Cationic HDL mimetics enhance in vivo delivery of self-replicating mRNA.

In vivo delivery of large RNA molecules has significant implications for novel gene therapy, biologics delivery, and vaccine applications. We have developed cationic nanolipoprotein particles (NLPs) to enhance the complexation and delivery of large self-amplifying mRNAs (replicons) in vivo. NLPs are high-density lipoprotein (HDL) mimetics, comprised of a discoidal lipid bilayer stabilized by apolipoproteins that are readily functionalized to provide a versatile delivery platform. Herein, we systematically screened NLP assembly with a wide range of lipidic and apolipoprotein constituents, using biophysical metrics to identify lead candidates for in vivo RNA delivery. NLPs formulated with cationic lipids successfully complexed with RNA replicons encoding luciferase, provided measurable protection from RNase degradation, and promoted replicon in vivo expression. The NLP complexation of the replicon and in vivo transfection efficiency were further enhanced by modulating the type and percentage of cationic lipid, the ratio of cationic NLP to replicon, and by incorporating additive molecules.

Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development.

Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with Chlamydia trachomatis are initially asymptomatic, symptoms can arise if left undiagnosed. Long-term infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. Chlamydia infection, therefore, constitutes a significant public health threat, underscoring the need for a Chlamydia-specific vaccine. Chlamydia strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein for vaccine development are limited by poor solubility, low yield, and protein misfolding. Here, we used an Escherichia coli-based cell-free system to express a MOMP protein from the mouse-specific species Chlamydia muridarum (MoPn-MOMP or mMOMP). The codon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1 (Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal 49 amino acids were removed. This co-translation process produced mMOMP supported within a telodendrimer nanolipoprotein particle (mMOMP-tNLP). The cell-free expressed mMOMP-tNLPs contain mMOMP multimers similar to the native MOMP protein. This cell-free process produced on average 1.5 mg of purified, water-soluble mMOMP-tNLP complex in a 1-ml cell-free reaction. The mMOMP-tNLP particle also accommodated the co-localization of CpG oligodeoxynucleotide 1826, a single-stranded synthetic DNA adjuvant, eliciting an enhanced humoral immune response in vaccinated mice. Using our mMOMP-tNLP formulation, we demonstrate a unique approach to solubilizing and administering membrane-bound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity.

Development of a nanoparticle-based influenza vaccine using the PRINT technology.

Historically it is known that presentation of vaccine antigens in particulate form, for a wide range of pathogens, has clear advantages over the presentation of soluble antigen alone [J.C. Aguilar, E.G. Rodriguez, Vaccine adjuvants revisited. Vaccine 25 (2007) 3752-3762, M. Singh, D. O'Hagan, Advances in vaccine adjuvants. Nature Biotechnology 17 (1999) 1075-1081]. Herein we describe a novel particle-based approach, which independently controls size, shape, and composition to control the delivery and presentation of vaccine antigen to the immune system. Highly uniform particles were produced using a particle molding technology called PRINT (Particle Replication in Non-wetting Templates) which is an off-shoot of imprint lithography [J Am Chem Soc 127 (2005) 10096-10100, J Am Chem Soc 126 (2004) 2322-2323, Chem Soc Rev 35 (2006) 1095-1104, J Am Chem Soc 130 (2008) 5008-5009, J Am Chem Soc 130 (2008) 5438-5439, Polymer Reviews 47 (2007) 321-327, Acc Chem Res 41 (2008) 1685-1695, Acc Chem Res 44 (10) (2011) 990-998]. Cylindrical (diameter [d]=80 nm, height [h]=320 nm) poly (lactide-co-glycolide) (PLGA) based PRINT particles were designed to electrostatically bind commercial trivalent injectable influenza vaccine. In a variety of blended PLGA formulations, these particles were safe and showed enhanced responses to influenza hemagglutinin in murine models. FROM THE CLINICAL EDITOR: Shape is one of the determining factors in interactions of nanoparticles with their biologic environment. PRINT technology is able to fabricate nearly uniform nanoparticles and this technology is tested here in murine models to effectively deliver influenza vaccine.

Rendering protein-based particles transiently insoluble for therapeutic applications.

