key: cord-0795403-qczqbwe3 authors: Roldão, A.; Silva, A. C.; Mellado, M.C.M.; Alves, P. M.; Carrondo, M.J.T. title: 1.47 Viruses and Virus-Like Particles in Biotechnology: Fundamentals and Applications ☆ date: 2017-12-31 journal: Comprehensive Biotechnology DOI: 10.1016/b978-0-12-809633-8.09046-4 sha: b7bf262f26d5b7587025e9bb2acd529b129e8dd8 doc_id: 795403 cord_uid: qczqbwe3 This is a reprint of A. Roldão, A.C. Silva, M.C.M. Mellado, P.M. Alves, M.J.T. Carrondo, Viruses and Virus-Like Particles in Biotechnology: Fundamentals and Applications, Reference Module in Life Sciences, Elsevier, 2017. Glossary Downstream process Technical steps, including all purification steps, of a bioprocess from the product harvest to the purified product. Metabolic engineering The practice of optimizing genetic and regulatory processes within cells to increase the cells' production of a certain substance. Prophylactic A medication or a treatment designed and used to prevent a disease from occurring. Transduction The process of introducing foreign DNA deliberately into cells using viral vectors. Transfection The process of introducing foreign DNA deliberately into cells using nonviral methods. Upstream process Technical steps of a bioprocess from the cell development to product harvest including all cell culture steps. Viruses are microscopic infectious agents and exclusively intracellular organisms that depend on animal, plant, or bacterial cells to multiply. All viruses have one nucleic acid, RNA or DNA, enclosed within a protein coat that protects the viral genes. Many viruses also have a viral envelope covering the nucleocapsids, consisting of host cell membrane lipids and proteins, and viral glycoproteins that protect viruses from degradation outside the cell and help their attachment to host cell membranes. Since the initial discovery of tobacco mosaic virus by Martinus Beijerinck in 1898 [1] , more than 5000 viruses have been described in detail. Nonetheless, significant progress in virus identification and propagation was possible only in the 1950s upon the establishment of cell culture technology [2, 3] . With this development, animal cell cultures gradually replaced live animals in the preparation of viral antigens for vaccines. More recently, advances in virology and molecular biology allowed the development of platforms for the production of virus-like particles (VLPs) as vaccines against emergent diseases and viral vectors for gene therapy. VLPs are composed of viral structural proteins that, when expressed in recombinant systems, form multiprotein structures mimicking the organization and conformation of authentic native viruses but lacking the viral genome. Several applications have been proposed for viruses and VLPs: vaccination, gene therapy, drug delivery, nanotechnology, and bioweapons, among others. This chapter aims at reviewing the fundamentals, applications, and production strategies of viruses and VLPs, as well as the challenges and perspectives for the future. classification system for viruses and does not consider viruses with DNA intermediates as RNA viruses. These viruses are included in group VI and possess ssRNA genomes that replicate using reverse transcriptase. Viruses such as the human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), and Rous sarcoma virus (RSV) (members of the Retroviridae familysee Fig. 1 (B)) are included in this group. dsRNA viruses represent a large group of pathogens whose genome can be monopartite or segmented up to 12 fragments. These viruses do not release the free dsRNA genome into infected cells and require that transcription and synthesis of new dsRNA genomes take place in confined environments. Reovirus and rotavirus, members of the Reoviridae family, are included in this group ( Table 2) . In contrast with the dsRNA or the (À) ssRNA viruses, the genomes of (þ) ssRNA viruses are infectious. Icosahedral (þ) ssRNA viruses represent a large fraction of all viruses known and include important human pathogens: poliovirus, human rhinovirus, hepatitis A virus, Norwalk virus, astrovirus, alphavirus, and members of the Flaviviridae family that carry an RNA-containing nucleocapsid are some examples. Unlike (À) ssRNA viruses, the nucleoproteins responsible for protecting the genome from non-specific cellular RNA binding are not expressed in (þ) ssRNA viruses. Thus, the synthesis of progeny viruses requires that the capsid proteins of these viruses specifically package the viral RNA genome while excluding the ubiquitous cellular RNA. Group IV includes the Flaviviridae (hepatitis C virus -HCV), Coronaviridae (severe acute respiratory syndrome virus -SARS virus), Picornaviridae, Astroviridae, Togaviridae, and Caliciviridae families ( Table 2) . characterized by nonsegmented genomes. The remaining three have genomes comprising 2, 3, and 6-8 (À) sense RNA segments, respectively. The large group of (À) sense RNA viruses includes (1) highly prevalent human pathogens such as respiratory syncytial virus, influenza, and human parainfluenza viruses; (2) two of the most deadly human pathogens, namely Ebola and Marburg viruses; and (3) viruses with a major economic impact on the poultry and cattle industries, namely the Newcastle disease virus (NDV) and rinderpest virus ( Table 2 ). VLPs are multimeric protein complexes composed of viral structural proteins that assemble spontaneously when expressed in recombinant systems. These structures mimic the organization and conformation of authentic native viruses but lack the viral genome. To date, different types of viruses have been mimicked by VLPs: viruses with single or multiple capsid proteins and with or without lipid envelopes ( Table 3) . In most nonenveloped viruses, the nucleocapsids are formed by a single, virally encoded protein. Thus, VLPs of these viruses are relatively easy to generate as the assembly process relies solely on the expression levels of a single protein. Some examples are presented in Table 3 . One of the most studied VLPs of structurally simple viruses is the human papillomavirus (HPV)-VLP. Although the native virus contains the major and minor capsid proteins of HPV, L1 and L2, respectively [27, 60] , the HPV-VLP is formed just by L1 protein organized in 72 pentameric capsomers. Canine parvovirus and porcine parvovirus (PPV)-VLPs are also formed by a single protein, VP2, the major structural protein in both viruses. These VLPs are normally expressed in insect cells and induce high immunogenic responses [49] . In the case of PPV-VLPs, large-scale production is doable [61] . Norwalk virus (NV)-VLPs, VLPs of hepatitis E virus (HEV), and chimeric VLPs from simian virus 40 (SV40) constitute other examples of expression in insect cells. NV-VLPs have been extremely useful as a source of diagnostic antigen to monitor disease outbreaks since the native virus has limited growth in cell culture [60] . These particles have also been shown to be effective at stimulating IgG, IgA, and humoral responses in mice [62] . Preliminary phase I trials in humans have confirmed that they are safe and effectively stimulate IgG and IgA responses [43] . A truncated form of HEV capsid readily assembles into a VLP in insect cells, but this has not yet been tested as a vaccine. In mice immunization studies, these VLPs were able to induce systemic and mucosal immune responses following oral administration [22] . Finally, by genetically manipulating the major capsid protein of SV40 (VP1), it is possible to accommodate foreign peptides on the surface of this protein in such a way that assembled chimeric SV40-VLPs display these peptides on their surface. This confers to chimeric SV40-VLPs the essential features to be used as a controlled, cell type-specific gene delivery system [58] . Many pathogenic viruses are surrounded by an envelope consisting of host cell membrane lipids and proteins, and viral glycoproteins. These proteins are the targets of neutralizing antibodies and essential components of a vaccine. Due to the inherent properties of the lipid envelope, assembly of enveloped VLPs is technically complex. Some examples are presented in Table 3 . The first VLP vaccine to be produced and characterized consisted of spherical particles with diameter between 17 and 25 nm formed by the surface antigen of the hepatitis B virus (HBsAg) co-assembled with host cellular membranes [63] . Meanwhile, several other enveloped VLPs have been successfully developed. The HCV-VLP, produced in the baculovirus/insect cell system by co-expression of core E1 and E2 proteins, and VLPs of Ebola and Marburg virus, vaccine candidates against these emergent diseases, are two examples. HCV-VLPs have been tested in mice and baboons and shown to be effective at stimulating both cellular and humoral immune responses [20, 64] . VLPs of Ebola and Marburg virus have been shown to protect small laboratory animals as well as nonhuman primates against lethal challenge by Ebola and/or Marburg viruses [8] . HSV-VLP [34] , NDV-VLP [40] , Hantaan-VLP [13] , and RVFV-VLP [54] exemplify other enveloped VLPs successfully expressed and assembled in the baculovirus/insect cell system. Substantial efforts were put forward to recreate the envelope of SARS coronavirus [55] and the envelope of viruses from Retroviridae family in a form that permits the efficient induction of broadly neutralizing