Killed but metabolically active microbes: a new vaccine paradigm for eliciting effector T-cell responses and protective immunity.

We developed a new class of vaccines, based on killed but metabolically active (KBMA) bacteria, that simultaneously takes advantage of the potency of live vaccines and the safety of killed vaccines. We removed genes required for nucleotide excision repair (uvrAB), rendering microbial-based vaccines exquisitely sensitive to photochemical inactivation with psoralen and long-wavelength ultraviolet light. Colony formation of the nucleotide excision repair mutants was blocked by infrequent, randomly distributed psoralen crosslinks, but the bacterial population was able to express its genes, synthesize and secrete proteins. Using the intracellular pathogen Listeria monocytogenes as a model platform, recombinant psoralen-inactivated Lm DeltauvrAB vaccines induced potent CD4(+) and CD8(+) T-cell responses and protected mice against virus challenge in an infectious disease model and provided therapeutic benefit in a mouse cancer model. Microbial KBMA vaccines used either as a recombinant vaccine platform or as a modified form of the pathogen itself may have broad use for the treatment of infectious disease and cancer.

Infection of human dendritic cells by a sindbis virus replicon vector is determined by a single amino acid substitution in the E2 glycoprotein.

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 p55(Gag) 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.

Delivery systems for gene-based vaccines.

Along with the elucidation of the role of cytotoxic T lymphocytes in the immune responses against a number of pathogens and cancer, and with the increased understanding of the cellular processing mechanisms of antigens for generation of these cells, has come an increased focus on vaccines that can generate cellular immunity along with antibodies. Promising approaches based on the delivery of genes, either as plasmid DNA or by viral vectors, have been extensively evaluated pre-clinically and in early-phase clinical trials. Although the first generation of DNA plasmid vaccines were broadly effective in animal disease models, early clinical immunogenicity pointed towards the need for increased potency. This manuscript reviews recent developments for gene-based vaccines, specifically, new approaches for formulating and delivering plasmid DNA and alphaviral replicon vectors, all of which have resulted in increased potency of gene-based vaccines.

Replicon vectors derived from Sindbis virus and Semliki forest virus that establish persistent replication in host cells.

Alphavirus replicon vectors are well suited for applications where transient, high-level expression of a heterologous gene is required. Replicon vector expression in cells leads to inhibition of host macromolecular synthesis, culminating in eventual cell death by an apoptotic mechanism. For many applications, including gene expression studies in cultured cells, a longer duration of transgene expression without resulting cytopathic effects is useful. Recently, noncytopathic Sindbis virus (SIN) variants were isolated in BHK cells, and the mutations responsible were mapped to the protease domain of nonstructural protein 2 (nsP2). We report here the isolation of additional variants of both SIN and Semliki Forest virus (SFV) replicons encoding the neomycin resistance gene that can establish persistent replication in BHK cells. The SIN and SFV variant replicons resulted from previously undescribed mutations within one of three discrete regions of the nsP2 gene. Differences among the panel of variants were observed in processing of the nonstructural polyprotein and in the ratios of subgenomic to genomic RNAs. Importantly, high-level expression of a heterologous gene was retained with most replicons. Finally, in contrast to previous studies, efficient packaging was obtained with several of the variant replicons. This work expands the utility of noncytopathic replicons and the understanding of how alphavirus replicons establish persistent replication in cultured cells.

VSV-G pseudotyped lentiviral vector particles produced in human cells are inactivated by human serum.

Lentiviral vectors transduce dividing and postmitotic cells and thus are being developed toward therapies for many diseases affecting diverse tissues. One essential requirement for efficacy will be that vector particles are resistant to inactivation by human serum complement. Most animal studies with lentiviral vectors have utilized VSV-G pseudotyped envelopes. Here we demonstrate that VSV-G pseudotyped HIV and FIV vectors produced in human cells are inactivated by human serum complement, suggesting that alternative envelopes may be required for therapeutic efficacy for many clinical applications of lentiviral vectors.

Generation of retroviral packaging and producer cell lines for large-scale vector production and clinical application: improved safety and high titer.

