Duodenal endoluminal barrier sleeve alters gut microbiota of ZDF rats.

BACKGROUND/OBJECTIVES: The combination of energy dense diets and reduced energy expenditure in modern society has escalated the prevalence of obesity and obesity-related comorbidities. Among these disease states, type-2 diabetics (T2D) are disproportionately associated with obesity, suggesting a shared etiology. In conjunction with defects in hormonal and inflammatory states, obesity and T2D are also characterized by dysbiosis. METHODS: We have recently described the beneficial effects of duodenal nutrient exclusion, as induced by the duodenal endoluminal sleeve (DES); including body weight loss, prevented fat mass accumulation, and improved glucose tolerance in the ZDF rat, a rodent model of obesity and type-2 diabetes (T2D). To assess the relative role of DES on hindgut microbiota in the context of these metabolic changes, we analyzed cecal samples from rats implanted with a duodenal endoluminal sleeve (DES), or a sham control of this procedure. A group of pair-fed (pf) sham controls was also included to account for changes induced by reduced body weight and food intake. RESULTS: Analysis of hindgut microbiota following DES in the ZDF rat elucidated discrete changes in several microbial populations including a reduction in Paraprevotella family members of the Clostridiales order along with an increase in Akkermansia muciniphila and species of the Allobaculum and Bifidobacterium genera. CONCLUSIONS: Altogether, these observations suggest that like Roux-en Y gastric bypass (RYGB) and Metformin, regulation of gut microbiota may be a contributing factor to the therapeutic effects of DES.

Fecal metabolomics in pediatric spondyloarthritis implicate decreased metabolic diversity and altered tryptophan metabolism as pathogenic factors.

We have previously shown alterations in the composition of the gut microbiota in children with enthesitis-related arthritis (ERA). To explore the mechanisms by which an altered microbiota might predispose to arthritis, we performed metabolomic profiling of fecal samples of children with ERA. Fecal samples were collected from two cohorts of children with ERA and healthy control subjects. Nano-liquid chromatography-mass spectroscopy (LC-MS) was performed on the fecal water homogenates with identification based upon mass: charge ratios. Sequencing of the 16S ribosomal DNA (rDNA) on the same stool specimens was performed. In both sets of subjects, patients demonstrated lower diversity of ions and under-representation of multiple metabolic pathways, including the tryptophan metabolism pathway. For example, in the first cohort, out of 1500 negatively charged ions, 154 were lower in ERA patients, compared with only one that was higher. Imputed functional annotation of the 16S ribosomal DNA sequence data demonstrated significantly fewer microbial genes associated with metabolic processes in the patients compared with the controls (77 million versus 58 million, P=0.050). Diminished metabolic diversity and alterations in the tryptophan metabolism pathway may be a feature of ERA.

Treatment rates and healthcare costs of patients with fragility fracture by site of care: a real-world data analysis.

In a characterization of treatment rates and healthcare costs among patients with an osteoporotic-related fragility fracture overall and by site of care, costs were high and treatment rates were low. PURPOSE: Osteoporotic fractures can be debilitating, even fatal, among older adults. The cost of osteoporosis and related fractures is projected to increase to more than $25 billion by 2025. The objective of this analysis is to characterize disease-related treatment rates and healthcare costs of patients with an osteoporotic fragility fracture overall and by site of fracture diagnosis. METHODS: In this retrospective analysis, individuals with fragility fractures were identified in the Merative MarketScan® Commercial and Medicare Databases among women 50 years of age or older and diagnosed with fragility fracture between 1/1/2013 and 6/30/2018 (earliest fracture diagnosis = index). Cohorts were categorized by clinical site of care where the diagnosis of fragility fracture was made and were continuously followed for 12 months prior to and following index. Sites of care were inpatient admission, outpatient office, outpatient hospital, emergency room hospital, and urgent care. RESULTS: Of the 108,965 eligible patients with fragility fracture (mean age 68.8), most were diagnosed during an inpatient admission or outpatient office visit (42.7%, 31.9%). The mean annual healthcare costs among patients with fragility fracture were $44,311 (± $67,427) and were highest for those diagnosed in an inpatient setting ($71,561 ± $84,072). Compared with other sites of care at fracture diagnosis, patients diagnosed during an inpatient admission also had highest proportion of subsequent fractures (33.2%), osteoporosis diagnosis (27.7%), and osteoporosis therapy (17.2%) during follow-up. CONCLUSION: The site of care for diagnosis of fragility fracture affects treatment rates and healthcare costs. Further studies are needed to determine how attitude or knowledge about osteoporosis treatment or healthcare experiences differ at various clinical sites of care in the medical management of osteoporosis.