antibodies. Although none of the retrovirus-derived VLPs have gone into clinical trials yet, initial experiments with HIV-VLP [65] and SIV-VLP [56] in animal models look promising. Chimeric VLPs containing the gag capsid protein from SIV and the envelope protein from HIV [66] , RSV-VLPs formed by in vitro assembly of gag proteins [67] , and VLPs for influenza A virus subtypes H3N2 [68] and H5N1 [69] are also part of the group of successfully produced VLPs with lipid envelope. VLPs composed of multiple interacting capsid proteins are more challenging to produce than those formed by one or two major capsid proteins, due to the site of protein expression: proteins encoded by multiple discrete mRNAs and not processed from a single polyprotein tend to be differentially localized in the cell, significantly affecting the efficiency of the assembly process. Thus, it is essential to guarantee that all interacting capsid proteins are expressed in the vicinity of each other and within the same cell [60] . Assembly of VLPs by expressing more than one structural viral protein has been achieved for various members of Picornaviridae (poliovirus [47] and enterovirus 71 [11] ) and Birnaviridae (infectious bursal disease virus [35, 36] ) families. The adeno-associated virus type 2 VLPs are also included in the category of VLPs with multiple-protein layers. These particles are produced by co-infecting insect cells with recombinant baculovirus coding for adenovirus capsid proteins VP1, VP2, and VP3 [4] . VLPs have been efficiently produced for the members of the Reoviridae family. These viruses are considerably complex to mimic as they are characterized by multiple concentric layers and different capsid proteins. The bluetongue virus (BTV)-VLP and rotavirus-like particles (RLPs) are two such cases. Intact and biologically active BTV-VLPs are produced in insect cells by simultaneously expressing all four structural proteins of the BTV (VP2, VP3, VP5, and VP7) using a multicistronic recombinant baculovirus [70] ; RLPs are also formed in a similar way. The rotavirus itself consists of three concentric protein layers: an inner core of VP2, a middle layer of the polymorphic protein VP6, and an external layer formed by the glycoprotein VP7, with 60 spikes of VP4. When treated with trypsin, VP4 is cleaved into VP5 and VP8, which allows virus binding and entry to cells [71] . Inside the core, small amounts of VP1 and VP3 exist, constituting less than 3% of the total viral protein [72] . Although VP4 is considered to play a key role in RLP stabilization upon assembly, it is consensual that VP2, VP6, and VP7 are sufficient to form a triple-layered particle structurally similar to the native virus and which is biologically functional [52, 53, 73, 74] . RLPs are normally expressed in the baculovirus/insect cell system by co-expression of the three rotavirus structural proteins mentioned above. The diversity of structures normally observed at the end of the culture indicates that the assembly process is highly inefficient (Fig. 2) . These particles are extensively used to study virus structure, role of protein in viral morphogenesis, protein function and biochemical properties, virus interaction with the mammalian host cell, and proteinprotein interactions, as reviewed by Palomares and Ramirez [75] . RLPs are also vaccine candidates against rotavirus disease. They are considered safe and induce a robust antibody response and protection in animals if they are engineered to include one or both of the outer capsid proteins VP4 and VP7, properly formulated with a potent adjuvant, and administered intramuscularly [51, 74, 76] . Even intrarectal immunization, inducing a local mucosal response, has been reported as sufficient for protection from rotavirus infection [77] . It is possible that RLPs may provide a viable alternative to the existing live virus vaccines, which have recently raised efficacy concerns in developing countries such as Bangladesh and South Africa [78] . Production of viruses was initially performed using the natural hosts of the viruses. Upon the establishment of cell culture technology in the 1950s, animal cell cultures gradually replaced live animals in the preparation of viral antigens for vaccines. The observation by Enders and co-workers [2] that non-nervous tissue culture could be used to replicate and produce poliovirus paved the way to large-scale production of vaccines. This discovery led to the development of the first commercial product generated using mammalian cell cultures (primary monkey kidney cells), the poliovirus vaccine. Regrettably, primary monkey kidney cells presented many drawbacks such as the relatively high risk of contamination with adventitious agents (contamination by various monkey viruses), shortage of donor animals, use of endangered animals as cell source, use of uncharacterized or insufficiently characterized cell substrates for virus production, limited expansion, and obligatorily adherent cell growth [80] [81] [82] [83] . In the 1960s, human diploid fibroblast cells, WI-38 [84] and MRC-5 [85] , and baby hamster kidney cells (BHK-21 (C13)) were established and used for the production of a vaccine against rabies virus [86] and foot-and-mouth disease [87] , respectively. Nowadays, there are several licensed human viral vaccines produced using cell substrates (see Table 4 ) or under clinical trials such as the influenza vaccine produced using Vero [88] [89] [90] , Madin-Darby canine kidney (MDCK) [91, 92] , or PER.C6 [93, 94] cells among others [95] . Despite these significant advances in vaccine manufacturing, there are vaccines that are still produced in eggs such as the recently approved influenza A (H1N1) 2009 monovalent vaccines from CSL, MedImmune LLC, or Novartis Vaccines and Diagnostics. Viruses may also serve purposes other than being vaccine production platforms. In fact, viruses such as adenovirus, retrovirus, lentivirus, and adenoassociated virus are commonly used as gene delivery vectors for gene therapy among other applications (see Section 1.47.5). The most common and well-documented packaging cell line for adenovirus production is the human embryonic kidney 293 (HEK 293) cell line, which contains the E1 region of the adenovirus [96] . Homologous recombination between the left terminus of firstgeneration adenovirus vector or helper virus DNA and partially overlapping E1 sequences in the genome of HEK 293 cells normally leads to the generation of replicative-competent adenoviruses (RCAs) [97] . The presence of RCAs is clearly undesirable as they may replicate in an uncontrolled manner. In recombinant adenovirus batches to be used in human patients, RCA is potentially hazardous, especially in immunocompromised patients, being associated with inflammatory responses [98] . Alternative host cell lines have been developed to overcome this problem either by reducing the overlapping sequences or by eliminating any overlap, as is the case of N52.E6 [99] and PER.C6 [100] cells. The PER.C6 cell line, derived from human embryonic retinal cells, has been established for industrial applications (eg, full traceability available) due to its reduced propensity to generate RCAs and its capacity to achieve high yields of adenovirus vectors. Another disadvantage of human adenovirus vectors is their limited clinical use; 90% of the population has developed preexisting humoral and cellular immunity to those vectors [101] . Sustainable platforms for the generation of vectors from different human serotypes or of those derived from nonhuman adenovirus at titers similar or greater than those obtained with human adenovirus vectors and free of detectable levels of RCAs are required [102, 103] . One example is the production of canine adenovirus type 2 (CAV-2) vectors in dog kidney cells [101, [104] [105] [106] . With these vectors, the risks associated with RCAs are diminished, if not completely eliminated, because CAV-2 vectors do not propagate in human cells; also, CAV-2 vectors transduce human-derived cells at an efficiency similar to that of human adenovirus type 5 and are amenable to in vivo use [101] . Novel cell lines, with emphasis on MDCK cells, for the production of CAV-2 vectors are under evaluation 107,108]. The NIH 3T3 mouse embryonic fibroblast cell line, a ferret brain cell line, the human cell lines HT1080, TE671, HEK293 (which can grow in suspension), and CEM (which is an obligatory suspension cell line) all have been used for the establishment of retrovirus vector producer cell lines; other detailed examples can be found elsewhere [109] . Recently, highly versatile producer cell lines such as Flp293A and 293 have been developed. Based on HEK 293 cells and equipped with flippase recognition target sites containing a murine leukemia virus (MLV)-green fluorescent protein (GFP) vector, these cells allow the efficient Flp recombinase-mediated cassette exchange of MLV vectors; thus, after cassette exchange, the tagged retrovirus producer cell clone is capable of producing vectors containing the transgene of choice at levels similar to those observed for the mother producer cell line [110, 111] . The major problem in the production of lentiviruses has been the development of a packaging cell line. Stable expression of lentivirus particles has proven to be more difficult than that of oncoviruses [112, 