of bluetongue virus and its Culicoides vectors in Sicily

During the recent Mediterranean epizootic of bluetongue, an extensive programme of serological and vector (Culicoides biting midges (Diptera: Ceratopogonidae)) surveillance was carried out across Sicily. This paper presents the analysis of 911 light trap catches collected at the times of peak Culicoides abundance (summer to autumn 2000-2002) in 269 sites, in order to produce detailed maps of the spatial distribution of the main European vector, Culicoides imicola Kieffer and that of potential novel vectors. Whereas C. imicola was found at only 12% of sites, potential novel vectors, Culicoides obsoletus group Meigen, Culicoides pulicaris Linnaeus and Culicoides newsteadi Austen were present at over 50% of sites. However, the spatial distribution of C. imicola showed the closest correspondence to that of the 2000 and 2001 bluetongue (BT) outbreaks and its presence and abundance were significant predictors of the probability of an outbreak, suggesting that it was the main vector during these years. Although C. imicola may have played a role in transmission in several sites near Paternó, it was absent from the majority of sites at which outbreaks occurred in 2002 and from all sites in the province of Messina. All three potential novel vectors were widespread across sites at which outbreaks occurred during 2002. Of these, C. newsteadi was an unlikely candidate, as it was significantly less prevalent in outbreak vs. non-outbreak sites in Messina. It is hypothesized that the yearly distribution and intensity of outbreaks is directly attributable to the distribution and abundance of the vectors involved in transmission during each year. When C. imicola operated as the main vector in 2000 and 2001, outbreaks were few in number and were restricted to coastal regions due to low abundance and prevalence of this species. In 2002, it is hypothesized that BTV transmission was handed over to more prevalent and abundant novel vector species, leading to numerous and widespread outbreaks and probably to overwintering of the virus between 2001 and 2002. Based on catch ranges in outbreak vs. non-outbreak sites, it is tentatively suggested that nightly catches of 400 or more C. obsoletus and 150 or more C. pulicaris allow BTV transmission at a site, and provide a strategy for a fuller examination of the relationship between BTV transmission and the abundance and distribution of different vector species.     

Indoor and outdoor winter activity of Culicoides biting midges,vectors of bluetongue virus,in Italy

Indoor and outdoor winter activity of Culicoides spp. (Diptera: Ceratopogonidae) in central Italy was investigated in order to evaluate whether indoor activity might account for the overwintering of bluetongue virus, as has been hypothesized by some authors. Weekly Culicoides collections were performed at three farms over three consecutive winter seasons. At each farm, two black‐light traps were operated simultaneously, indoors and outdoors. Culicoides were identified using both morphological and molecular means. The Culicoides obsoletus group accounted for 98.2% of sampled specimens. Within this group, C. obsoletus s.s. accounted for 56.8% and Culicoides scoticus for 43.2% of samples. Nulliparous, parous and engorged females were caught throughout the entire winter, both indoors and outdoors. At times, indoor catch sizes outnumbered outdoor collections. A significant inverse correlation was found between minimum temperature and the proportion of indoor Culicoides of the total midge catch, thus indicating that lower outdoor temperatures drive Culicoides midges indoors. High rates of engorged females were recorded indoors, possibly as the result of the propensity of C. obsoletus females to feed indoors. Higher proportions of parous females were found in indoor than in outdoor catches, indicating higher survival rates indoors and, consequently, higher vectorial capacities of midges sheltering indoors compared with those remaining outdoors.     

Recombinant BCG approach for development of vaccines: cloning and expression of immunodominant antigens of M. tuberculosis

In spite of major advances in our understanding of the biology and immunology of tuberculosis, the incidence of the disease has not reduced in most parts of the world. In an attempt to improve the protective efficacy of Mycobacterium bovis bacille Calmette-Guérin (BCG), we have developed a generic vector system, pSD5, for expression of genes at varying levels in mycobacteria. In this study, we have cloned and overexpressed three immunodominant secretory antigens of M. tuberculosis, 85A, 85B and 85C, belonging to the antigen 85 complex. All the genes were cloned under the control of a battery of mycobacterial promoters of varying strength. The expression was analysed in the fast-growing strain M. smegmatis and the slow-growing vaccine strain M. bovis BCG. The recombinant BCG constructs were able to express the antigens at high levels and the majority of the expressed antigens was secreted into the medium. These results show that by using this strategy the recombinant BCG approach can be successfully used for the development of candidate vaccines against infections associated with mycobacteria as well as other pathogens.     