For many applications, human clinical therapies using retroviral vectors still require many technological improvements in key areas of vector design and production. These improvements include higher unprocessed manufacturing titers, complement-resistant vectors, and minimized potential to generate replication-competent retrovirus (RCR). To address these issues, we have developed a panel of human packaging cell lines (PCLs) with reduced homology between retroviral vector and packaging components. These reduced-homology PCLs allowed for the use of a novel high multiplicity of transduction ("high m.o. t.") method to introduce multiple copies of provector within vector-producing cell lines (VPCLs), resulting in high-titer vector without the generation of RCR. In a distinct approach to increase vector yields, we integrated manufacturing parameters into screening strategies and clone selection for large-scale vector production. Collectively, these improvements have resulted in the development of diverse VPCLs with unprocessed titers exceeding 2 x 10(7) CFU/ml. Using this technology, human Factor VIII VPCLs yielding titers as high as 2 x 10(8) CFU/ml unprocessed supernatant were generated. These cell lines produce complement-resistant vector particles (N. J. DePolo et al., J. Virol. 73: 6708-6714, 1999) and provide the basis for an ongoing Factor VIII gene therapy clinical trial.

Transduction of murine cerebellar neurons with recombinant FIV and AAV5 vectors.

Our data demonstrate that vectors derived from recombinant feline immunodeficiency virus (rFIV) and adeno-associated virus type 5 (rAAV5) transduce cerebellar cells following direct injection into the cerebellar lobules of mice. Both recombinant viruses mediated gene transfer predominantly to neurons, with up to 2500 and 1500 Purkinje cells transduced for rAAV5 or rFIV-based vectors, respectively. The vectors also transduced stellate, basket and Golgi neurons, with occasional transduction of granule cells and deep cerebellar nuclei. rAAV5 also spread outside the cerebellum to the inferior colliculus and ventricular epithelium, while rFIV demonstrated the ability to undergo retrograde transport to the physically close lateral vestibular nuclei. Thus, AAV5 and FIV-based vectors show promise for targeting neurons affected in the hereditary spinocerebellar ataxias. These vectors could be important tools for unraveling the pathophysiology of these disorders, or in testing factors which may promote neuronal survival.

Enhancement of tumor-specific immune response with plasmid DNA replicon vectors.

To enhance the immunogenicity of nucleic acid vaccines, we used plasmid DNA vectors that contained replicons derived from the prototype alphavirus, Sindbis, and another alphavirus, Semliki Forest virus. When transfected into cells or injected directly into animal muscle, these plasmids launch a self-replicating RNA vector (replicon) which in turn directs the expression of a model tumor antigen. Immunization with plasmid DNA replicons elicited immune responses at doses 100 to 1000-fold lower than conventional DNA plasmids and effectively treated mice bearing an experimental tumor expressing the model antigen. Significantly, replicon-based DNA plasmids did not produce a greater quantity of antigen; instead, antigen production differed qualitatively. Plasmid DNA replicons mediated antigen production that was homogeneous in all transfected cells and associated with the apoptotic death of the host cells. Because of their safety and efficacy, plasmid DNA replicons may be useful in the development of recombinant vaccines for infectious diseases and cancer.

Alphavirus DNA and particle replicons for vaccines and gene therapy.

Alphaviruses have several features that make them attractive as gene delivery platforms, and vectors derived principally from Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEE), are currently being developed as prophylactic and therapeutic vaccines for infectious diseases and cancer. Alphavirus vectors, termed "replicons", retain the nonstructural protein genes encoding the viral replicase, that in turn programme high level cytoplasmic amplification of the vector RNA. We have developed plasmid DNA and recombinant vector particle delivery systems derived from the prototype alphavirus, SIN. Each system uses RNA polymerase II-based expression of alphavirus genome components and both vector formats are highly efficacious towards inducing robust antigen-specific immune responses in vaccinated animals. To increase the potency of SIN vector particles, which are not known to be lymphotropic, the tropism was re-directed for efficient infection of dendritic cells, both in vitro and in vivo.