Herein, we report the fabrication of protein (bovine serum albumin, BSA) particles which were rendered transiently insoluble using a novel, reductively labile disulfide-based cross-linker. After being cross-linked, the protein particles retain their integrity in aqueous solution and dissolve preferentially under a reducing environment. Our data demonstrates that cleavage of the cross-linker leaves no chemical residue on the reactive amino group. Delivery of a self-replicating RNA was achieved via the transiently insoluble PRINT protein particles. These protein particles can provide new opportunities for drug and gene delivery.

Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and antitumor effects.

We recently demonstrated that Venezuelan equine encephalitis virus-based replicon particle (VRPs) encoding tumor antigens could break tolerance in the immunomodulatory environment of advanced cancer. We hypothesized that local injection of VRP-expressing interleukin-12 (IL-12) at the site of injections of VRP-based cancer vaccines would enhance the tumor-antigen-specific T cell and antibody responses and antitumor efficacy. Mice were immunized with VRP encoding the human tumor-associated antigen, carcinoembryonic antigen (CEA) (VRP-CEA(6D)), and VRP-IL-12 was also administered at the same site or at a distant location. CEA-specific T cell and antibody responses were measured. To determine antitumor activity, mice were implanted with MC38-CEA-2 cells and immunized with VRP-CEA with and without VRP-IL-12, and tumor growth and mouse survival were measured. VRP-IL-12 greatly enhanced CEA-specific T cell and antibody responses when combined with VRP-CEA(6D) vaccination. VRP-IL-12 was superior to IL-12 protein at enhancing immune responses. Vaccination with VRP-CEA(6D) plus VRP-IL-12 was superior to VRP-CEA(6D) or VRP-IL-12 alone in inducing antitumor activity and prolonging survival in tumor-bearing mice. Importantly, local injection of VRP-IL-12 at the VRP-CEA(6D) injection site provided more potent activation of CEA-specific immune responses than that of VRP-IL-12 injected at a distant site from the VRP-CEA injections. Together, this study shows that VRP-IL-12 enhances vaccination with VRP-CEA(6D) and was more effective at activating CEA-specific T cell responses when locally expressed at the vaccine site. Clinical trials evaluating the adjuvant effect of VRP-IL-12 at enhancing the immunogenicity of cancer vaccines are warranted.

Androgen ablation augments prostate cancer vaccine immunogenicity only when applied after immunization.

BACKGROUND: Androgen ablation (AA) causes apoptosis of normal and neoplastic prostate cells. It is a standard treatment for advanced prostate cancer. Androgen ablation-mediated immunological effects include bone marrow hyperplasia, thymic regeneration, T and B cell lymphopoeisis and restoration of age-related peripheral T cell dysfunction. Androgens also regulate the transcription of several cytokines. Dendritic cells (DC) are the most potent antigen presenting cells that can activate antigen-specific naïve T cells. Despite myriad clinical trials involving DC-based prostate cancer immunotherapies, the effects of AA on DC function remain largely uncharacterized. Therefore, we investigated the effects of AA on DC and whether it could improve the efficacy of prostate cancer immunotherapy. METHODS: Cytokine expression changes due to AA were quantified by multiplex ELISA. Flow cytometry was used to assess AA-mediated effects on DC maturation and expression of costimulatory markers. Mixed leukocyte reactions and cell-mediated lysis assays elucidated the role of androgens in DC function. The effect of AA on the efficacy of vaccination against a prostate tumor-associated antigen was tested using Elispot assays. RESULTS: Androgen ablation increased dendritic cell maturation and costimulatory marker expression, but had no effect on DC costimulatory function. However, DC isolated from castrated mice increased the expression of key cytokines by antigen-experienced T cells while decreasing their expression in naïve cells. Finally, androgen ablation improved immune responses to vaccination only when applied after immunization. CONCLUSION: Androgen ablation causes differential effects of DC on primary and secondary T cell responses, thus augmenting vaccine immunogenicity only when applied after immunization.

In vivo effects of vaccination with six-transmembrane epithelial antigen of the prostate: a candidate antigen for treating prostate cancer.

