Alphavirus expression vectors and their use as recombinant vaccines: a minireview

Alphavirus vectors have become widely used in basic research to study the structure and function of proteins and for protein production purposes. Development of a variety of vectors has made it possible to deliver foreign sequences as naked RNA or DNA, or as suicide virus particles produced using helper vector strategies. Preliminary reports also suggest that these vectors may be useful for in vivo applications where transient, high-level protein expression is desired, such as recombinant vaccines. The initial studies have already shown that alphavirus vaccines can induce strong humoral and cellular immune responses with good immunological memory and protective effects.     

Retrovirus vectors and their uses in molecular biology

Retroviral vectors utilize the biochemical processes unique to retroviruses, to transfer genes with high efficiency into a wide variety of cell types in tissue culture and in living animals. With such vectors, the effect of newly introduced genes and the mechanism of gene expression can be studied in cell types so far refractory to other methods of transfer.     

Parvovirus-like particles as vaccine vectors.

A wide array of systems have been developed to improve "classic" vaccines. The use of small polypeptides able to elicit potent antibody and cytotoxic responses seems to have enormous potential in the design of safer vaccines. While peptide coupling to large soluble proteins such as keyhole limpet hemocyanin is the current method of choice for eliciting antibody responses and insertion in live viruses for cytotoxic T-lymphocyte responses, alternative cheaper and/or safer methods will clearly be required in the future. Virus-like particles constitute very immunogenic molecules that allow for covalent coupling of the epitopes of interest in a simple way. In this article, we detail the methodology employed for the preparation of efficient virus vectors as delivery systems. We used parvovirus as the model for the design of new vaccine vectors. Recently parvovirus-like particles have been engineered to express foreign polypeptides in certain positions, resulting in the production of large quantities of highly immunogenic peptides, and to induce strong antibody, helper-T-cell, and cytotoxic T-lymphocyte responses. We discuss the different alternatives and the necessary steps to carry out this process, placing special emphasis on the flow of decisions that need to be made during the project.     

Lactobacilli as live vaccine delivery vectors: progress and prospects

Evidence is accumulating that lactobacilli influence the immune response in a strain-dependent manner. This immunomodulatory capacity is important for the development of the immune response, and also identifies Lactobacillus as a potent oral vaccine carrier. Most of our current knowledge of the use of lactobacilli for vaccination purposes has been obtained with tetanus toxin fragment C (TTFC) as the model antigen. This knowledge, together with our ever-increasing understanding of the immune system and recent developments in cloning and expression techniques, should enable the utilisation of antigens other than TTFC and has made the development of lactobacilli as live vaccines a realistic prospect.     

Attenuated vesicular stomatitis viruses as vaccine vectors

We showed previously that a single intranasal vaccination of mice with a recombinant vesicular stomatitis virus (VSV) expressing an influenza virus hemagglutinin (HA) protein provided complete protection from lethal challenge with influenza virus (A. Roberts, E. Kretzschmar, A. S. Perkins, J. Forman, R. Price, L. Buonocore, Y. Kawaoka, and J. K. Rose, J. Virol. 72:4704-4711, 1998). Because some pathogenesis was associated with the vector itself, in the present study we generated new VSV vectors expressing HA which are completely attenuated for pathogenesis in the mouse model. The first vector has a truncation of the cytoplasmic domain of the VSV G protein and expresses influenza virus HA (CT1-HA). This nonpathogenic vector provides complete protection from lethal influenza virus challenge after intranasal administration. A second vector with VSV G deleted and expressing HA (DeltaG-HA) is also protective and nonpathogenic and has the advantage of not inducing neutralizing antibodies to the vector itself.     

Synthesis of polyallylamine derivatives and their use as gene transfer vectors in vitro.