113] , partly due to the expression of proteins such as rev and viral proteases which appear to be toxic to cells [114] . Consequently, lentiviral vectors have been produced by transient transfection of high-expressing cell lines such as COS [115] and 293T [116, 117] , generally using four different plasmids (gagpol, env, rev, and lv-vector). Lentiviral vectors can also be produced by transduction of 293T cells with baculoviruses; sustained transgene expression was observed after lentivirus transduction of HeLa cells [118] . Recombinant adeno-associated viral (rAAV) vectors can be produced using stable cell lines containing the required genes or by transient transfection. Transient transfection employs the use of 293 or A549 cells co-transfected with two plasmids containing the rAAV vector and the rep and cap genes, followed by an infection with helper virus to induce the replication of rAAV. Another possibility is to co-transfect cells with three plasmids containing the rAAV vector, the rep and cap genes, and the adenovirus helper genes [119, 120] . Stable cell lines, on the other hand, require only the presence of a helper virus to initiate rAAV production since they already contain the rAAV vector and the rep and cap genes of adeno-associated virus. It is possible to use HeLa (the most common), 293, and A549 cells as stable cell lines. This system is better suited for large-scale production of rAAV than transient transfection; nevertheless, generation of such stable cell lines can be tedious and time consuming. It has been estimated that 10 12 -10 14 rAAV particles are required for clinical human use [121] . Independent of the production strategy, maximum rAAV titers are typically around 10 7 infectious particles (IP) per mL, clearly insufficient to fulfill the needs. In order to overcome this limitation, recent studies have focused on producing rAAV vectors in insect cell cultures, using the recombinant baculovirus system [122] . Production of rAAV particles is achieved by coinfecting Spodoptera frugiperda (Sf)-9 cells with three baculovirus vectors, BacRep, BacCap, and Bac-rAAV; these encode the respective components of the rAAV production machinery. This system lends itself to large-scale production under serum-free conditions, as Sf-9 cells are grown in suspension. The most popular expression system for the production of VLPs is the yeast system due to its easy protein expression, ability to scale up, and cost of production. However, appropriate posttranslational modifications (PTMs) such as protein glycosylation and correct protein folding, protein assembly, and codon optimization may dictate alternative production systems. Within those, mammalian cell lines (either transiently or stably transfected or transduced with viral expression vectors), the baculovirus/insect cell system, and various species of bacteria and plant cells have been receiving special attention ( Table 5) . , polyomavirus-VLP [12, 123] , norovirus (No)-VLP [41] , and HCV-VLP [124] , are under investigation. VLPs produced in bacteria (ie, Escherichia coli) have not yet reached the market. In fact, little or no immunogenic information is available for HBV-VLP [18] , HPV-VLP [30, 32] , HCV-VLP [19] , Ebola-VLP [125] , SV40-VLP [59] , RSV-VLP [67] , infectious hypodermal and hematopoietic necrosis virus-VLP [126] , No-VLP [127] , and Indian peanut clump virus-VLP [37] . This is due to the inability of the proteins expressed in prokaryotic cells to undergo PTM such as protein glycosylation, a key feature in most VLP-derived vaccines. Another common difficulty of these cells is the expression of a soluble and full-length product free of toxins, heat shock proteins (HSPs), and chaperone proteins. Since limited solubility of recombinant viral proteins promotes the formation of inclusion bodies [30, 32] , the downstream processing is significantly compromised. Consequently, the process route becomes potentially more expensive than the eukaryotic route. Even in downstream processing of soluble fractions, the VLP precursors often require separation of HSPs and molecular chaperone proteins (eg, GroEL) that can remain attached to the capsomeres, creating a significant bioseparation problem [59] . Protease degradation and codon bias can also be a source of reduced manufacturing precision, contributing to lower yields and possibly to nonhomogeneity of the final VLP architecture [128] . To overcome these limitations, expression systems such as the baculovirus/insect cell system should be used instead of bacteria or yeast. Another alternative is the disassembly and reassembly of VLPs in vitro [129] , a method described in detail in the following sections. VLPs formed by several proteins require simultaneous expression of multiple proteins. A versatile and efficient system for the production of these recombinant proteins is the baculovirus/insect cell system. Insect cells are initially grown to a desired cell concentration after which they are infected with one or several recombinant baculoviruses containing the gene or genes coding for the proteins of interest. The construction of recombinant baculovirus is simple and fast, providing a high versatility to the expression system. This rapid construction is very important when producing vaccines for rapidly changing viruses, a fundamental requirement in an efficient approach to contend with potential pandemics in a timely manner. For instance, an influenza vaccine production campaign based on the baculovirus/insect cell system can be completed within 1.5 months after having identified the particular circulating viral strain, whereas an egg-based or other cell culture-based platform would require 7-9 months [130] . A list of VLPs produced using this system is shown in Tables 3 and 6. Proteins expressed in mammalian cell culture systems can undergo complex PTMs by copying nature, a significant advantage over other systems that are limited to high-mannose glycoprotein modifications (baculovirus/insect cell system and yeast) or incapable of any type of PTM (bacteria). Thus, the assembly of these proteins into VLPs closely resembles the formation of native virus particles. The main disadvantages of this system are its low controllability and high production costs. Different mammalian cells can be used for VLP production; for example, BSC-1 [35] , SW480 [28] , BHK-21, and Vero E6 cells [13] are able to produce VLPs when infected with recombinant vaccinia vectors carrying the genes coding for viral proteins of different viruses. VLPs have also been produced by stably or transiently transfecting CHO [17] and 293T cells [7, 26] . Wang et al. [21] have used recombinant baculovirus to transduce HuH-7, HepG2, HeLa, BHK, and primary rat articular chondrocyte cells. Recent advances in plant biotechnology have made possible the use of transgenic plants as potentially viable alternatives to cell culture systems for the production of recombinant edible subunit vaccines [162] . HBV-VLP and NV-VLP are the most studied VLPs produced by three different species of transgenic plants: Solanum tuberosum (potato) [163, 164] , Lycopersicon esculentum (tomato) [165] , and Nicotiana benthamiana (tobacco) [37, 165, 166] . Edible plants offer a palatable oral delivery system that would preclude the costly purification process of injectable vaccines. In theory, the scale-up of production would not require large investments in hardware and culture media. Few resources would be needed such as agricultural practice, a stable transgenic line, and acreage for cultivation. The main disadvantages of using transgenic plants are the low expression levels obtained and antigen degradation taking place during in vivo delivery. The main areas of application of animal, bacterial, plant, and algal viruses include vaccine development and gene therapy. Vaccines based on live viruses have been traditionally effective and relatively easy to produce. The elimination of smallpox was accomplished through mass vaccination with the live vaccinia virus, a mildly pathogenic animal virus related to smallpox. Likewise, live attenuated vaccines are well tolerated and highly immunogenic. The live attenuated poliovirus vaccine developed by Dr. Albert Sabin in 1961 eradicated poliomyelitis disease in the Western hemisphere and drastically reduced its incidence rate worldwide. Vaccines against infectious diseases such as yellow fever, typhoid fever, mumps, and shigella are also based on live attenuated viruses. The attenuation of viruses is accomplished through one of the following methods: (1) attenuation of the pathogen by physical means and (2) selection of naturally occurring mutants that lead to infection with abortive replication of the pathogen while retaining its immunogenicity. Inactivated (killed) vaccines can also stimulate a protective immune response. The inactivated poliovirus vaccines (IPOL, Sanofi Pasteur SA), influenza vaccines (Fluarix, GlaxoSmithKline), and typhoid fever vaccines (Typhim Vi, Sanofi Pasteur MSD) constitute some examples. The disease-causing organism is inactivated with chemicals such as formaldehyde; the main drawback of these vaccines is that they require boosting for continuous, efficient immune response. Nowadays, using molecular biology and DNA manipulation methods, it is possible to express protective proteins in adequate live vectors