The susceptibility of Culicoides imicola and other South African livestock-associated Culicoides species to infection with bluetongue virus serotype 8

In 2006, a strain of bluetongue virus serotype 8 (BTV-8) of sub-Saharan origin was responsible for the first outbreaks in recorded history of clinical bluetongue disease (BT) in northern Europe. In this study, we examine the oral susceptibility of Culicoides (Avaritia) imicola Kieffer (Diptera: Ceratopogonidae) and other livestock-associated Culicoides species from southern Africa to infection with several strains of BTV-8. Following feeding using an artificial membrane-based method and incubation, virus was found in <1% of C. imicola individuals tested. Higher rates of susceptibility were found, however, for a variety of other South African species, including Culicoides (Avaritia) bolitinos Meiswinkel. Although these results do not preclude the role of C. imicola as a vector of BTV-8, its low susceptibility to BTV indicates that other less abundant Culicoides species may have the potential to play decisive roles in the epidemiology of this virus and should not be excluded from risk assessment studies.     

Advancements in molecular design and bioprocessing of recombinant adeno-associated virus gene delivery vectors using the insect-cell baculovirus expression platform

Despite rapid progress in the field, scalable high-yield production of adeno-associated virus (AAV) is still one of the critical bottlenecks the manufacturing sector is facing. The insect cell-baculovirus expression vector system (IC-BEVS) has emerged as a mainstream platform for the scalable production of recombinant proteins with clinically approved products for human use. In this review, we provide a detailed overview of the advancements in IC-BEVS for rAAV production. Since the first report of baculovirus-induced production of rAAV vector in insect cells in 2002, this platform has undergone significant improvements, including enhanced stability of Bac-vector expression and a reduced number of baculovirus-coinfections. The latter streamlining strategy led to the eventual development of the Two-Bac, One-Bac, and Mono-Bac systems. The one baculovirus system consisting of an inducible packaging insect cell line was further improved to enhance the AAV vector quality and potency. In parallel, the implementation of advanced manufacturing approaches and control of critical processing parameters have demonstrated promising results with process validation in large-scale bioreactor runs. Moreover, optimization of the molecular design of vectors to enable higher cell-specific yields of functional AAV particles combined with bioprocess intensification strategies may also contribute to addressing current and future manufacturing challenges.     

Stability of <Emphasis Type="Italic">Potato virus X</Emphasis> expression vectors is related to insert size: implications for replication models and risk assessment

We investigated the stability of expression constructs based on Potato virus X (PVX) as a function of insert length. Five different inserts ranging in length from 261 to 1,758 bp (human proinsulin, murine interleukin-10, HIV-1 nef, petunia expansin-1 and human gad65) were expressed using a PVX vector in Nicotiana benthamiana plants for three sequential passages. Using a competitive RT-PCR approach we demonstrated that insert–deletion could occur in the first infection cycle for all inserts, but that this was much more likely to be the case for longer ones. This suggested a negative correlation between insert length and vector stability. Sequence analysis of the deleted constructs suggested that recombination usually occurred at sites close to the duplicated sub-genomic promoter, but in a smaller number of cases the foreign gene itself was probably involved, resulting in partially deleted constructs containing transgene fragments. The implications of these results in the context of recombinant protein expression and its risks are discussed.     

Use of Hydrocyclones for Mammalian Cell Retention: Separation Efficiency and Cell Viability (Part 1)