Feline immunodeficiency virus vectors persistently transduce nondividing airway epithelia and correct the cystic fibrosis defect.

Several problems limit the application of gene transfer to correct the cystic fibrosis (CF) Cl(-) transport defect in airway epithelia. These include inefficient transduction with vectors applied to the apical surface, a low rate of division by airway epithelial cells, failure of transgene expression to persist, and immune responses to vectors or vector-encoded proteins. To address these issues, we used a feline immunodeficiency virus-based (FIV-based) vector. FIV vector formulated with a calcium chelator transduced fully differentiated, nondividing human airway epithelia when applied to the apical surface. FIV-based vector encoding the cystic fibrosis transmembrane conductance regulator cDNA corrected the Cl(-) transport defect in differentiated CF airway epithelia for the life of the culture (>3 months). When this approach was applied in vivo, FIV vector expressing beta-galactosidase transduced 1-14% of adult rabbit airway epithelia. Transduced cells were present in the conducting airways, bronchioles, and alveoli. Importantly, gene expression persisted, and cells with progenitor capacity were targeted. FIV-based lentiviral vectors may be useful for the treatment of genetic lung diseases such as CF. This article may have been published online in advance of the print edition.

Alphavirus vectors for gene expression and vaccines.

Alphavirus expression vectors are finding novel uses in research. They are showing increasing promise as vaccines and are being developed for diagnostic assays of other viruses. Some highlights over the past couple of years include improvements in packaging of replicons, targeting of Sindbis virus replicons, stable cell lines that can be induced to produce replicons, and the isolation of noncytopathic variants of Sindbis virus replicons. Reports that alphavirus vectors can efficiently infect neurons in rat hippocampal slices should increase their use in neurobiological studies.

The resistance of retroviral vectors produced from human cells to serum inactivation in vivo and in vitro is primate species dependent.

The ability to deliver genes as therapeutics requires an understanding of the vector pharmacokinetics similar to that required for conventional drugs. A first question is the half-life of the vector in the bloodstream. Retroviral vectors produced in certain human cell lines differ from vectors produced in nonhuman cell lines in being substantially resistant to inactivation in vitro by human serum complement (F. L. Cosset, Y. Takeuchi, J. L. Battini, R. A. Weiss, and M. K. Collins, J. Virol. 69:7430-7436, 1995). Thus, use of human packaging cell lines (PCL) may produce vectors with longer half-lives, resulting in more-efficacious in vivo gene therapy. However, survival of human PCL-produced vectors in vivo following systemic administration has not been explored. In this investigation, the half-lives of retroviral vectors packaged by either canine D17 or human HT1080 PCL were measured in the bloodstreams of macaques and chimpanzees. Human PCL-produced vectors exhibited significantly higher concentrations of circulating biologically active vector at the earliest time points measured (>1, 000-fold in chimpanzees), as well as substantially extended half-lives, compared to canine PCL-produced vectors. In addition, the circulation half-life of human PCL-produced vector was longer in chimpanzees than in macaques. This was consistent with in vitro findings which demonstrated that primate serum inactivation of vector produced from human PCL increased with increasing phylogenetic distance from humans. These results establish that in vivo retroviral vector half-life correlates with in vitro resistance to complement. Furthermore, these findings should influence the choice of animal models used to evaluate retroviral-vector-based therapies.

Stable alphavirus packaging cell lines for Sindbis virus and Semliki Forest virus-derived vectors.

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 vector-encoded 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 units/ml, 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 replication-competent virus, suggesting utility for production of large-scale vector preparations. Furthermore, both Sindbis virus-based and Semliki Forest virus-based vectors could be packaged with similar efficiency, indicating the possibility of developing a single PCL for use with multiple alphavirus-derived vectors.

DNA immunization against herpes simplex virus: enhanced efficacy using a Sindbis virus-based vector.