Gut microbial differences in breast and prostate cancer cases from two randomised controlled trials compared to matched cancer-free controls.

Implicated in several chronic diseases, the gastrointestinal microbiome is hypothesised to influence carcinogenesis. We compared faecal microbiota of newly diagnosed treatment-naïve overweight and obese cancer patients and matched controls. Cases were enrolled in presurgical weight-loss trials for breast (NCT02224807) and prostate (NCT01886677) cancers and had a body mass index (BMI) ≥25 kg/m2. Cancer-free controls were matched 1:1 by age (±5 years), race, gender, and BMI (±5 kg/m2). All participants provided faecal samples; isolated bacterial DNA were PCR amplified at the V4 region of the 16S rRNA gene and analysed using the QIIME pipeline. Tests compared cases versus controls, then separately by gender. Microbial alpha-diversity and beta-diversity were assessed, and relative abundance of Operational Taxonomic Units (OTU's) were compared at the genus level, with false discovery rate (FDR) correction. 22 overweight and obese cancer patients were matched with 22 cancer-free controls, with an average BMI of 30.5±4.3 kg/m2, age 54.4±5.3 years, and 54.5% were black. Fourteen matches were made between breast cancer cases and healthy female controls, and 8 matches were made with prostate cancer cases and healthy male controls. Comparison of all cases and controls revealed no differences in alpha diversity, though prostate cancer patients had higher Chao1 (P=0.006) and Observed Species (P=0.036) than cancer-free males. Beta-diversity metrics were significantly different between cases and controls (P<0.03 for all tests in whole sample and in men), though only unweighted Unifrac was different in women (P=0.005). Kruskal Wallis tests indicated significant differences among 16 genera in all matches, 9 in female, and 51 in male. This study suggests the faecal microbiota of treatment-naive breast and prostate cancer patients differs from controls, though larger samples are needed to substantiate these findings. Trial registration: NIH Clinical Trials, NCT01886677, NCT02224807, registered 26 June 2013, 25 Aug 2014 (respectively) - retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT01886677; https://clinicaltrials.gov/ct2/show/NCT02224807.

Examining the Role of Microbiota in Emotional Behavior: Antibiotic Treatment Exacerbates Anxiety in High Anxiety-Prone Male Rats.

Intestinal microbiota are essential for healthy gastrointestinal function and also broadly influence brain function and behavior, in part, through changes in immune function. Gastrointestinal disorders are highly comorbid with psychiatric disorders, although biological mechanisms linking these disorders are poorly understood. The present study utilized rats bred for distinct emotional behavior phenotypes to examine relationships between emotionality, the microbiome, and immune markers. Prior work showed that Low Novelty Responder (LR) rats exhibit high levels of anxiety- and depression-related behaviors as well as myriad neurobiological differences compared to High Novelty Responders (HRs). Here, we hypothesized that the divergent HR/LR phenotypes are accompanied by changes in fecal microbiome composition. We used next-generation sequencing to assess the HR/LR microbiomes and then treated adult HR/LR males with an antibiotic cocktail to test whether it altered behavior. Given known connections between the microbiome and immune system, we also analyzed circulating cytokines and metabolic factors to determine relationships between peripheral immune markers, gut microbiome components, and behavioral measures. There were no baseline HR/LR microbiome differences, and antibiotic treatment disrupted the microbiome in both HR and LR rats. Antibiotic treatment exacerbated aspects of HR/LR behavior, increasing LRs' already high levels of anxiety-like behavior while reducing passive stress coping in both strains. Our results highlight the importance of an individual's phenotype to their response to antibiotics, contributing to the understanding of the complex interplay between gut microbes, immune function, and an individual's emotional phenotype.

Microbial community changes in a female rat model of Rett syndrome.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder that is predominantly caused by alterations of the methyl-CpG-binding protein 2 (MECP2) gene. Disease severity and the presence of comorbidities such as gastrointestinal distress vary widely across affected individuals. The gut microbiome has been implicated in neurodevelopmental disorders such as Autism Spectrum Disorder (ASD) as a regulator of disease severity and gastrointestinal comorbidities. Although the gut microbiome has been previously characterized in humans with RTT compared to healthy controls, the impact of MECP2 mutation on the composition of the gut microbiome in animal models where the host and diet can be experimentally controlled remains to be elucidated. By evaluating the microbial community across postnatal development as behavioral symptoms appear and progress, we have identified microbial taxa that are differentially abundant across developmental timepoints in a zinc-finger nuclease rat model of RTT compared to WT. We have additionally identified p105 as a key translational timepoint. Lastly, we have demonstrated that fecal SCFA levels are not altered in RTT rats compared to WT rats across development. Overall, these results represent an important step in translational RTT research.

