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.

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.

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.

T-cell lymphomas emerging as epineoplasms in mice bearing transplanted polyoma virus-induced salivary gland tumors.

A subset of salivary epithelial tumors induced by mouse polyoma virus (PyV) has been designated lymphoepithelioma on the basis of a prominent lymphocytic component. Serial transplantation of this variant has previously been observed to result in lymphoma development. A recent repetition of this phenomenon allowed us to characterize the lymphoma cell populations with regard to phenotypic markers and PyV content. Lymphomas emerged in recipients of the third, fifth, sixth, and seventh transplant generations of the lymphoepithelioma. Most lymphomas were widely disseminated in hematopoietic and lymphoreticular tissues, and other sites as well. Flow cytometric analysis of lymphocyte populations from lymphomas in six recipients revealed that, while all lymphomas expressed phenotypic markers of immature cortical thymocytes, i.e., Thy-1, Pgp-1, Jlld, and CD5, they were not uniform with regard to other T-cell markers, notably CD4 and CD8. Varying levels of T-cell receptor markers CD3 and alpha/beta, as well as interleukin 2 receptor, were also noted. DNA blot analysis failed to detect PyV in lymphoma cells at a sensitivity level capable of detecting less than one intact copy per cell. It appears improbable the lymphoma was directly induced by PyV. Hypotheses invoking other mechanisms of lymphoma development are outlined.

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.

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.

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.

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.

The primary site of replication alters the eventual site of persistent infection by polyomavirus in mice.

Using DNA blot analysis, we monitored the course of polyomavirus infection in mice receiving an intranasal inoculation and compared this with the course of infection in mice receiving an intraperitoneal inoculation. Intranasal infection was characterized by an initial primary replication phase in the respiratory tract, followed by a systemic infection of the visceral organs. At 12 days postinfection, there was partial clearing of viral DNA in all organs; by 22 days postinfection, viral DNA persisted only in the lungs and kidneys, and the level of DNA slowly decreased during the next 3 months. Lungs have been a previously unrecognized site for polyomavirus persistent infection. In contrast to intranasal infection, intraperitoneal infection of mice was characterized by only three phases: an initial systemic phase in which viral DNA was found in the same respiratory and visceral organs as during intranasal infection, clearing of the virus from the organs, and ultimately, a persistent infection in the kidneys but not in the lungs. Thus, different organs became persistently infected when mice were inoculated via these different routes.

Direct transfection of viral and plasmid DNA into the liver or spleen of mice.

A method for the direct transfection of polyoma viral DNA and polyoma-plasmid recombinant DNA into the liver or spleen of newborn or adult mice was developed. Calcium phosphate-precipitated DNA was injected directly into mouse organs in combination with hyaluronidase and collagenase. Transfected DNA was shown to replicate at moderate efficiency, relative to direct infection of organs with virus. Transfection with viral DNA rapidly led to an acute infection. A polyoma-bacterial plasmid recombinant DNA also was shown to replicate when transfected into mice. With this plasmid, however, genomic-length polyoma DNA rapidly recombined away from the bacterial component and replicated as viral DNA. This method should allow the direct determination of the biological activity of a cloned DNA within a mouse organ.

Detection of DNA and RNA virus genomes in organ systems of whole mice: patterns of mouse organ infection by polyomavirus.

A technique which detects viral DNA or RNA in situ in the organ systems of whole mice is described. Frozen thin sections from whole mice were transferred directly to nitrocellulose and hybridized to labeled viral DNA, allowing the detection of viral DNA or RNA. By this procedure, polyomavirus infection of newborn mice inoculated intranasally was followed. We found that the initial inoculum could be detected in the nasal cavity, lungs, and stomach lining after a 5-h absorption period. Primary replication of virus was observed in the nasal cavity, submaxillary gland, and lungs, followed by a systemic phase of infection in which the liver, spleen, kidney, and large colon also became infected. Viral RNA as well as DNA could also be detected as shown by infecting mice intracerebrally with vesicular stomatitis virus. Vesicular stomatitis virus-specific RNA was observed only in the brains of these mice. It is most likely that this technique can be applied to general molecular studies of mice. With this method we should be able to detect all viruses, bacteria, plasmids, and organ-specific transcripts to which a cloned probe exists.

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.

Polyomavirus replication in mice: influences of VP1 type and route of inoculation.

Patterns of polyomavirus replication and spread have been studied following inoculation of virus into newborn mice. Levels of virus replication in different tissues were followed in situ by using whole mouse section blots and immunoperoxidase staining for the major capsid protein VP1, as well as by tissue extraction and direct quantitation of viral DNA and infectious virus. Patterns of replication and spread were compared between the "high tumor" strain (inducing a high incidence of tumors) PTA and and the "low tumor" strain (inducing a low incidence of tumors) RA, following different routes of inoculation. The ability to induce a high tumor profile correlated with the ability to establish disseminated productive infection, with the kidney as a major site of amplification. Furthermore, results with PTA-RA recombinant viruses and site-directed mutants showed that the VP1 specificity of PTA, demonstrated earlier to be a critical determinant for induction of a high tumor profile (R. Freund, A. Calderone, C. J. Dawe, and T. L. Benjamin, J. Virol. 65:335-341, 1991), is also critical for amplification in the kidney and for establishment of disseminated infections.

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.

Necrotizing arterial lesions in mice-bearing tumors induced by polyoma virus.

In the course of determining tumor profiles for wild-type and recombinant mouse polyoma viruses (MPyV's), we fortuitously discovered two types of necrotizing arterial disease in polyoma tumor-bearing C3H/BiDa mice. One type, designated BLAND, consisted of foci of necrosis unaccompanied by inflammatory reaction, in the muscular coat of aorta, pulmonary arterial trunk, and primary or occasionally secondary branches of these vessels. BLAND lesions contained MPyV capsid antigen VP1 as shown by immunocytochemistry, and appeared to be the result of viral cytolytic infection within artery walls. Lesions of the second type are designated PANoid in view of their resemblance to polyarteritis nodosa in humans. PANoid lesions had much the same distribution in the arterial tree as BLAND lesions and were also focal, but had the histologic properties of highly destructive acute inflammatory reactions. Specifically, there were dense infiltrations of polymorphonuclear leukocytes in any and often all coats of the arterial wall, acute fibrinoid necrosis, endothelial proliferations, intravascular thrombosis, and in one example, rupture of the intimal and medial coats with microaneurysm formation. In the acute phase, PANoid lesions exhibited attenuation, fragmentation, and loss of elastic laminae, and in the healing phase, intimal and medial fibrosis with varying degrees of lumenal occlusion. PANoid lesions gave negative immunocytochemical reactions for MPyV capsid antigen VP1, indicating either that the antigen was not present, or that it was masked in complexes with antibody and C3. BLAND lesions were found in 51% of 459 MPyV-infected mice, while PANoid lesions were found in 11%. There was no sex predilection for either type lesion, and practically all mice with lesions fell within ages 60-200 days. We suspect the PANoid lesions are examples of immune-complex arteritis related to persistent MPyV infection, but support for this hypothesis is presently tenuous, resting entirely on the coexistence of BLAND and PANoid lesions in MPyV-infected mice and the histological resemblance of PANoid lesions to naturally occurring and experimentally induced immune complex arteritis.

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.

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.