Alphavirus replicon particle-based vaccine vectors derived from Sindbis virus (SIN), Semliki Forest virus,and Venezuelan equine encephalitis virus (VEE) have been shown to induce robust antigen-specific cellular, humoral, and mucosal immune responses in many animal models of infectious disease and cancer. However, since little is known about the relative potencies among these different vectors, we compared the immunogenicity of replicon particle vectors derived from two very different parental alphaviruses, VEE and SIN, expressing a human immunodeficiency virus type 1 p55Gag antigen. Moreover, to explore the potential benefits of combining elements from different alphaviruses, we generated replicon particle chimeras of SIN and VEE. Two distinct strategies were used to produce particles with VEE-p55 gag replicon RNA packaged within SIN envelope glycoproteins and SIN-p55 gag replicon RNA within VEE envelope glycoproteins. Each replicon particle configuration induced Gag-specific CD8 ؉ T-cell responses in murine models when administered alone or after priming with DNA. However, Gag-specific responses varied dramatically, with the strongest responses to this particular antigen correlating with the VEE replicon RNA, irrespective of the source of envelope glycoproteins. Comparing the replicons with respect to heterologous gene expression levels and sensitivity to alpha/ beta interferon in cultured cells indicated that each might contribute to potency differences. This work shows that combining desirable elements from VEE and SIN into a replicon particle chimera may be a valuable approach toward the goal of developing vaccine vectors with optimal in vivo potency, ease of production, and safety.

We have expressed and characterized the severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein in cDNA-transfected mammalian cells. The full-length spike protein (S) was newly synthesized as an endoglycosidase H (endo H)-sensitive glycoprotein (gp170) that is further modified into an endo H-resistant glycoprotein (gp180) in the Golgi apparatus. No substantial proteolytic cleavage of S was observed, suggesting that S is not processed into head (S1) and stalk (S2) domains as observed for certain other coronaviruses. While the expressed full-length S glycoprotein was exclusively cell associated, a truncation of S by excluding the C-terminal transmembrane and cytoplasmic tail domains resulted in the expression of an endoplasmic reticulum-localized glycoprotein (gp160) as well as a Golgi-specific form (gp170) which was ultimately secreted into the cell culture medium. Chemical cross-linking, thermal denaturation, and size fractionation analyses suggested that the full-length S glycoprotein of SARS-CoV forms a higher order structure of ϳ500 kDa, which is consistent with it being an S homotrimer. The latter was also observed in purified virions. The intracellular form of the C-terminally truncated S protein (but not the secreted form) also forms trimers, but with much less efficiency than full-length S. Deglycosylation of the full-length homotrimer with peptide N-glycosidase-F under native conditions abolished recognition of the protein by virus-neutralizing antisera raised against purified virions, suggesting the importance of the carbohydrate in the correct folding of the S protein. These data should aid in the design of recombinant vaccine antigens to prevent the spread of this emerging pathogen.