Cytokine production in motor neurons by poliovirus replicon vector gene delivery.

Poliovirus replicon vectors transiently express foreign proteins selectively in motor neurons of the anterior horn of the spinal cord. Here we intraspinally inoculated mice transgenic for the poliovirus receptor (PVR) with replicons encoding murine tumor necrosis factor alpha (mTNF-alpha). We detected high-level expression of mTNF-alpha in the spinal cords of these animals at 8-12 h post inoculation; this returned to background by 72 h. The mice exhibited ataxia and tail atony, whereas animals given a replicon encoding green fluorescent protein (GFP) exhibited no neurological symptoms. Histology of spinal cords from mice given the replicon encoding mTNF-alpha revealed neuronal chromatolysis, reactive astrogliosis, decreased expression of myelin basic protein, and demyelination. These animals recovered with only slight residual damage. This study shows that replicon vectors have potential for targeted delivery of therapeutic proteins to the central nervous system and provide a new approach for treatment of spinal cord trauma and neurological disease.

Comparative effectiveness of faecal microbiota transplant by route of administration.

The optimal route of delivery for faecal microbiota transplant (FMT) is unknown. This observational single-centre study analysed the two-week cure rates for all patients who received FMT from 2013 to 2016 according to route of delivery. Overall, nasogastric delivery of FMT was less effective than lower endoscopic delivery. When patients were stratified by illness severity, nasogastric delivery achieved similar cure rates in healthier individuals, whereas lower endoscopic delivery was preferred for relatively ill individuals. Nasogastric delivery may be less effective than lower endoscopic delivery; however, when taking the cost, preparation and potential risk into account, this difference may not be clinically significant for patients with mild disease.

Helicobacter pylori infection is associated with an altered gastric microbiota in children.

The intestinal microbiome in early life influences development of the mucosal immune system and predisposition to certain diseases. Because less is known about the microbiome in the stomach and its relationship to disease, we characterized the microbiota in the stomachs of 86 children and adults and the impact of Helicobacter pylori infection on the bacterial communities. The overall composition of the gastric microbiota in children and adults without H. pylori infection was similar, with minor differences in only low abundance taxa. However, the gastric microbiota in H. pylori-infected children, but not infected adults, differed significantly in the proportions of multiple high abundance taxa compared with their non-infected peers. The stomachs of H. pylori-infected children also harbored more diverse microbiota, smaller abundance of Firmicutes, and larger abundance of non-Helicobacter Proteobacteria and several lower taxonomic groups than stomachs of H. pylori-infected adults. Children with restructured gastric microbiota had higher levels of FOXP3, IL10, and TGFß expression, consistent with increased T-regulatory cell responses, compared with non-infected children and H. pylori-infected adults. The gastric commensal bacteria in children are altered during H. pylori infection in parallel with more tolerogenic gastric mucosae, potentially contributing to the reduced gastric disease characteristic of H. pylori-infected children.

Essential regions of the tRNA primer required for HIV-1 infectivity.

Human immunodeficiency virus (HIV), like all retroviruses, requires a cellular tRNA as a primer for initiation of reverse transcription. In a previous study, we demonstrated that an HIV-1 with a primer binding site complementary to yeast tRNA(Phe) (psHIV-Phe) was not infectious unless yeast tRNA(Phe) was supplied in trans. This unique in vivo complementation system has now been used to define the elements of the tRNA required for HIV-1 replication. Mutant tRNA(Phe) with deletions in TPsiC stem-loop, anticodon stem-loop or D stem-loop of the tRNA were generated and assessed for the capacity to rescue psHIV-Phe. Mutant tRNA(Phe) with disrupted TPsiC stem-loop did not rescue psHIV-Phe. In contrast, a mutant tRNA(Phe) without the D stem-loop was fully functional for the rescue. The tRNA anticodon stem-loop region was found to be important for efficient complementation. The results of our studies demonstrate for the first time the importance of specific structural and sequence elements of the tRNA primer for HIV-1 reverse transcription and define new targets for interruption of HIV-1 replication.

RNA replicons derived from poliovirus are directly oncolytic for human tumor cells of diverse origins.