Cationic polymers possessing primary amine groups are inefficient in transferring nucleic acids into eukaryotic cells. With appropriate chemical modification, namely glycolylation of the amine groups of polylysine and polyallylamine, the actual number of free amino groups was decreased, hydrophilic residues were introduced, and the cytotoxicity of both polymers decreased significantly. Furthermore, in the case of polyallylamine, its ability to mediate gene transfer into cells increased by several orders of magnitude. Transfection efficiency was found to be dependent on the substitution level of amino groups and reached highest levels in the presence of lysosomotropic and/or fusogenic agents. At optimal conditions, glycolylated PAM was shown to be as efficient as the linear polyethylenimine of 22 kDa.     

Pathogenic bacteria as vaccine vectors: teaching old bugs new tricks

As our scientific knowledge of bacteria grows, so does our ability to manipulate these bacteria to protect rather than infect mammalian hosts from a diverse group of diseases. The old axiom that the best way to protect from a disease is to get infected in the first place is not feasible in the face of the diverse group of pathogens that infect humans. Therefore, reprogramming bacteria to protect against diverse bacterial, viral, and parasitic diseases as well as cancer is a new reality in the field of vaccines.     

Internal ribosome entry site biology and its use in expression vectors.

Internal ribosome entry sites (IRESs) are cis-acting elements that recruit the small ribosomal subunits to an internal initiator codon in the mRNA with the help of cellular trans-acting factors. The recent discovery of the IRES recognition site of the eIF4G initiation factor is beginning to shed some light into how IRES elements are recognized by the translational machinery. Additionally, the progress made in the understanding of the parameters that influence start codon selection will be instrumental in establishing the rational design of bicistronic expression vectors.     

Comparative analysis of macrophage associated vectors for use in genetic vaccine

Background

Antigen presentation by non professional antigen presenting cells (APC) can lead to anergy. In genetic vaccines, targeting the macrophages and APC for efficient antigen presentation might lead to balanced immune response. One such approach is to incorporate APC specific promoter in the vector to be used.

Methods

Three promoters known to be active in macrophage were selected and cloned in mammalian expressing vector (pAcGFP1-N1) to reconstruct (pAcGFP-MS), (pAcGFP-EMR) and (pAcGFP-B5I) with macrosialin, EmrI and Beta-5 Integrin promoters respectively. As a positive control (pAcGFP-CMV) was used with CMV promoter and promoterless vector (pAcGFP-NIX) which served as a negative control. GFP gene was used as readout under the control of each of the promoter. The expression of GFP was analyzed on macrophage and non-macrophage cell lines using Flow cytometry and qRT-PCR with TaqMan probe chemistries.

Results

All the promoters in question were dominant to macrophage lineage cell lines as observed by fluorescence, Western blot and quantitative RT-PCR. The activity of macrosialin was significantly higher than other macrophage promoters. CMV promoter showed 1.83 times higher activity in macrophage cell lines. The expression of GFP driven by macrosialin promoter after 24 hours was 4.40 times higher in macrophage derived cell lines in comparison with non macrophage cell lines.

Conclusions

Based on this study, macrosialin promoter can be utilized for targeting macrophage dominant expression. In vivo study needs to be carried out for its utility as a vaccine candidate.     

The conundrum between immunological memory to adenovirus and their use as vectors in clinical gene therapy

In the context of clinical gene transfer using viral vectors, the risk of memory antivector immunity is often poorly appreciated. The immunological past of the patient, the site of injection, and the vector dose will play intertwined and decisive roles in the safety and efficacy of treatment. To circumvent the drawbacks due to the ubiquitous human adenovirus (HAd) memory immunity, we believe that vectors derived from canine adenovirus type 2 (CAV-2) will be more clinically useful than those derived from HAds based, in part, on the potential lack of immunological memory. CAV-2 is not a human pathogen in spite of the approx 100,000 yr of cohabitation of humans with dogs. During the last 8 yr, we found that CAV-2 vectors preferentially transduced neurons in the central nervous system (CNS) of several species, and had a surprisingly efficient level of axoplasmic transport. CAV-2 vectors also lead to greater than 1 yr transgene expression in the immunocompetent rat CNS-without immunosuppression. However, more immediate harm can be caused to a patient via an acute and/or chronic vector-induced cellular infiltration in the CNS than by the normal progression of most neurodegenerative disorders. In this context, we continue to assess the clinical potential of CAV-2. This mini-review addresses our analysis of the interaction of CAV-2 vectors with human memory immunity and monocyte-derived dendritic cells.     

