Actinic keratosis (AK) and cutaneous squamous cell carcinoma (SCC) are two of the most common dermatologic conditions in Western countries and cause substantial morbidity worldwide. The role of human papillomaviruses under these conditions has been well studied yet remains inconclusive. One PCR-based study has investigated bacteria in the etiology of these conditions; however, no study has investigated the microbiomes of AK and SCC more broadly. We longitudinally profiled the microbiomes of 112 AK lesions, profiled cross sections of 32 spontaneously arising SCC lesions, and compared these to matching nonlesional photodamaged control skin sites. We identified commonly occurring strains of Propionibacterium and Malassezia at higher relative abundances on nonlesional skin than in AK and SCC lesions, and strains of Staphylococcus aureus were relatively more abundant in lesional than nonlesional skin. These findings may aid in the prevention of SCC.

We have previously developed replicon vectors derived from the Australian flavivirus Kunjin that have a unique noncytopathic nature and have been shown to direct prolonged high-level expression of encoded heterologous genes in vitro and in vivo and to induce strong and long-lasting immune responses to encoded immunogens in mice. To facilitate further applications of these vectors in the form of virus-like particles (VLPs), we have now generated a stable BHK packaging cell line, tetKUNCprME, carrying a Kunjin structural gene cassette under the control of a tetracycline-inducible promoter. Withdrawal of tetracycline from the medium resulted in production of Kunjin structural proteins that were capable of packaging transfected and self-amplified Kunjin replicon RNA into the secreted VLPs at titers of up to 1.6 ؋ 10 9 VLPs per ml. Furthermore, secreted KUN replicon VLPs from tetKUNCprME cells could be harvested continuously for as long as 10 days after RNA transfection, producing a total yield of more than 10 Subgenomic replicon vectors of positive-strand RNA viruses offer great potential as gene expression vectors and for development of vaccines (12). They possess a number of unique advantages over other viral vector systems, which are used for vaccine development. These are the following: (i) high-level expression of encoded heterologous proteins due to efficient amplification of replicon RNA; (ii) no DNA intermediates and replication exclusively in the cell cytoplasm, thereby eliminating any problems associated with integration into the cell genome; (iii) a single infection cycle, and thus, replicon RNA cannot escape from cells they are delivered to, eliminating any possibility of uncontrolled virus spread in vaccinees; and (iv) the relative ease of constructing replicon-based vaccines.Kunjin virus (KUN) is a member of the Japanese encephalitis subgroup of the genus Flavivirus in the family Flaviviridae (41). Unlike the majority of other human pathogens from the Flavivirus genus, KUN appears to be naturally attenuated in the human population, with infections nearly always asymptomatic. KUN is endemic only in the northern parts of Australia and neighboring islands, with no preexisting immunity in the rest of the world. In addition, KUN is genetically stable, and its epidemiology and virus replication strategy are well understood (38), making KUN an attractive candidate for vaccine vector development. The KUN genome consists of a positive-polarity single-stranded RNA encoding a large polyprotein that is cotranslated and posttranslationally cleaved into three structural proteins, i.e., core (C), premembrane (prM), and envelope (E), and seven nonstructural proteins, i.e., NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (3, 13). We previously constructed the first flavivirus replicons based on KUN cDNA by deleting the majority of the genomic region encoding structural genes and retaining only the first 20 codons of the C gene and the last 22 codons of the E gene (14). KUN replicons have been extensively used as vectors for...

Replicon-based vectors of positive-strand RNA viruses are becoming more and more popular for development of antiviral and anticancer vaccines (reviewed in reference 24). Several features make these vectors a desirable choice for development of highly efficient and safe vaccines. These include (i) a high level of expression of encoded heterologous genes (HGs) due to the ability of replicon RNA to amplify itself; (ii) exclusively cytoplasmic replication, which eliminates any possible complications associated with nuclear splicing and/or chromosomal integration; (iii) inability of the replicon RNA to escape from the transfected (or infected) cell, thus limiting the spread of the vaccine vector in the immunized subject, which makes these vectors biologically safe; and (iv) relatively small genome size (7 to 9 kb), allowing easy manipulations with the cDNA and generation of recombinants. An additional beneficial feature of replicon vectors is a variety of modalities that can be used to deliver replicon RNA into cells (Fig. 1A) (24). It can be either delivered directly as naked RNA transcribed in vitro (RNA based), packaged first into virus-like particles (VLPs) and then delivered via infection with these VLPs, or delivered in the form of plasmid DNA, which encodes replicon cDNA placed under the control of a mammalian expression promoter (DNA based), allowing the production of functional replicon RNA in vivo by the cellular RNA polymerase II (Fig. 1A). Replicon-based expression vectors have been developed for representatives of most positive-strand RNA virus families, including alphaviruses, picornaviruses, and flaviviruses (reviewed in reference 24). However, the great majority of the data on immunogenic properties of replicon vectors in laboratory animals have been accumulated using replicons of alphaviruses such as Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEE) (see reference 47 for a review and references 8, 10, 11, and 39 for more recent studies). In general, these studies showed that alphavirus replicon vectors induced strong antibody and CD8 ϩ T-cell responses to encoded immunogens and in most cases protected immunized animals from appropriate virus or tumor challenges. All three delivery modalities were effective; however, VLP delivery was shown to be the most efficient. In comparative studies of conventional (nonreplicating) plasmid DNA vectors and alphavirus DNA-based replicon vectors, the latter generally induced stronger immune responses and at significantly lower DNA concentrations than did conventional vectors (4, 17). However, in some studies the efficiency of immune responses induced by DNA-based alphavirus replicons appeared to depend on the nature of the encoded immunogen, and the immunity to some immunogens was shown to be similar to or lower than that induced by the same amount of conventional plasmid DNA vector (10,22).We have been developing replicon vectors derived from the flavivirus Kunjin (KUN) (25,27,52,53). The KUN replicon expression system,...