Immunotherapy may provide an alternative treatment for cancer patients, especially when tumors overexpress antigens that can be recognized by immune cells. The identification of markers and therapeutic targets that are up-regulated in prostate cancer has been important to design new potential treatments for prostate cancer. Among them, the recently identified six-transmembrane epithelial antigen of the prostate (STEAP) is considered attractive due to its overexpression in human prostate cancer tissues. Our study constitutes the first assessment of the in vivo effectiveness of STEAP-based vaccination in prophylactic and therapeutic mouse models. Two delivery systems, cDNA delivered by gene gun and Venezuelan equine encephalitis virus-like replicon particles (VRP), both encoding mouse STEAP (mSTEAP) and three vaccination strategies were used. Our results show that mSTEAP-based vaccination was able to induce a specific CD8 T-cell response against a newly defined mSTEAP epitope that prolonged the overall survival rate in tumor-challenged mice very significantly. This was achieved without any development of autoimmunity. Surprisingly, CD4 T cells that produced IFNgamma, tumor necrosis factor-alpha (TNF-alpha), and interleukin-2 (IL-2) played the main role in tumor rejection in our model as shown by using CD4- and CD8-deficient mice. In addition, the presence of high IL-12 levels in the tumor environment was associated with a favorable antitumor response. Finally, the therapeutic effect of STEAP vaccination was also assessed and induced a modest but significant delay in growth of established, 31 day old tumors. Taken together, our data suggest that vaccination against mSTEAP is a viable option to delay tumor growth.

Restricted and selective tropism of a Venezuelan equine encephalitis virus-derived replicon vector for human dendritic cells.

Dendritic cells (DCs) consist of heterogeneous phenotypic populations and have diverse immunostimulatory functions dependent on both lineage and functional phenotype, but as exceptionally potent antigen-presenting cells, they are targets for generating effective antigen-specific immune responses. A promising replicon particle vector derived from Venezuelan equine encephalitis virus (VEE) has been reported to transduce murine footpad DCs. However, the receptive DC subset, the degree of restriction for this tropism, and the extent of conservation between rodents and humans have not been well characterized. Using fresh peripheral blood DCs, mononuclear cells, monocyte-derived macrophages, and monocyte-derived DCs, our results demonstrate conservation of VEE replicon particle (VRP) tropism for DCs between humans and rodents. We observed that a subset of immature myeloid DCs is the target population, and that VRP-transduced immature DCs retain intact functional capacity, for example, the ability to resist the cytopathic effects of VRP transduction and the capacity to acquire the mature phenotype. These studies support the demonstration of selective VRP tropism for human DCs and provide further insight into the biology of the VRP vector, its parent virus, and human DCs.

Digital-to-biological converter for on-demand production of biologics.

Manufacturing processes for biological molecules in the research laboratory have failed to keep pace with the rapid advances in automization and parellelization. We report the development of a digital-to-biological converter for fully automated, versatile and demand-based production of functional biologics starting from DNA sequence information. Specifically, DNA templates, RNA molecules, proteins and viral particles were produced in an automated fashion from digitally transmitted DNA sequences without human intervention.

Phage Therapy in the Era of Synthetic Biology.

For more than a century, bacteriophage (or phage) research has enabled some of the most important discoveries in biological sciences and has equipped scientists with many of the molecular biology tools that have advanced our understanding of replication, maintenance, and expression of genetic material. Phages have also been recognized and exploited as natural antimicrobial agents and nanovectors for gene therapy, but their potential as therapeutics has not been fully exploited in Western medicine because of challenges such as narrow host range, bacterial resistance, and unique pharmacokinetics. However, increasing concern related to the emergence of bacteria resistant to multiple antibiotics has heightened interest in phage therapy and the development of strategies to overcome hurdles associated with bacteriophage therapeutics. Recent progress in sequencing technologies, DNA manipulation, and synthetic biology allowed scientists to refactor the entire bacterial genome of Mycoplasma mycoides, thereby creating the first synthetic cell. These new strategies for engineering genomes may have the potential to accelerate the construction of designer phage genomes with superior therapeutic potential. Here, we discuss the use of phage as therapeutics, as well as how synthetic biology can create bacteriophage with desirable attributes.