Characterization of putative rolling-circle plasmids from the Gram-negative bacterium Xylella fastidiosa and their use as shuttle vectors

This study was initiated to characterize a small Xylella fastidiosa (X. fastidiosa) plasmid and attempt to create a X. fastidosa/Escherichia coli shuttle vector that was stable in planta. Restriction enzyme analysis of a 1.3kb plasmid DNA from a grape-infecting strain of X. fastidiosa (UCLA) revealed the presence of three similar, but genetically distinct, plasmids, pUCLAs. Evidence that suggests the pUCLA plasmids replicate via a rolling-circle (RC) mechanism include: (i) the presence of ssDNA in X. fastidiosa cells; (ii) the presence of conserved motifs in the predicted ORF1 that are typical of initiator (Rep) proteins associated with RC replication; (iii) high amino acid identity between the putative Rep proteins of pUCLAs and Pf3, a filamentous bacteriophage of Pseudomonas aeruginosa that replicates by a RC mechanism; and (iv) the presence of a putative origin of replication upstream of ORF1 that has the potential to form secondary hairpin structures. One DNA motif present in pUCLA shared sequence similarity to known nicking sites in the origins of replication of other RC plasmids and phages. The shuttle vector, pXF001, successfully transformed grape X. fastidiosa strains and was found to be present as autonomous, structurally unchanged DNA molecules in X. fastidiosa. However, pXF001 was not stably maintained in X. fastidiosa without antibiotic selection.     

Polypeptide composition of rotavirus empty capsids and their possible use as a subunit vaccine.

Two types of empty capsid particles that differed with respect to the presence of the two outer shell proteins were isolated from MA-104 cells infected with bovine rotavirus V1005. Three previously uncharacterized polypeptides, I, II, and III, migrating between VP2 and VP6, were detected in empty capsids but not in single- and double-shelled rotavirus particles. Peptide mapping revealed that all three proteins were related to VP2. Polypeptides I, II, and III could be generated by in vitro trypsin digestion of empty capsids not exposed to trypsin in the infection medium. Labeled polypeptides appeared in empty capsids before they were detected in intracellular single- or double-shelled rotavirus particles. Empty capsids were also observed in MA-104 cells infected with bovine rotaviruses UK and NCDV, simian rotavirus SA11, and human rotavirus KU. VP7-containing empty capsid is the minimal subunit vaccine for cows; we failed to induce a substantial neutralizing antibody increase with VP7 purified under denaturating or nondenaturating conditions or with synthetic peptides corresponding to two regions of VP7.     

Construction of plasmids integrating into the Bacillus subtilis chromosome through homologous recombination and their use as integration vectors

We constructed a number of plasmids which integrate into the chromosome of Bacillus subtilis through homology recombination. Plasmids consist of pBR322 replicon, different fragments of Bac. subtilis chromosomal DNA, Cm resistance marker from pBD64 plasmid. Frequency of transformation was 10(-4) per bacterial cell. Foreign DNA (genes for tryptophan metabolism of Bac. mesentericus) was introduced into the chromosome of Bac. subtilis with the help of these plasmids.     

Systems biology of persistent infection: tuberculosis as a case study

The human immune response does an excellent job of clearing most of the pathogens that we encounter throughout our lives. However, some pathogens persist for the lifetime of the host. Despite many years of research, scientists have yet to determine the basis of persistence of most pathogens, and have therefore struggled to develop reliable prevention and treatment strategies. Systems biology provides a new and integrative tool that will help to achieve these goals. In this article, we use Mycobacterium tuberculosis as an example of how systems-biology approaches have begun to make strides in uncovering important facets of the host-pathogen interaction.