Alphavirus vectors, derived principally from Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus, are widely used for gene expression studies in vitro and are being developed for both vaccine and gene therapy applications (25). Typically, these vectors are constructed in a format known as a replicon, due to the selfamplifying nature of the vector RNA (30). Replicons contain both the cis and trans alphavirus genetic elements required for RNA replication, as well as heterologous gene expression via the native subgenomic promoter. Upon introduction into cells, replicon RNA is translated to produce four nonstructural proteins (nsPs), which together comprise the alphaviral replicase. Replication proceeds through a minus-strand RNA intermediate and subsequently generates two distinct positive-strand RNA species, corresponding to a genomic-length vector RNA and an abundant subgenomic RNA encoding the heterologous gene (27). The replicon RNA can be packaged into virion-like particles by providing the structural proteins in trans, from in vitro-transcribed defective helper RNA (4, 15-17) or using packaging cell lines (16). Alternatively, the replicon RNA can be introduced directly into cells as plasmid DNA (2,6,8,13).In most mammalian cells, host macromolecular synthesis is inhibited following the introduction of alphavirus replicons, leading to eventual cell death by an apoptotic mechanism (11, 25). Thus, application of these vectors for some gene therapy applications and extended gene expression studies in cultured cells is limited. Given the many other attractive features of the alphavirus replicon system, it would be useful to extend the utility of these vectors to include long-term expression and reduced cytopathogenicity options.Under appropriate conditions, alphaviruses and alphavirusderived vectors can establish persistence in cultured cells (14,26,29) or exhibit delayed onset of cytopathic effects (9). The establishment of SIN replicon persistence in BHK cells has been associated with mutation of the protease domain of nsP2 (7, 10), and studies have suggested that the use of such mutants for long-term expression may be possible (1, 3). It remains to be determined whether mutation of other alphavirus nsPs or nsP2 domains can provide a noncytopathic phenotype by a similar or alternative mechanism.To expand the utility of the noncytopathic replicon and further explore how persistence is established, we isolated additional SIN replicons with this phenotype, as well as SFV replicons with a similar phenotype. Mutations that conferred the establishment of persistent replication were mapped to several regions of nsP2 for both SIN and SFV replicons, in addition to the same residue 726 mutation identified previously (7, 10). These mutations had various effects on the levels of genomic and subgenomic replicon RNA and, in some cases, processing of the nonstructural polyprotein.Selection of replicons that establish persistent replication. To select alphavirus replicon variants capable of...

Alphavirus vectors are being developed for possible human vaccine and gene therapy applications. We have sought to advance this field by devising DNA-based vectors and approaches for the production of recombinant vector particles. In this work, we generated a panel of alphavirus vector packaging cell lines (PCLs). These cell lines were stably transformed with expression cassettes that constitutively produced RNA transcripts encoding the Sindbis virus structural proteins under the regulation of their native subgenomic RNA promoter. As such, translation of the structural proteins was highly inducible and was detected only after synthesis of an authentic subgenomic mRNA by the vectorencoded replicase proteins. Efficient production of biologically active vector particles occurred after introduction of Sindbis virus vectors into the PCLs. In one configuration, the capsid and envelope glycoproteins were separated into distinct cassettes, resulting in vector packaging levels of 10 7 infectious units͞ml, but reducing the generation of contaminating replication-competent virus below the limit of detection. Vector particle seed stocks could be amplified after low multiplicity of infection of PCLs, again without generating replicationcompetent virus, suggesting utility for production of largescale vector preparations. Furthermore, both Sindbis virusbased and Semliki Forest virus-based vectors could be packaged with similar efficiency, indicating the possibility of developing a single PCL for use with multiple alphavirusderived vectors.The use of virus-derived expression vectors for gene therapy and vaccine applications increasingly is being pursued, with a number of diverse virus types and approaches. Alphaviruses are attractive candidates for such applications because of their high levels of replication and gene expression, their ability to infect a variety of diverse cell types, and the ability to manipulate cDNA clones from which infectious viral RNA may be transcribed (for review, see refs. 1 and 2). The alphavirus genome is a single-stranded, positive-sense RNA of approximately 11.7 kb and is encapsidated within an icosahedral capsid protein shell (for review, see ref.3). Nucleocapsids, in turn, are surrounded by a host-derived lipid envelope from which the viral spike glycoproteins E1 and E2 protrude. Cytoplasmic replication of the RNA genome is mediated by four viral-encoded nonstructural proteins and proceeds through a full-length negative-sense intermediate. Subsequent positive-strand RNA synthesis results in both progeny genome RNA and an abundant, internally initiated subgenomic mRNA. The virus structural proteins are translated from the subgenomic mRNA as a polyprotein that is processed into the individual components of the virion.The general strategy for construction of alphavirus-based expression vectors has been to substitute the viral structural protein genes with a heterologous gene, maintaining transcriptional control via the highly active subgenomic RNA promoter (1, 2, 4). As such, these vector r...