with the purpose of designing live vaccines against various types of pathogens. In addition, the development of reverse genetics systems for the recovery of viruses from cDNA has made it possible to rapidly generate recombinant attenuated derivatives. Table 4 lists some examples of current licensed viral vaccines. In the late 1970s and the early 1980s, the emergence of techniques for subcloning mammalian genes into prokaryotic plasmids and bacteriophage was correctly foreseen as the stepping-stone to precursors of techniques for human gene therapy. Parallel investigations on the biology of avian and murine onco-retroviruses led to the development of retroviral vectors, which began to be used in the mid-1980s as a tool for gene transfer into mammalian cells. The first human trial of gene transfer was carried out in the late 1980s for the treatment of patients with advanced metastatic cancer. The process consisted in introducing the gene coding for neomycin resistance into human tumor-infiltrating lymphocytes by retroviral-mediated gene transduction, before their infusion into patients, thus, using the new gene as a marker for the infused cells [167] . Since then, there has been a remarkable expansion in the number of vector systems available to express human genes directly associated with disease states for therapeutic purposes. Gene therapy trials using retroviral vectors to treat X-linked severe combined immunodeficiency (X-SCID) constitute the most successful application of gene therapy to date. SCID is a disease in which the patient has neither cell-mediated immune responses nor the ability to generate antibodies. A high rate of immune system reconstitution was observed in patients treated in the X-SCID gene therapy trials [168] , but 5 out of more than 20 patients developed a leukemia-like illness, of which 4 fully recovered after conventional anti-leukemia treatment [169, 170] . Despite these results, gene therapy trials to treat SCID due to deficiency of the adenosine deaminase enzyme continue with relative success in the United States, France, the United Kingdom, Italy, and Japan. In the last decade, the process of retroviral vector production has been under considerable investigation as it presents many difficulties, mainly due to vector instability and low cell productivities hampering the attainment of high viral titers. Strategies based on the manipulation of sugar carbon sources [171, 172] , lipids [173] , temperature [174] , or osmotic pressure [175] used in bioreaction and on the establishment of pioneering packaging cell lines such as 293 FLEX [176] and Flp293A [111] show potential to increase the yields of infectious retroviral vectors. This will allow the generation of high-quality clinical preparations for gene therapy applications. Adenovirus vectors are also efficient vehicles for delivering nucleic acids into mammalian cells. The human adenovirus type 2 and 5 are the most used vector backbones for adenovirus-mediated gene transfer. However, due to a number of significant disadvantages such as the need to immunosuppress or tolerize patients to a potentially debilitating virus [101, 106] , vectors from different serotypes or those derived from nonhuman adenovirus (bovine, sheep, and birds) were developed [102, 103] . Recently, CAV-2 vectors, produced in dog kidney cells, have been gaining increasing attention due to their emerging potential for the study of the pathophysiology and potential treatment of neurodegenerative diseases like Parkinson's, Alzheimer's, and Huntington's, among others [177] . The first clinical trial using recombinant adenovirus was carried out in 1993 in cystic fibrosis (CF) patients [178] . Two years later, the first recombinant adeno-associated virus trial was initiated in CF patients [179] ; trials in hemophilia B patients commenced shortly after [180] . Inevitably, lentivirus and recombinant herpesvirus vectors have also entered into clinical trials [181] [182] [183] . All the four viral vector systems mentioned above are highly efficient systems for gene transfer and expression in vivo in nondividing cells. In fact, more than 50% of all viral vectors currently undergoing clinical trials are adenovirus (24.1%), retrovirus (21.2%), adeno-associated virus (4.4%), or HSV (3.3%). A detailed description of the cell lines used for the production of these vectors and the respective production strategies are presented in Table 7 . Due to their inability to replicate and absence of toxicity in mammalian cells, baculoviral vectors have emerged as gene therapy vehicles for the treatment of a wide range of human diseases. Recently, a genetically modified recombinant baculovirus encoding for a cherry-red fluorescent protein under the control of a strong mammalian cell promoter (cytomegalovirus promoter) proved to be effective in transducing a human liver carcinoma cell line, HepG2 [208] . Other studies indicate that baculoviruses show promising gene expression efficiencies in liver [209] , skeletal muscle [210] , brain [211] , and eye [212] . Importantly, baculoviral vectors present efficiencies similar to those of adenoviral vectors in transducing human smooth muscle cells, human cardiomyocytes, and fibroblasts [213] . The major challenge is the production of high titers of recombinant baculovirus [214] . Although metabolic engineering approaches have shown to improve baculovirus titers at high cell densities [215, 216] , platforms for the production of baculoviral vectors to be used in gene therapy clinical trials have not yet been implemented. VLPs can be used as prophylactic or therapeutic vaccines against a wide variety of diseases. The recent developments in molecular biology and virology renewed the interest in VLPs as versatile systems for gene and drug delivery. VLPs used for vaccination are normally devoid of any DNA of viral or cellular origin. VLPs have been produced from the capsid or envelope proteins of a wide variety of viruses for the purpose of studying viral assembly and for developing vaccines. While HBV-VLPs and HPV-VLPs are successful vaccines, VLP-based vaccines against pathogens that directly affect immune cells and successfully evade the immune system, such as HIV-1 and HCV, are proving to be extremely difficult to develop. Another important and forthcoming application of VLPs is their use in the generation of immune responses against foreign protein epitopes by fusing or by coupling them to VLPs of different origins, resulting in the so-called chimeric VLP ( Table 6 ; [217] ). The vaccine against HPV infection is an example of a chimeric VLP, in which the L2 protein epitopes are inserted into the L1 protein [132] to confer protection against a broader range of HPV types. In the end, tailoring of VLPs depends on their final application as vaccine (prophylactic or therapeutic) and may require adaptations in their structure (particle size, envelope structure, etc.), target host (dendritic cells, mucosal surfaces, etc.), and route of administration (intranasal, intramuscular, etc.) to achieve the desired immune response [27] . In cancer immunotherapy, a T-cell response is often more desirable than a B-cell response. Additionally, a T-cell epitope localized inside the VLP avoids interference with VLP uptake by the natural receptors of the native virus. Thus, a VLP vaccine candidate does not necessarily have to display on its surface-specific epitopes in order to be recognized by the immune system. This is the case for HPV 16 VLPs and HBV-VLPs. In the first case, the HPV 16 E6 and/or E7 peptide containing a T-cell epitope was fused to HPV L1 protein and inserted into the VLP [217] . In the second case, HPV 16 E7 epitopes were inserted into the surface or core proteins of HBV-VLPs [137] . The HPV-VLP was shown to induce E7-specific cytotoxic T cells and to protect mice against a challenge with an HPV 16-transformed tumor [149] . Results on purified chimeric HBV-VLPs have also confirmed their high immunogenicity in mice [218] . Most of these VLPs have a common feature: to induce an immune response against a non-self-antigen (the only exception being conjugated VLPs). However, for immune therapy of cancers of nonviral origin, the strategy is to induce an immune response against a self-antigen. In one study, a CD8þT-cell epitope derived from ovalbumin, a well-studied melanoma tumor antigen, was inserted directly into murine polyomavirus-VLP [151] ; VLPs carrying this ovalbumin epitope were then injected into mice in a therapeutic setting, with two injections after the melanoma tumor challenge. Complete protection against tumor development was obtained and induction of T cells specific for the ovalbumin epitope was demonstrated. Many capsid proteins used for VLP formation, such as HPV L1 and polyomavirus VP1, have the ability to bind non-specifically to viral or cellular DNA [219] . In 1983, it was demonstrated that murine polyomavirus-VLP could package viral DNA and transduce it into cells in vitro, resulting in expression of the viral gene products [220] . Later on, experiments with plasmid DNA demonstrated the feasibility of using these VLPs for gene transfer [221] . Since then, a number of studies have been performed in order to evaluate and optimize DNA packaging and transduction by VLPs derived from different members of Polyomaviridae family [222] . An example