A hydrocyclone with a volume of 2.56 cm³ was studied as a potential cell retention device for mammalian cell cultures (6 L volume). For the feasible operation range (0.9 to 1.6 L/min flow corresponding to pressure drops of 0.4 to 1.3 bar) the hydrocyclone was characterized with regard to flow split (underflow‐to‐overflow ratio) and flow ratio (underflow to supply). Cultures of BHK and HeLa cells (with low cell concentrations) were applied to measure separation efficiency and cell viability for a hydrocyclone operation period of 3 min corresponding to a cell suspension throughput of 2.7 to 4.8 L. Cell separation efficiencies ranged from 0.77 to 0.97 and cell viability was not affected except for BHK cells in the overflow at the highest pressure drop (1.3 bar). As the overflow is commonly used for product harvest and cells are discarded, the application of the hydrocyclone has no detrimental effect on the reactor perfusion system. The results indicate that only cells passing from the primary vortex downwards into the inner secondary vortex and from there upwards could be damaged. Evidence for this hypothesis is obtained from operating the hydrocyclone with closed overflow (only centrifugal forces acting) for a period of 3 h. In these studies no significant effect on cell viability could be detected for HeLa and CHO cells. Hence, the results indicate that the hydrocyclone can be appropriately used for cell retention and separation in perfusion cultures. Application at higher pressures is recommended whereby separation efficiencies of 0.97 without any loss in viability can be achieved.     

金黄色葡萄球菌组分疫苗研究进展

临床上,耐药性的金黄色葡萄球菌日益增多,极其耐药的菌株甚至获得了超级耐药细菌的称谓,难以找到有效的抗菌素控制其感染,因而研制有效的疫苗来防治金黄色葡萄球菌感染显得更加重要。对研制开发的金黄色葡萄球菌荚膜多糖疫苗、纤维粘连结合蛋白疫苗、RNAⅢ激活蛋白疫苗以及核酸疫苗等进行了综述。     

Co-expression vs. co-infection using baculovirus expression vectors in insect cell culture: Benefits and drawbacks

The baculovirus expression vector system (BEVS) is a versatile and powerful platform for protein expression in insect cells. With the ability to approach similar post-translational modifications as in mammalian cells, the BEVS offers a number of advantages including high levels of expression as well as an inherent safety during manufacture and of the final product. Many BEVS products include proteins and protein complexes that require expression from more than one gene. This review examines the expression strategies that have been used to this end and focuses on the distinguishing features between those that make use of single polycistronic baculovirus (co-expression) and those that use multiple monocistronic baculoviruses (co-infection). Three major areas in which researchers have been able to take advantage of co-expression/co-infection are addressed, including compound structure-function studies, insect cell functionality augmentation, and VLP production. The core of the review discusses the parameters of interest for co-infection and co-expression with time of infection (TOI) and multiplicity of infection (MOI) highlighted for the former and the choice of promoter for the latter. In addition, an overview of modeling approaches is presented, with a suggested trajectory for future exploration. The review concludes with an examination of the gaps that still remain in co-expression/co-infection knowledge and practice.     

First detection of endosymbiotic bacteria in biting midges Culicoides pulicaris and Culicoides punctatus,important Palaearctic vectors of bluetongue virus

Heritable bacteria have been highlighted as important components of vector biology, acting as required symbionts with an anabolic role, altering competence for disease transmission, and affecting patterns of gene flow by altering cross compatibility. In this paper, we tested eight U.K. species of Culicoides (Diptera: Ceratopogonidae) midge for the presence of five genera of endosymbiotic bacteria: Cardinium (Bacteroidales: Bacteroidaceae); Wolbachia (Rickettsiales: Rickettsiaceae); Spiroplasma (Entomoplasmatales: Spiroplasmataceae); Arsenophonus (Enterobacteriales: Enterobacteriaceae), and Rickettsia (Rickettsiales: Rickettsiaceae). Cardinium spp. were detected in both sexes of Culicoides pulicaris and Culicoides punctatus, two known vectors of bluetongue virus. Cardinium spp. were not detected in any other species, including the Culicoides obsoletus group, the main vector of bluetongue and Schmallenberg viruses in northern Europe. The other endosymbionts were not detected in any Culicoides species. The Cardinium strain detected in U.K. Culicoides species is very closely related to the Candidatus Cardinium hertigii group C, previously identified in Culicoides species in Asia. Further, we infer that the symbiont is not a sex ratio distorter and shows geographic variation in prevalence within a species. Despite its detection in several species of Culicoides that vector arboviruses worldwide, the absence of Cardinium in the C. obsoletus group suggests that infections of these symbionts may not be necessary to the arboviral vector competence of biting midges.     