Phylogenetic Distribution of CRISPR-Cas Systems in Antibiotic-Resistant Pseudomonas aeruginosa.

UNLABELLED: Pseudomonas aeruginosa is an antibiotic-refractory pathogen with a large genome and extensive genotypic diversity. Historically, P. aeruginosa has been a major model system for understanding the molecular mechanisms underlying type I clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas)-based bacterial immune system function. However, little information on the phylogenetic distribution and potential role of these CRISPR-Cas systems in molding the P. aeruginosa accessory genome and antibiotic resistance elements is known. Computational approaches were used to identify and characterize CRISPR-Cas systems within 672 genomes, and in the process, we identified a previously unreported and putatively mobile type I-C P. aeruginosa CRISPR-Cas system. Furthermore, genomes harboring noninhibited type I-F and I-E CRISPR-Cas systems were on average ~300 kb smaller than those without a CRISPR-Cas system. In silico analysis demonstrated that the accessory genome (n = 22,036 genes) harbored the majority of identified CRISPR-Cas targets. We also assembled a global spacer library that aided the identification of difficult-to-characterize mobile genetic elements within next-generation sequencing (NGS) data and allowed CRISPR typing of a majority of P. aeruginosa strains. In summary, our analysis demonstrated that CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributed P. aeruginosa isolates. IMPORTANCE: P. aeruginosa is both an antibiotic-refractory pathogen and an important model system for type I CRISPR-Cas bacterial immune systems. By combining the genome sequences of 672 newly and previously sequenced genomes, we were able to provide a global view of the phylogenetic distribution, conservation, and potential targets of these systems. This analysis identified a new and putatively mobile P. aeruginosa CRISPR-Cas subtype, characterized the diverse distribution of known CRISPR-inhibiting genes, and provided a potential new use for CRISPR spacer libraries in accessory genome analysis. Our data demonstrated the importance of CRISPR-Cas systems in modulating the accessory genomes of globally distributed strains while also providing substantial data for subsequent genomic and experimental studies in multiple fields. Understanding why certain genotypes of P. aeruginosa are clinically prevalent and adept at horizontally acquiring virulence and antibiotic resistance elements is of major clinical and economic importance.

Rapidly produced SAM(®) vaccine against H7N9 influenza is immunogenic in mice.

The timing of vaccine availability is essential for an effective response to pandemic influenza. In 2009, vaccine became available after the disease peak, and this has motivated the development of next generation vaccine technologies for more rapid responses. The SAM(®) vaccine platform, now in pre-clinical development, is based on a synthetic, self-amplifying mRNA, delivered by a synthetic lipid nanoparticle (LNP). When used to express seasonal influenza hemagglutinin (HA), a SAM vaccine elicited potent immune responses, comparable to those elicited by a licensed influenza subunit vaccine preparation. When the sequences coding for the HA and neuraminidase (NA) genes from the H7N9 influenza outbreak in China were posted on a web-based data sharing system, the combination of rapid and accurate cell-free gene synthesis and SAM vaccine technology allowed the generation of a vaccine candidate in 8 days. Two weeks after the first immunization, mice had measurable hemagglutinin inhibition (HI) and neutralizing antibody titers against the new virus. Two weeks after the second immunization, all mice had HI titers considered protective. If the SAM vaccine platform proves safe, potent, well tolerated and effective in humans, fully synthetic vaccine technologies could provide unparalleled speed of response to stem the initial wave of influenza outbreaks, allowing first availability of a vaccine candidate days after the discovery of a new virus.

An alphavirus vector overcomes the presence of neutralizing antibodies and elevated numbers of Tregs to induce immune responses in humans with advanced cancer.