is the SV40-VLP produced in the baculovirus/insect cell system. Using an efficient methodology for in vitro packaging of plasmids with less than 17.7 kb, SV40-VLPs [161] have proven to be efficient for gene delivery in vivo [223] . Another example is the JC polyomavirus-VLP produced in the baculovirus/insect cell system; by VLP disassembly and VP1 pentamer reassembly in vitro, it was possible to insert a 1.6 kb DNA fragment coding for enhanced green fluorescent protein (EGFP) inside a JC polyomavirus-VLP [150] . VLP transduction results in TC-620 cells, measured by flow cytometry, showed that 70% of cells were expressing EGFP. Protein epitopes or other small molecules can be attached to the surface of already formed purified VLPs [158, 224] . This presents some advantages over VLP-forming methods that directly insert the epitopes into the VLP proteins. One advantage is that the already formed VLP can be used as a basis for the attachment of a number of different drugs, either proteins or smaller molecules, providing a flexible platform for drug delivery. Second, the attached molecule may provide a stronger and more efficient immune response due to its localization in the VLP surface. One example is the attachment of peptides or small molecules to VLPs consisting of the coat protein from the bacteriophage Qb or AP205; these VLPs have been developed for the generation of antibody responses against nicotine and angiotensin II [153, 158] . In phase I trials, healthy nonsmokers vaccinated with nicotine-conjugated Qb-VLPs [158] demonstrated a strong nicotine-specific IgM/IgG response. Noteworthy is that nicotine per se does not induce an antibody response. Only when conjugated with a VLP, nicotine potentially helps smokers quit smoking by reducing the satisfaction derived from nicotine intake. In phase II clinical trials, an increase in long-term abstinence was observed in smokers who attained high antibody levels following vaccination [155] . This same platform has been used to couple IL-1b molecules and Ab1-6 peptide to VLPs for rheumatoid arthritis and Alzheimer's treatment, respectively [154, 156, 159] . Peacey et al. [160] genetically modified rabbit hemorrhagic disease virus (RHDV)-VLP with various short peptide sequences that are capable of being presented to and recognized by immune cells. However, this approach does have limitations, including the time involved in engineering each chimeric VLP, size constraints imposed on introduced peptides, increased instability of modified capsids leading to limited yield, and the inaccessibility and altered conformation of introduced residues. To overcome these difficulties, a chemical linker was used to covalently conjugate both small peptides and whole protein to the RHDV-VLP scaffold. This rapid approach enabled surface conjugation of a substantial range of antigens without the constraints imposed by subunit folding and VLP formation. Attachment of antigen to RHDV-VLP conferred the immuno-stimulatory properties of the underlying viral shell to the conjugated antigen, and so enabled the initiation of both antigen-specific humoral and cell-mediated immune responses [160] . The results demonstrated that RHDV-VLP can be utilized as a versatile molecular scaffold in many applications, from vaccine development to biological nanotechnology. Drug delivery may also be achieved with a VLP carrying the desired drug inside the capsid. This can be accomplished, for example, with steps of in vitro disassembly and reassembly. Lee and Tan [18] have successfully encapsulated GFP inside HBV-VLP by disassembling the VLP into monomers with urea followed by reassembly using dialysis with GFP molecule. Viruses and VLPs have been successfully produced in vivo or in vitro since the beginning of the 20th century and in the late 1980s, respectively [225] [226] [227] [228] . The production of these bioproducts involves several bioengineering issues that must be carefully addressed in order to control upstream and downstream processing, and to maximize performance and reduce production cost. Product yields are strongly dependent on the production strategy chosen. Batch, fed-batch, continuous, and perfusion strategies are normally used for the production of viruses and VLPs. In batch production, it is important to control the accumulation of toxic products as well as the depletion of essential nutrients for cellular growth [215, 229] . Depletion can be avoided by selective addition of nutrients (glucose and glutamine) and/or amino acids, and complete medium addition [214, [230] [231] [232] using fed-batch strategies. The yields are significantly improved but the scale-up becomes difficult and expensive since culture medium is inefficiently used. The alternative, a continuous system, presents a short throughput time and a small number of production steps. Nonetheless, continuous reactors operated for long periods (>1 month) are prone to generating defective viruses that either directly reduce viral yields or indirectly impact on VLP yields by competing with the host cell for the protein expression machinery [233, 234] . In addition, the complexity of the bioproducts produced in this system is generally low, which constitutes a major drawback compared to other systems. Perfusion strategies enhance cellular growth and product yields [235, 236] . However, perfusion requires the use of large volumes of media, significantly increasing the production cost. In addition, the devices used for separating cells from the medium are difficult to scale up and normally induce cellular damage, impacting negatively on cellular growth rate and subsequently on viral or VLP production rates. 1.47.6.2 The "Envirome" The "envirome" affects viral replication and VLP production at the level of cellular growth and metabolic state, DNA transcription and replication, mRNA translation, and protein PTMs. Among others, the "envirome" enfolds the dissolved oxygen concentration, pH, temperature, agitation rate, cell and substrate concentration, inlet gas flow and composition, volume, pressure, fluid dynamics, power input, and osmolarity. Most of these variables differ from process to process and are possible to monitor and control in situ by continuously adjusting bioreactor parameters to certain predetermined set points [237, 238] . For example, recombinant protein production in insect cells is maintained constant at osmolarities between 300 and 380 mOsm [239] . If osmolarity falls out of this range by 30 mOsm, productivities are significantly reduced. Depending on the sensitivity of the cell line used, the stress generated while sparging and by bubble entrainment during agitation may impact negatively on viral and VLP productivities [23, 48] . Sparging-related stress can be alleviated using head space aeration, albeit the productivities achieved in these culture systems are remarkably lower; a more valid solution is the use of nonionic copolymers such as the Pluronic F-68, which lowers the culture medium surface tension and impedes the attachment of cells to bubbles, which liberate lethal energy during bursting [190, 240] . Pluronic F-68 also interacts with the cell membrane, increasing its rigidity and making it more resistant to hydrodynamic forces. Oxygen limitation or excess can induce protease synthesis and subsequent degradation of the product of interest [241] . Concomitantly, oxygen-derived free radicals present at high levels of dissolved oxygen tension can cause oxidative stress to cells or oxidative damage to proteins [242] . It is also important to bear in mind that temperature fluctuation induces different oxygen solubility levels: increasing temperatures induce lower oxygen solubility. The absence of contaminant proteins and DNA (host or viral), the absence of incorrectly assembled macrostructures, and endotoxin levels below those specified by regulatory agencies (Food and Drug Administration (FDA) and European Medicines Agency (EMEA)) ensure the quality and efficacy of the downstream process and are critical for the success of the process technology used. This is particularly a challenging task as in most cases produced viruses and VLPs do not differ significantly in size and molecular weight from other protein complexes or defective viruses that need to be removed. The nature of the bioproduct, either extracellular or associated with cellular structures, also influences the purification strategy [52, 208, [243] [244] [245] [246] [247] . Products that are secreted into the media are easily processed since they do not require a high number of purification steps. If produced intracellularly, extraction prior to purification is essential. In both the cases, fusion tags can be used to facilitate monitoring and downstream processing [248, 249] . Nonetheless, care must be taken when placing these fusion tags as they may affect virus maturation and protein expression, indirectly compromising their biological activity [250, 251] . In medium containing serum, the major problem is the high protein content that complicates the downstream processing [252, 253] . The use of serum-free medium is recommended as it does not contain animal-derived supplements, which pose safety concerns, and facilitates downstream operations. The nature of the outer protein of viruses and VLPs confers to these bioproducts specific and unique characteristics