We sought to develop a retroviral vector system that would produce secretion of high levels of bone morphogenetic protein (BMP)-4 by optimizing the expression construct and developing an improved retroviral vector. Replacement of the propeptide domain of BMP4 with that of BMP2 increased the secretion level of mature BMP4 protein in transduced cells. The intact BMP2 pro-peptide sequence was essential, as deletion of a small part of the propeptide sequence of BMP2 from the BMP2/4 hybrid construct diminished BMP4 expression and secretion. Addition of a hemaglutinin tag to the carboxy terminus of BMP4 abolished the bioactivity of secreted BMP4. Transduction of rat marrow stromal cells (and fibroblasts) with an MFG-based retroviral vector pseudotyped with VSV-G envelope containing this BMP2/4 hybrid expression construct led to secretion of very high levels of mature BMP4 in conditioned medium (up to 1 microg/10(6) cells/24 hours). The secreted BMP4 was biologically active, as it induced alkaline phosphatase expression in C2C12 cells. The transduced rat marrow stromal cells expressing mature BMP4 induced de novo ectopic bone formation in syngenic immune-competent rats. We have developed an MFG-based retroviral vector system that causes secretion of high levels of functionally active human BMP4 protein.

After more than 25 years of human immunodeficiency virus (HIV) research, a prophylactic vaccine able to control or prevent the worldwide spread of HIV/AIDS remains an elusive goal. Recent results in Thailand with the recombinant canary pox (ALVAC-HIV, vCP1521; Sanofi-Pasteur) prime-gp120 (AIDSVAX B/E) protein boost vaccine approach give us hope that such a vaccine is achievable (45). Nevertheless, the results from this trial as well as the disappointing outcome of the Step Study trial (7, 29, 46) vividly highlight the need to better understand the immune correlates of protection and the immune responses engendered by the diverse new vaccine technologies currently under evaluation (13,18,20,49). In the case of viral vectors, this is particularly critical, as the spectrum of immune responses elicited in animal models does not necessarily predict those eventually observed in human clinical trials and will require more thorough evaluations in order to identify the most predictive models. At the moment, nonhuman primate models, such as simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) infection of macaques appear to be the most informative for guiding vaccine development (3,24,47,55), and more rigorous application of these models has begun to yield new and encouraging insights into protective immunity (5,19,27,56). Moreover, as most HIV transmissions occur through mucosal membranes, understanding the correlates of protection, following successful vaccinations, against mucosal challenge is of strong interest.Alphaviruses are positive-sense single-stranded 11.5-kb RNA viruses in the Togaviridae family. They are relatively simple enveloped viruses of approximately 60-nm diameter that have a cytoplasmic RNA-based life cycle and mature at the plasma membranes of infected cells. Recombinant alphavirus replicon particles used for vaccine applications are composed of a replicon vector that encodes the viral replicases (nonstructural proteins [NSPs]) and the vaccine antigen of interest and two packaging vectors that encode the major viral structural proteins (capsid and glycoproteins E1 and E2) required for particle formation. The chimeric (VEE/SIN) alpha-* Corresponding author. Mailing address: Novartis Vaccines and Diagnostics,