Previously we reported the development of a plasmid DNA expression vector system derived from Sindbis virus (T. W. Dubensky, Jr., et al., J. Virol. 70:508-519, 1996). In vitro, such vectors exhibit high-level heterologous gene expression via self-amplifying cytoplasmic RNA replication. In the present study, we demonstrated the in vivo efficacy of the Sindbis virus-based pSIN vectors as DNA vaccines. A single intramuscular immunization of BALB/c mice with pSIN vectors expressing the glycoprotein B of herpes simplex virus type 1 induced a broad spectrum of immune responses, including virus-specific antibodies, cytotoxic T cells, and protection from lethal virus challenge in two different murine models. In addition, dosing studies demonstrated that the pSIN vectors were superior to a conventional plasmid DNA vector in the induction of all immune parameters tested. In general, 100- to 1,000-fold-lower doses of pSIN were needed to induce the same level of responsiveness as that achieved with the conventional plasmid DNA vector. In some instances, significant immune responses were induced with a single dose of pSIN as low as 10 ng/mouse. These results indicate the potential usefulness of alphavirus-based vectors for DNA immunization in general and more specifically as a herpes simplex virus vaccine.

Plasmid DNA-based alphavirus expression vectors for nucleic acid immunization.

Alphavirus-derived vectors are being developed for vaccine, gene therapy and recombinant protein production applications, based in part on observations of transient, high level expression of heterologous genes in eukaryotic cells. Efficient means for launching the RNA alphavirus genome from RNA polymerase II expression cassettes have been developed, obviating the need for transcription in vitro of long cDNA templates. One system being developed from this technology is a layered plasmid DNA vector which, when inoculated directly into animal muscle, launches a self-amplifying alphavirus vector, resulting in subsequent induction of comparatively robust immune responses specific for the expressed antigen.

Sindbis virus DNA-based expression vectors: utility for in vitro and in vivo gene transfer.

Several DNA-based Sindbis virus vectors were constructed to investigate the feasibility and potential applications for initiating the virus life cycle in cells transfected directly with plasmid DNA. These vectors, when transfected into mammalian cells, have been used to produce virus, to express heterologous genes, and to produce infectious vector particles. This approach involved the conversion of a self-replicating vector RNA (replicon) into a layered DNA-based expression system. The first layer includes a eukaryotic RNA polymerase II expression cassette that initiates nuclear transcription of an RNA which corresponds to the Sindbis virus vector replicon. Following transport of this RNA from the nucleus to the cytoplasm, the second layer, autocatalytic amplification of the vector, proceeds according to the Sindbis virus replication cycle and results in expression of the heterologous gene. The Sindbis virus DNA vectors expressed reporter genes in transfected cells at levels that were comparable to those of in vitro-transcribed RNA replicons and were approximately 10-fold higher than the levels produced by conventional RNA polymerase II-dependent plasmids in which the promoter and reporter gene were linked directly. Reporter gene expression was also observed in rodent muscle following injection with Sindbis virus DNA vectors. In a second application, packaged vector particles were produced in cells cotransfected with complementing replicon and defective helper DNAs. The Sindbis virus-derived DNA vectors described here increase the utility of alphavirus-based vector systems in general and also provide a vector with broad potential applications for genetic immunization.

The phylogenetic relationship and complete nucleotide sequence of human papillomavirus type 35.

The 7851-bp nucleotide sequence of human papillomavirus (HPV) type 35 was determined. HPV 35 is associated with high-grade cervical intraepithelial neoplasia and invasive carcinomas. From the HPV 35 sequence, open reading frames encoding putative proteins E6, E7, E1, E2, E4, E5, L2, and L1, common to other mucosal HPV types, were identified. Structural and control elements present in the long control region (LCR) conserved among other mucosal HPV types were also present in HPV 35. Analysis of the LCR revealed an additional 20-bp sequence element present in all HPV types associated with malignant proliferation. To further classify HPV 35 with regard to oncogenic potential, phylogenetic analysis of the E6 and E7 proteins from the anogenital HPV types 6, 11, 16, 18, 31, 33, 35, 39, 43, 44, 45, and 51 was performed. This analysis indicated three distinct HPV subgroups; those associated with benign lesions and two branches of those HPV types more often associated with malignant proliferation. HPV 35 is most closely related to HPV types 31 and 16.