that strongly impact on the design of the downstream strategy. In the end, there is a clear trend in downstream processing from classical laboratorial purification methods like sucrose or cesium chloride gradient centrifugations toward more sophisticated techniques like tangential flow filtration, gel permeation chromatography, liquid chromatography, ion exchange chromatography, affinity chromatography, and sizeexclusion chromatography, including the use of newer, disposable membrane technology [245, 254, 255 ]. Heterologous proteins, the basis of viral particles and VLPs, are normally expressed by transfection in transient or stable systems or by transduction using viral expression vectors. The availability of stable packaging cell lines capable of continuously expressing a specific gene represents a step toward the scaled-up production of viral vector stocks to be used as drug delivery systems or in applications such as gene therapy [256] . Although the productivities of some viral vector producer cell lines remain lower than expected [257] , the generation of acceptable viral yields and the expression of secreted and insoluble proteins are normally favored by these stable transfected cell lines. The use of a constitutively active promoter that integrates into the genome and does not require infection for its activation is essential [258] . In applications such as protein characterization and high-throughput screening of gene functions, transient transfection is more appropriate [200, 259] . The use of viral expression vectors for virus and/or protein synthesis is another alternative. Although it requires the additional and always fiddly step of viral infection, viral yields can be as high as 10 10 IP mL À1 as reported for baculovirus or adenovirus production [214, 260] and protein expression levels can be between 1 and 500 mg L À1 [53, [261] [262] [263] . The strength of the promoter(s) controlling protein(s) expression for either virus encapsulation or VLP assembly, as well as the time at which the promoter(s) become(s) active, drives the productivity levels and thus can be optimized [264, 265] . The promoter strength must be carefully evaluated as it is common that very strong promoters overwhelm the processing capacity of the endoplasmic reticulum, thus reducing the final yields. Indeed, the secretion and complete complex glycosylation of recombinant proteins in the baculovirus/insect cell system improve when genes are under the control of the p10 promoter instead of the stronger polyhedrin promoter [266] . The use of early instead of late or very late promoters is a difficult choice; with very late promoters, the expression of proteins occurs toward the end of the culture when cells are in the death phase and proteases influence the correct protein PTM, negatively impacting final yields. On the other hand, early promoters induce lower protein productivities, as in most cases the enzymes and transcriptional factors necessary for protein expression are not yet fully active at such an early stage. The combination of promoter strength with the correct time for promoter activation is essential to assess high product yields, and mathematical modeling of intracellular events can be used to identify best strategies to obtain the optimal stoichiometry and thermodynamic conditions for VLP assembly [267] . is determinant for attaining optimal yields and robust production systems. The MOI to be used depends on the target product, the production process, and the dimension of viral stocks, and normally relies on predictions of the Poisson distribution [263, [268] [269] [270] . Low MOIs (0.01-1 virus per cell) have the advantage of requiring low concentrations of viruses. The number of viruses is normally insufficient to infect all the cells; thus, a high percentage of cells remain healthy and grow upon initial infection. A steep increase in infected cell concentration is observed as a second generation of viruses start to infect the uninfected cell population [263] . At the end of bioreaction, the concentration of infected cells is sufficiently high to sustain the production of viruses and/ or VLPs to high levels. The main drawback is the action of proteases. Since the overall process (infection plus viral and/or protein synthesis) is slow, the bioproduct is exposed to cellular proteases for long periods of time, which may compromise the quality and quantity of the final product. On the contrary, high MOIs (>1 virus per cell) require large viral stocks and favor the selection of fastreplicative defective virus [271] . In addition, volumetric yields are considerably lower than those achieved at low MOIs as a result of lower concentration of infected cells at the end of bioreaction. In theory, such differences could be compensated by increasing the CCI. However, at high CCI, the change in cell energetic state upon infection induces a significant drop in cell-specific productivity [215] . Medium replacement at TOI and the use of fed-batch or perfusion cultures are strategies capable of maintaining specific productivities similar to those reported at low CCI; the major inconvenience is that they are neither practical nor necessarily economical at large scale. This creates an opportunity for the development of novel techniques for process optimization based on metabolic engineering and systems biology. Another important parameter is the TOH. Delayed harvest times increase the exposure of viruses and VLPs to intracellular or extracellular proteases [272] and induce a more pronounced cell lysis. Additionally, the release of contaminant proteins (degraded or not), host and viral DNA, cell compartments, and viral or protein macrostructures to the extracellular medium will complicate the downstream processing of the product of interest. Optimal harvest times are normally between 72 and 120 hpi (hours postinfection) (40 and 70% cell viability) [24, 230, 273] . The interplay between all the abovementioned parameters is complex and normally requires substantial experimentation. To avoid this, mathematical models can be used. They significantly reduce the amount of work required and can assist the definition of optimal process-related parameters such as MOI, TOI, TOH, and CCI in complex biological systems for maximization of process performance. The commercial success of a bioproduct requires a controlled and monitored process and a well-characterized product. Unfortunately, most monitoring systems and characterization techniques are complicated to handle, extremely costly, semiquantitative, and in many cases non-existent, which significantly compromises the robustness and scalability of the process. In viral quantification, there is no accurate method to measure both infectious and total particles. Titration assays such as plaque-forming unit assay, growth cessation and cell size assay, alamar blue assay, tissue culture infectious dose 50, microculture tetrazolium assay, reverse transcriptase activity assay, and electron microscopy directly or indirectly assess viral titers of either infectious or total particles, but not both simultaneously [274] [275] [276] . Other methods such as flow cytometry, real-time quantitative polymerase chain reaction (Q-PCR), immunoblotting [277] [278] [279] , and high-performance liquid chromatography (HPLC) [280, 281] are semiquantitative. In the end, the most appropriate titration method depends on the type of virus and the detection system available. The quantification of VLPs involves a higher degree of complexity. Most methods are based on immunoassays such as Western blot, enzyme-linked immunosorbent assay (ELISA), and bicinchoninic acid (BCA) protein quantification assay [48, 53, 282] . Electron microscopy and real-time q-PCR are alternatives. However, all these methods are semiquantitative. They are unable to differentiate proteins that are part of a correctly assembled VLP from others that are in incomplete VLPs, viral particles, or other macromolecular structures. Thus, overestimated or underestimated VLP yields are frequently obtained [262] . Recently developed methods such as gelpermeation HPLC [262] , sodium dodecyl sulfate-capillary gel electrophoresis [75] , new application of intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) methodology [283] , and capillary zone electrophoresis [284] may allow a simple, fast, and low-cost quantification of viral proteins and VLPs in purified and bulk samples. In the near future, with the development of novel detection sensors based on acoustic resonance, turbidity, and fluorescence, it will be possible to monitor the kinetics of VLP formation in situ and online, as is already possible with a wide variety of culture constituents. One of those techniques is the two-dimensional (2D) fluorometry that, using optical fiber technology, allows the simultaneous monitoring of several compounds present outside (envirome) and inside (metabolome) the cells [285, 286] . The assembly of viral particles, VLPs, and other spherical polymersclosed structures composed of several protein subunitsis poorly understood. There is little experimental information describing intra-and intersubunit binding energies, rates and orders for assembly reactions, and formation of nucleating structures [287] . During the last decade, mathematical models have become a prominent