The ability to target antigen-presenting cells with vectors encoding desired antigens holds the promise of potent prophylactic and therapeutic vaccines for infectious diseases and cancer. Toward this goal, we derived variants of the prototype alphavirus, Sindbis virus (SIN), with differential abilities to infect human dendritic cells. Cloning and sequencing of the SIN variant genomes revealed that the genetic determinant for human dendritic cell (DC) tropism mapped to a single amino acid substitution at residue 160 of the envelope glycoprotein E2. Packaging of SIN replicon vectors with the E2 glycoprotein from a DC-tropic variant conferred a similar ability to efficiently infect immature human DC, whereupon those DC were observed to undergo rapid activation and maturation. The SIN replicon particles infected skin-resident mouse DC in vivo, which subsequently migrated to the draining lymph nodes and upregulated cell surface expression of major histocompatibility complex and costimulatory molecules. Furthermore, SIN replicon particles encoding human immunodeficiency virus type 1 p55Gag elicited robust Gag-specific T-cell responses in vitro and in vivo, demonstrating that infected DC maintained their ability to process and present replicon-encoded antigen. Interestingly, human and mouse DC were differentially infected by selected SIN variants, suggesting differences in receptor expression between human and murine DC. Taken together, these data illustrate the tremendous potential of using a directed approach in generating alphavirus vaccine vectors that target and activate antigen-presenting cells, resulting in robust antigen-specific immune responses.Dendritic cells (DC) are the most potent antigen-presenting cell population and play a major role in the activation of both memory and naïve T cells. Immature DC capture antigen in the periphery and migrate to the draining lymph nodes, where they undergo maturation. Presentation of acquired antigen by mature DC is critical for induction of antigen-specific immune responses (1, 9, 13, 36) and stimulation of protective T-cell responses (3, 10). Transduction of autologous cultured DC ex vivo with gene delivery vectors encoding a desired antigen, followed by adoptive transfer, has been shown to stimulate antigen-specific T-cell responses in vivo (45,46). Unfortunately, the ability to target the DC cell population in vivo has been quite limited or has been shown to interfere with DC function or development (5,17,20,23,32,39). We rationalized that enhanced delivery of antigen to immature DC may provide an opportunity for improvement of vaccines, particularly for gene-based vaccination approaches.Toward a goal of improving DC-targeting approaches, we have focused on alphavirus-based vectors. The use of alphavirus vectors for vaccine and gene therapy applications is a rapidly emerging field (15, 42, 44). These RNA-based vectors, known as "replicons" because they retain the replicase functions necessary for RNA self-amplification and high-level expression, can be launched in vi...

Almost all vaccinations today are delivered through parenteral routes. Mucosal vaccination offers several benefits over parenteral routes of vaccination, including ease of administration, the possibility of self-administration, elimination of the chance of injection with infected needles, and induction of mucosal as well as systemic immunity. However, mucosal vaccines have to overcome several formidable barriers in the form of significant dilution and dispersion; competition with a myriad of various live replicating bacteria, viruses, inert food and dust particles; enzymatic degradation; and low pH before reaching the target immune cells. It has long been known that vaccination through mucosal membranes requires potent adjuvants to enhance immunogenicity, as well as delivery systems to decrease the rate of dilution and degradation and to target the vaccine to the site of immune function. This review is a summary of current approaches to mucosal vaccination, and it primarily focuses on adjuvants as immunopotentiators and vaccine delivery systems for mucosal vaccines based on protein, DNA or RNA. In this context, we define adjuvants as protein or oligonucleotides with immunopotentiating properties co-administered with pathogenderived antigens, and vaccine delivery systems as chemical formulations that are more inert and have less immunomodulatory effects than adjuvants, and that protect and deliver the vaccine through the site of administration. Although vaccines can be quite diverse in their composition, including inactivated virus, virus-like particles and inactivated bacteria (which are inert), protein-like vaccines, and non-replicating viral vectors such as poxvirus and adenovirus (which can serve as DNA delivery systems), this review will focus primarily on recombinant protein antigens, plasmid DNA, and alphavirus-based replicon RNA vaccines and delivery systems. This review is not an exhaustive list of all available protein, DNA and RNA vaccines, with related adjuvants and delivery systems, but rather is an attempt to highlight many of the currently available approaches in immunopotentiation of mucosal vaccines.Key words: adjuvant, delivery system, immunomodulator, mucosal, vaccine. Recombinant protein vaccinesTraditional vaccines have comprised live-attenuated microbes, inactivated microorganisms, purified microbial components, polysaccharide-carrier protein conjugates or recombinant proteins. The first of the latter type of vaccine was derived from the diphtheria and tetanus toxoids, and was developed in the first half of the twentieth century. The two toxins were chemically detoxified to produce the non-toxic toxoids. Conventional approaches to vaccine development have been based on biochemical, immunological and microbiological methods that have been labourious, time-consuming and have allowed identification of the most abundant antigens of any given pathogen. Recent progress in DNA sequencing and subsequently in bioinformatics have resulted in advances in vaccine development. When the whole sequence of ...