Therapeutic anticancer vaccines are designed to boost patients' immune responses to tumors. One approach is to use a viral vector to deliver antigen to in situ DCs, which then activate tumor-specific T cell and antibody responses. However, vector-specific neutralizing antibodies and suppressive cell populations such as Tregs remain great challenges to the efficacy of this approach. We report here that an alphavirus vector, packaged in virus-like replicon particles (VRP) and capable of efficiently infecting DCs, could be repeatedly administered to patients with metastatic cancer expressing the tumor antigen carcinoembryonic antigen (CEA) and that it overcame high titers of neutralizing antibodies and elevated Treg levels to induce clinically relevant CEA-specific T cell and antibody responses. The CEA-specific antibodies mediated antibody-dependent cellular cytotoxicity against tumor cells from human colorectal cancer metastases. In addition, patients with CEA-specific T cell responses exhibited longer overall survival. These data suggest that VRP-based vectors can overcome the presence of neutralizing antibodies to break tolerance to self antigen and may be clinically useful for immunotherapy in the setting of tumor-induced immunosuppression.

Prostate cancer immunotherapy yields superior long-term survival in TRAMP mice when administered at an early stage of carcinogenesis prior to the establishment of tumor-associated immunosuppression at later stages.

Prostate cancer immunotherapy clinical trials have been performed, but often in immunocompromised patients with limited clinical success. The study aim was to determine whether the stage of prostate cancer development at which immunization occurs affects vaccine efficacy, and if so which tumor-associated immunosuppressive mechanisms may be involved at later stages. Therapeutic vaccination of TRAMP mice with only precancerous PIN lesions confered superior protection to immunization after development of invasive carcinoma. The presence of Treg, upregulation of tumor indoleamine-2,3-dioxygenase and TGFbeta and an immunosuppressive intratumoral cytokine milieu were identified in more advanced prostate cancer. These results indicate that prostate cancer immunotherapy trials will be more successful if conducted in patients with less advanced disease.

Prostate stem cell antigen vaccination induces a long-term protective immune response against prostate cancer in the absence of autoimmunity.

Prostate stem cell antigen (PSCA) is an attractive antigen to target using therapeutic vaccines because of its overexpression in prostate cancer, especially in metastatic tissues, and its limited expression in other organs. Our studies offer the first evidence that a PSCA-based vaccine can induce long-term protection against prostate cancer development in prostate cancer-prone transgenic adenocarcinoma mouse prostate (TRAMP) mice. Eight-week-old TRAMP mice displaying prostate intraepithelial neoplasia were vaccinated with a heterologous prime/boost strategy consisting of gene gun-delivered PSCA-cDNA followed by Venezuelan equine encephalitis virus replicons encoding PSCA. Our results show the induction of an immune response against a newly defined PSCA epitope that is mediated primarily by CD8 T cells. The prostates of PSCA-vaccinated mice were infiltrated by CD4-positive, CD8-positive, CD11b-positive, and CD11c-positive cells. Vaccination induced MHC class I expression and cytokine production [IFN-gamma, tumor necrosis factor-alpha, interleukin 2 (IL-2), IL-4, and IL-5] within prostate tumors. This tumor microenvironment correlated with low Gleason scores and weak PSCA staining on tumor cells present in hyperplastic zones and in areas that contained focal and well-differentiated adenocarcinomas. PSCA-vaccinated TRAMP mice had a 90% survival rate at 12 months of age. In contrast, all control mice had succumbed to prostate cancer or had heavy tumor loads. Crucially, this long-term protective immune response was not associated with any measurable induction of autoimmunity. The possibility of inducing long-term protection against prostate cancer by vaccination at the earliest signs of its development has the potential to cause a dramatic paradigm shift in the treatment of this disease.

Analysis of Venezuelan equine encephalitis replicon particles packaged in different coats.

BACKGROUND: The Venezuelan equine encephalitis (VEE) virus replicon system was used to produce virus-like replicon particles (VRP) packaged with a number of different VEE-derived glycoprotein (GP) coats. The GP coat is believed to be responsible for the cellular tropism noted for VRP and it is possible that different VEE GP coats may have different affinities for cells. We examined VRP packaged in four different VEE GP coats for their ability to infect cells in vitro and to induce both humoral and cellular immune responses in vivo. METHODOLOGY/PRINCIPAL FINDINGS: The VRP preparations were characterized to determine both infectious units (IU) and genome equivalents (GE) prior to in vivo analysis. VRP packaged with different VEE GP coats demonstrated widely varying GE/IU ratios based on Vero cell infectivity. BALB/c mice were immunized with the different VRP based on equal GE titers and the humoral and cellular responses to the expressed HIV gag gene measured. The magnitude of the immune responses measured in mice revealed small but significant differences between different GP coats when immunization was based on GE titers. CONCLUSIONS/SIGNIFICANCE: We suggest that care should be taken when alternative coat proteins are used to package vector-based systems as the titers determined by cell culture infection may not represent accurate particle numbers and in turn may not accurately represent actual in vivo dose.