and reliable source of knowledge for understanding what is driving the building of a virus capsid or a polyhedral protein macrostructure. Many mathematical models have been proposed based on theoretical constants of association and dissociation (K n and K d,app , respectively), free energies associated with intersubunit contact (DG c ) and assembly pathways [36, [288] [289] [290] [291] [292] . The way in which these constants and energies relate with specific culture parameters such as pH, calcium concentration, ionic strength, and others needs to be better understood. In vitro experiments of assembly and disassembly of VLPs are helpful in assessing these kinds of relationships. A recent study of in vitro disassembly of RLPs (triple-layered 2/6/7 particles) into DLPs (doublelayered 2/6 particles) and the assembly of DLPs and VP7 monomers into RLPs addresses the effect of physicochemical parameters (pH, ionic strength, and temperature) on the formation and stability of RLPs and DLPs [293] . The results indicate that both particles are stable within a specific pH (3-7) and temperature range (5-25 C) . Outside those thresholds, particle aggregation (T˛ [35-45 C] ), disassembly (T >65 C), and instability (isoelectric point of RLPs¼3 and DLPs¼3.8) become evident. In addition, the reaction rates of RLP disassembly are correlated with the temperature and ionic strength; low temperatures and low ionic strengths induce low disassembly reaction rates. On the other hand, RLP assembly reaction rates decrease with the increase in pH, ionic strength, temperature, and calcium concentration. These findings clearly demonstrate that process optimization of complex protein macrostructures is feasible by manipulation of physicochemical parameters. Nonetheless, care must be taken when extrapolating in vitro results to in vivo experiments, as in most cases the best conditions for subunit interaction and particle formation are not the same due to the different environmental culture conditions of the two systems. Viruses and VLPs are protein-related macrostructures and preferential vehicles for many applications. As complex products, the design of efficient upstream and downstream strategies, the definition of appropriate quality control and monitoring methods, and the attainment of high productivities are as complicated as they are essential. Thus, it is imperative to constrain the "envirome" effect on the quality and quantity of the product, so that viral particles and VLPs can be further used in their respective areas of application. Viruses are widely used in gene therapy [294, 295] , high-throughput screening of gene functions [200, 259] , drug delivery [296] , in vitro assembly studies to design antiviral drugs [19] , recombinant protein production [53, 297] , and bioinsecticide [298] and bioweapon [299, 300] preparation. In materials science and engineering, they can be the building blocks for electronics, biosensors, and chemistry [301] . VLPs, on the other hand, are established as a powerful tool for vaccine development [51, 302, 303] . Furthermore, improved knowledge points out toward larger and promising applications of virus and VLP platforms to other areas of research in the near future, such as nanotechnology, where they can be used as tools for biomedical science [304] [305] [306] [307] . 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protein and ISCOMATRIX adjuvant in women with cervical intraepithelial neoplasia Insertion of a foreign sequence on capsid surface loops of human papillomavirus type 16 virus-like particles reduces their capacity to induce neutralizing antibodies and delineates a conformational neutralizing epitope Comparison of human papillomavirus type 16 L1 chimeric virus-like particles versus L1/L2 chimeric virus-like particles in tumor prevention Characterization of virus-like particle assembly for DNA delivery using asymmetrical flow field-flow fractionation and light scattering Beneficial therapeutic effects with different particulate structures of murine polyomavirus VP1-coat protein carrying self or non-self CD8 T cell epitopes against murine melanoma Parvovirus B19 empty capsids as antigen carriers for presentation of antigenic determinants of dengue 2 virus A vaccine for hypertension based on virus-like particles: Preclinical efficacy and phase I safety and immunogenicity S2-04-06: Safety, tolerability and immunogenicity of the Ab immunotherapeutic vaccine CAD106 in a first-in-man study in Alzheimer patients A vaccine against nicotine for smoking cessation: A randomized controlled trial O1-06-01: Immunization with Ab1-6 coupled to the virus-like particle Qb (CAD106) efficiently removes b-amyloid without inducing Ab-reactive T-cells Therapeutic vaccines for nicotine dependence A therapeutic vaccine for nicotine dependence: Preclinical efficacy, and Phase I safety and immunogenicity Active immunization with IL-1 displayed on virus-like particles protects from autoimmune arthritis Versatile RHDV virus-like particles: Incorporation of antigens by genetic modification and chemical conjugation SV40 pseudovirions as highly efficient vectors for gene transfer and their potential application in cancer therapy Edible plant vaccines: Applications for prophylactic and therapeutic molecular medicine Oral immunization with hepatitis B surface antigen expressed in transgenic plants Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato Virus-like particle expression and assembly in plants: Hepatitis B and Norwalk viruses Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice Gene transfer into humans -Immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy Activation of the T-cell oncogene LMO2 after gene therapy for X-linked severe combined immunodeficiency New insights and unresolved issues regarding insertional mutagenesis in X-linked SCID gene therapy Retrovirus producer cell line metabolism: Implications on viral productivity Effect of medium sugar source on the production of retroviral vectors for gene therapy Retroviral vector production under serum deprivation: The role of lipids Stabilization of gammaretroviral and lentiviral vectors: From production to gene transfer Effect of osmotic pressure on the production of retroviral vectors: Enhancement in vector stability 293 FLEX a new cell line for fast, reproducible and efficient production of retroviral gene therapy vectors Gene transfer to the central nervous system: Current state of the art of the viral vectors Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis A phase I study of an adeno-associated virus-CFTR gene vector in adult CF patients with mild lung disease Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector A phase I/II clinical trial of beta-globin gene therapy for beta-thalassemia Exploiting the neurotherapeutic potential of peptides: Targeted delivery using HSV vectors Gene therapy for the treatment of sensory neuropathy From the first to the third generation adenoviral vector: What parameters are governing the production yield? Two different serum-free media and osmolality effect upon human 293 cell growth and adenovirus production Development of a packaging cell line for propagation of replication-deficient adenovirus vector Production of adenovirus vector for gene therapy Scaleable production of adenoviral vectors by transfection of adherent PER.C6 cells Adenoviral vectors for gene transfer and therapy Large-scale propagation of a replication-defective adenovirus vector in stirred-tank bioreactor PER.C6 cell culture under sparging conditions Serum-free suspension cultivation of PER.C6(R) cells and recombinant adenovirus production under different pH conditions Exploration of critical parameters for transient retrovirus production Lentiviral and MLV based retroviral vectors for ex vivo and in vivo gene transfer Gene delivery by lentivirus vectors Simplified lentivirus vector production in protein-free media using polyethylenimine-mediated transfection Critical assessment of current adeno-associated viral vector production and quantification methods The state of the art of adeno-associated virus-based vectors in gene therapy Scalable production of adeno-associated virus type 2 vectors via suspension transfection Transfection of mammalian cells using linear polyethylenimine is a simple and effective means of producing recombinant adeno-associated virus vectors Production of lentiviral vectors by large-scale transient transfection of suspension cultures and affinity chromatography purification Replication-defective genomic herpes simplex vectors: Design and production Gene delivery using herpes simplex virus vectors Multiple applications for replication-defective herpes simplex virus vectors Delivery using herpes simplex virus: An overview Engineering cell lines for production of replication defective HSV-1 gene therapy vectors Effect of genetic background and culture conditions on the production of herpesvirus-based gene therapy vectors Effect of protease inhibitors on yield of HSV-1-based viral vectors Purification of recombinant baculoviruses for gene therapy using membrane processes Transient disruption of intercellular junctions enables baculovirus entry into nondividing hepatocytes In vivo gene transfer in mouse skeletal muscle mediated by baculovirus vectors Neuronal gene transfer by baculovirus-derived vectors