Evaluation of housing as a means to protect cattle from Culicoides biting midges, the vectors of bluetongue virus

The housing of animals at night was investigated as a possible means of protecting them from attack by Culicoides biting midges (Diptera: Ceratopogonidae), the vectors of bluetongue. Light-trap catches of Culicoides were compared inside and outside animal housing, in the presence and absence of cattle. A three-replicate, 4 × 4 Latin square design was used at four farms in Bala, north Wales, over 12 nights in May and June 2007, and the experiment repeated in October. In the two studies, respectively, >70 000 and >4500 Culicoides were trapped, of which 93% and 86%, respectively, were of the Culicoides obsoletus group. Across the four farms, in May and June, the presence of cattle increased catches of C. obsoletus by 2.3 times, and outside traps caught 6.5 times more insects than inside traps. Similar patterns were apparent in October, but the difference between inside and outside catches was reduced. Catches were strongly correlated with minimum temperature and maximum wind speed and these two variables explained a large amount of night-to-night variation in catch. Outside catches were reduced, to a greater extent than inside catches, by colder minimum temperatures and higher maximum wind speeds. These conditions occur more frequently in October than in May and June, thereby suppressing outside catches more than inside catches, and reducing the apparent degree of exophily of C. obsoletus in autumn. The results suggest that the risk of animals receiving bites from C. obsoletus is reduced by housing at both times of year and the benefit would be greatest on warm, still nights when outside catches are at their greatest.     

Effect of morphological integrity,period, and type of culture system on the in vitro development of isolated caprine preantral follicles

The aims of this study were the following: (1) to define an optimal period for the IVC of isolated caprine preantral follicles, (2) to verify the relationship between follicular morphology (intact, extruded, and degenerate follicles) and estradiol production, and (3) to evaluate the effects of the bidimensional (2D) and three-dimensional (3D) culture systems on the in vitro development of caprine preantral follicles. Three experiments were performed. In experiments 1 and 2, the isolated secondary follicles were cultured for 18, 24, and 30 days or 30, 36, and 42 days, respectively. In experiment 3, the optimal culture period from experiment 2 was used for 2D and 3D culture systems. After culture, the oocytes were submitted to IVM. The morphological integrity, antral cavity formation rates, follicular diameter, presence of healthy, grown oocytes (≥110 μm), rates of resumption of meiosis, and estradiol concentrations were evaluated. In experiment 1, the percentage of oocytes that resumed meiosis was higher in oocytes cultured for 30 days (48.84%) than in oocytes cultured for 18 and 24 days (15% and 20.93%, respectively). In experiment 2, the percentage of oocytes that resumed meiosis was significantly higher in oocytes cultured for 30 and 36 days (47.5% and 50%, respectively) than in oocytes cultured for 42 days (20%). The estradiol concentrations on Day 12 of culture were similar for normal and extruded follicles and higher than those observed in degenerate follicles at the end of the culture period. In conclusion, the 36-day culture period resulted in the highest rates of meiosis resumption. In addition, because the loss of follicular integrity affects the patterns of estradiol production, follicular integrity is a good predictor of follicular quality.     

Use of virus vectors for the expression in plants of active full-length and single chain anti-coronavirus antibodies

To extend the potential of antibodies and their derivatives to provide passive protection against enteric infections when supplied orally in crude plant extracts, we have expressed both a small immune protein (SIP) and a full-length antibody in plants using two different plant virus vectors based on potato virus X (PVX) and cowpea mosaic virus (CPMV). The alphaSIP molecule consisted of a single chain antibody (scFv) specific for the porcine coronavirus, transmissible gastroenteritis virus (TGEV) linked to the alpha-CH3 domain from human IgA. To express the full-length IgA, the individual light and heavy chains from the TGEV-specific mAb 6A.C3 were inserted into separate PVX constructs and plants were co-infected with both constructs. Western blot analysis revealed the efficient expression of both the SIP and IgA molecules. Analysis of crude plant extracts revealed that both the plant-expressed alphaSIP and IgA molecules could bind to and neutralize TGEV in tissue culture, indicating that active molecules were produced. Oral administration of crude extracts from antibody-expressing plant tissue to 2-day-old piglets showed that both the alphaSIP and full-length IgA molecules can provide in vivo protection against TGEV.