Influenza neuraminidase antibodies provide partial protection for chickens against high pathogenic avian influenza infection.

Protection of chickens against avian influenza (AI) is mostly attributed to production of antibodies against the viral glycoprotein hemagglutinin, whereas less is known about the protective role of antibodies to the other surface glycoprotein neuraminidase (NA). Therefore, vaccines encoding NA antigen (e.g., DNA and alphavirus-based virus like replicon particles (VRP)) or baculovirus-expressed recombinant NA (rN2) were tested for their ability to protect against highly pathogenic AI (HPAI) in chickens. Vaccination with A/Pheasant/Maryland/4457/93 (Ph/MD) rN2 protein produced significantly higher levels of NA-inhibition (NI) activity and 88% protection from HPAI H5N2 challenge than vaccination with Ph/MD N2 DNA (25% protection). Vaccination with Ph/MD N2 VRP a minimum of two times also produced high levels of NI activity and protection against HPAI challenge (63% protection). Vaccination with VRP encoding an N2 gene that was genetically distant from the challenge virus N2 failed to protect chickens. Vaccines producing higher levels of NI activity conferred partial protection, but failed to affect viral shedding. Consideration of the homology between vaccine and challenge virus isolate NA genes may provide improved immunity if high levels of NI activity are obtained.

VRP immunotherapy targeting neu: treatment efficacy and evidence for immunoediting in a stringent rat mammary tumor model.

The ability to overcome intrinsic tolerance to a strict "self" tumor-associated antigen (TAA) and successfully treat pre-existing tumor is the most stringent test for anti-tumor immunotherapeutic strategies. Although this capacity has been demonstrated in various models using complicated strategies that may not be readily translated into the clinical arena, straightforward antigen-specific immunotherapeutic strategies in the most stringent models of common epithelial cancers have largely failed to meet this standard. We employed an immunotherapeutic strategy using an alphavirus-based, virus-like replicon particle (VRP), which has in vivo tropism for dendritic cells, to elicit immune responses to the non-mutated TAA rat neu in an aggressive rat mammary tumor model. Using this VRP-based immunotherapeutic strategy targeting a single TAA, we generated effective anti-tumor immunity in the setting of pre-existing tumor resulting in the cure of 36% of rats over multiple experiments, P = 0.002. We also observed down-regulation of rat neu expression in tumors that showed initial responses followed by tumor escape with resumption of rapid tumor growth. These responses were accompanied by significant anti-tumor proliferative responses and CD8+ cellular tumor infiltrates, all of which were restricted to animals receiving the anti-neu immunotherapy. Together these data, obtained in a stringent "self" TAA model, indicate that the VRP-based antigen-specific immunotherapy elicits sufficiently potent immune responses to exert immunologic pressure, selection, and editing of the growing tumors, thus supporting the activity of this straightforward immunotherapy and suggesting that it is a promising platform upon which to build even more potent strategies.

Development and preclinical evaluation of an alphavirus replicon vaccine for influenza.

We used a propagation-defective, single-cycle, alphavirus replicon vector system to produce virus-like replicon particles (VRP) expressing the hemagglutinin (HA) and neuraminidase (NA) proteins from influenza A/Wyoming/03/2003 (H3N2). Efficient production methods were scaled to produce pilot lots of HA VRP and NA VRP and clinical lots of HA VRP. HA VRP-induced high-titered antibody responses in mice, rabbits and rhesus macaques, as measured by ELISA or hemagglutination inhibition (HI) assays, and robust cellular immune responses in mice and rhesus macaques, as measured by IFN-gamma ELISPOT. NA VRP also induced cellular immune responses in mice. A toxicology study with HA VRP and NA VRP in rabbits showed no adverse effects in any parameter. These studies support clinical testing of alphavirus replicon vaccines for influenza.