accommodating a neurone-specific promoter Baculovirus is an efficient vector for the transduction of the eye: Comparison of baculovirus-and adenovirusmediated intravitreal vascular endothelial growth factor D gene transfer in the rabbit eye Comparison between recombinant baculo-and adenoviral-vectors as transfer system in cardiovascular cells Baculovirus production for gene therapy: The role of cell density, multiplicity of infection and medium exchange Cell density effect in the baculovirus-insect cells system: A quantitative analysis of energetic metabolism Improving baculovirus production at high cell density through manipulation of energy metabolism Vaccination, immune and gene therapy based on virus-like particles against viral infections and cancer Mosaic hepatitis B virus core particles presenting the complete preS sequence of the viral envelope on their surface Characterization of the DNA binding properties of polyomavirus capsid protein Gene transfer by polyoma-like particles assembled in a cell-free system Polyoma virus pseudocapsids as efficient carriers of heterologous DNA into mammalian cells Murine polyomavirus virus-like particles (VLPs) as vectors for gene and immune therapy and vaccines against viral infections and cancer Liver-targeted gene therapy by SV40-based vectors using the hydrodynamic injection method Conjugation of a self-antigen to papillomavirus-like particles allows for efficient induction of protective autoantibodies Liquid vaccine virus Characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells Expression of rotavirus VP2 produces empty corelike particles Human hepatitis B vaccine from recombinant yeast Effect of ammonia production on intracellular pH: Consequent effect on adenovirus vector production Effect of re-feed strategies and non-ammoniagenic medium on adenovirus production at high cell densities Transcriptional profiling of batch and fed-batch protein-free 293-HEK cultures Virus-like particle production at low multiplicities of infection with the baculovirus insect cell system A continuous process for the production of baculovirus using insect cell cultures Continuous beta-galactosidase production in insect cells with a p10 gene based baculovirus vector in a two-stage bioreactor system Baculovirus expression system scaleup by perfusion of high-density Sf-9 cell cultures A high-yield and scaleable adenovirus vector production process based on high density perfusion culture of HEK 293 cells as suspended aggregates Virus-like particle and viral vector production using the baculovirus expression vector system/insect cell system Modeling retrovirus production for gene therapy. 1. Determination of optimal bioreaction mode and harvest strategy Monitoring and control of animal cell reactors: Biochemical Engineering considerations Scale up of insect cell cultures: Protective effects of Pluronic F-68 Kinetic analysis of alkaline protease activity, recombinant protein production and metabolites for infected insect (Sf9) cells under different DO levels Effects of oxygen on recombinant protein expression Rapid purification of extracellular and intracellular Moloney murine leukemia virus Purification of recombinant rotavirus VP7 glycoprotein for the study of in vitro rotavirus-like particles assembly Towards purification of adenoviral vectors based on membrane technology Scaleable purification process for gene therapy retroviral vectors Partial purification of intracellular murine sarcoma-leukemia virus RNA species by membrane filtration Mocr: A novel fusion tag for enhancing solubility that is compatible with structural biology applications Generation of chimeric baculovirus with histidine-tags displayed on the envelope and its purification using immobilized metal affinity chromatography Attachment of histidine tags to recombinant tumor necrosis factor-alpha drastically changes its properties Relative position of the hexahistidine tag effects binding properties of a tumor-associated single-chain Fv construct Serum-free and serum-containing media for growth of suspended BHK aggregates in stirred vessels Production of adenoviral vectors and its recovery Downstream processing of viral vectors and vaccines Anion-exchange membrane chromatography for purification of rotavirus-like particles Packaging cell lines for simian foamy virus type 1 vectors Impact of retroviral vector components stoichiometry on packaging cell lines: Effects on productivity and vector quality Use of early baculovirus promoters for continuous expression and efficient processing of foreign gene products in stably transformed lepidopteran cells High throughput creation of recombinant adenovirus vectors by direct cloning, green-white selection and I-Sce I-mediated rescue of circular adenovirus plasmids in 293 cells 293 Cell cycle synchronisation adenovirus vector production High-level recombinant protein production in bioreactors using the baculovirus-insect cell expression system Quantification of rotavirus-like particles by gel permeation chromatography Stochastic simulation of protein expression in the baculovirus/insect cells system Functional analysis of various promoters in lentiviral vectors at different stages of in vitro differentiation of mouse embryonic stem cells Promoter influence on baculovirus-mediated gene expression in permissive and nonpermissive insect cell lines Critical relationship between glycosylation of recombinant lutropin receptor ectodomain and its secretion from baculovirusinfected insect cells Modeling rotavirus-like particles production in a baculovirus expression vector system: Infection kinetics, baculovirus DNA replication, mRNA synthesis and protein production Modeling the population dynamics of baculovirus-infected insect cells: Optimizing infection strategies for enhanced recombinant protein yields Population kinetics during simultaneous infection of insect cells with two different recombinant baculoviruses for the production of rotavirus-like particles Production of parvovirus B19 vaccine in insect cells co-infected with double baculoviruses Baculovirus defective interfering particles are responsible for variations in recombinant protein production as a function of multiplicity of infection Modeling and optimization of the baculovirus expression vector system in batch suspension culture Baculovirus expression of the human papillomavirus type 16 capsid proteins: Detection of L1-L2 protein complexes Kinetic analyses of stability of simple and complex retroviral vectors Determination of infectious retrovirus concentration from colony-forming assay with quantitative analysis Error assessment in recombinant baculovirus titration: Evaluation of different methods Quantitation of MLV-based retroviral vectors using real-time RT-PCR A sensitive method for the detection of murine C-type retroviruses Murine leukemia virus nucleocapsid mutant particles lacking viral RNA encapsidate ribosomes High-performance liquid chromatography method for rapid assessment of viral particle number in crude adenoviral lysates of mixed serotype High-performance liquid chromatographic total particles quantification of retroviral vectors pseudotyped with vesicular stomatitis virus-G glycoprotein Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell suspension culture system: From shake flasks to a 20-L bioreactor Monitoring virus-like particle and viral protein production by intact cell MALDI-TOF mass spectrometry Assessment of RLPs production and assembly efficiency using capillary zone electrophoresis Advances in on-line monitoring and control of mammalian cell cultures: Supporting the PAT initiative In situ 2D fluorometry and chemometric monitoring of mammalian cell cultures Model-based analysis of assembly kinetics for virus capsids or other spherical polymers A kinetic and statistical-thermodynamic model for baculovirus infection and virus-like particle assembly in suspended insect cells An equilibrium model of the self assembly of polyhedral protein complexes Theoretical aspects of virus capsid assembly Mechanism of capsid assembly for an icosahedral plant virus How does your virus grow? Understanding and interfering with virus assembly Impact of physicochemical parameters on in vitro assembly and disassembly kinetics of recombinant triple-layered rotavirus-like particles Gene therapy: Use of viruses as vectors Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses Transplacental drug delivery: Gene and virus delivery to the trophoblast Production and purification of recombinant African swine fever virus attachment protein p12 Innovative applications for insect viruses: Towards insecticide sensitization Smallpox vaccines for biodefense: Need and feasibility Disrupting the transmission of influenza A: Face masks and ultraviolet light as control measures Viruses and their uses in nanotechnology Heterotypic protection and induction of a broad heterotypic neutralization response by rotavirus-like particles Influenza vaccines based on virus-like particles An engineered virus as a scaffold for three-dimensional self-assembly on the nanoscale Viruses as building blocks for materials and devices Use of recombinant rotavirus VP6 nanotubes as a multifunctional template for the synthesis of nanobiomaterials functionalized with metals Fluorescent signal amplification of carbocyanine dyes using engineered viral nanoparticles