The failure and/or toxicity of conventional therapies for many types of human cancers underscore the need for development of safe and effective alternative treatments. Toward this goal, we describe the direct oncolytic activity of RNA-based vectors derived from poliovirus, termed replicons, which are genetically incapable of producing infectious virus. These replicons are cytopathic in vitro for human tumor cells originating from brain, breast, lung, ovary, and skin (melanoma). The cytopathic effects in a malignant glioma cell line were associated with nuclear DNA condensation, indicative of cells undergoing apoptosis. Injection of replicons into established xenograft flank tumors in scid mice resulted in oncolytic activity and extended survival. Inoculation of replicons into established intracranial xenograft tumors in scid mice resulted in tumor infection within 8 h and extended survival. Histological analysis revealed that replicons had infected tumor cells at the site of inoculation and, most importantly, diffused to infect tumor cells that had metastasized from the initial site of implantation. The wide spectrum of cytopathic activity for human tumors combined with effective distribution after in vivo inoculation establishes the therapeutic potential of poliovirus replicons for a variety of cancers.

Characterization of poliovirus replicons encoding carcinoembryonic antigen.

Recombinant vaccines hold great promise for the prevention and therapy of infections diseases and cancer. We have explored the use of poliovirus as a recombinant vector to deliver genes into cells for the purpose of vaccination. For our studies, we have chosen to express the gene-encoding carcinoembryonic antigen (CEA) using a novel poliovirus vector. We have constructed a recombinant CEA-poliovirus replicon in which the CEA gene was substituted for the poliovirus capsid gene. Following in vitro transcription, the RNA was transfected into cells to demonstrate CEA expression. We found that a genome in which the region encoding the signal sequence of the CEA protein (amino acids 1-34) was removed was replication competent (i.e., referred to as a replicon). We encapsidated the CEA-poliovirus replicon by transfecting this RNA into cells previously infected with a recombinant vaccinia virus (VV-P1) which expresses the poliovirus capsid protein (P1). Serial passage in the presence of VV-P1 resulted in the generation of stocks of these encapsidated replicons. Infection of cells with the encapsidated replicon containing the CEA-poliovirus genome resulted in expression of the CEA protein. To test immunogenicity, mice susceptible to poliovirus were given three doses of the encapsidated replicons via the i.m. route. By the third administration, a CEA-specific antibody response was detected. Potential future use of the poliovirus replicon system as both a parenteral and oral vaccine vector is discussed.

Recombinant viruses as vectors for mucosal immunity.

The development and characterization of viral based vaccine vectors is extremely active research field. Much of this work has been facilitated by developments in molecular biology that allow work with large plasmid-based vectors, as well as the use of PCR. Several different vector systems are now available using RNA viruses and DNA viruses. Each vector system has its own strengths and weaknesses. Due to the differences and diversity between the viruses used as vectors, it is doubtful that a single system will be useful for all desired vaccines. However, the further development of existing, as well as potentially new systems, will provide a repertoire for vaccinologists to design the recombinant vaccine which will generate an optimal humoral and immune response for protection against infection or disease caused by pathogens that infect via mucosal surfaces.

Repetitive intrathecal injections of poliovirus replicons result in gene expression in neurons of the central nervous system without pathogenesis.

Poliovirus-based vectors (replicons) can be used for gene delivery to motor neurons of the CNS. In the current study, a replicon encoding green fluorescent protein (GFP) was encapsidated into authentic poliovirions, using established procedures. Intrathecal delivery of encapsidated replicons encoding GFP to the CNS of mice transgenic for the human poliovirus receptor did not result in any functional deficits as judged by behavioral testing. Histological analysis of the CNS of mice given a single intrathecal injection of poliovirus replicons encoding GFP revealed no obvious pathogenesis in neurons (or other cell types) within the CNS. The expression of GFP was confined to motor neurons throughout the neuroaxis; a time course of expression of GFP revealed that expression was detectable 24 hr postinoculation and returned to background levels by 120 hr postinoculation. A procedure was devised to allow repetitive inoculation of replicons within the same animal. Behavioral testing of animals that had received 6 to 13 independent inoculations of replicons revealed no functional deficits. Histological analysis of the CNS from animals that had received 6 to 13 sequential inoculations of replicons revealed no obvious abnormalities in neurons or other cell types in the CNS; expression of GFP was demonstrated in neurons 24 to 72 hr after the final inoculation of the replicon. Furthermore, there was no obvious inflammatory response in the CNS after the multiple inoculations. These studies establish the safety and efficacy of replicons for gene delivery to the CNS and are discussed with respect to use of replicons as new therapeutic strategies for spinal cord injuries and/or neurological diseases.

Construction and characterization of encapsidated poliovirus replicons that express biologically active murine interleukin-2.

Poliovirus genomes have been constructed in which the capsid genes have been substituted with the murine gene encoding interleukin-2 (IL-2) (referred to as replicons). One replicon contained the gene for IL-2 in place of the poliovirus capsid VP2 and VP3 genes, and a second replicon was constructed that contained the murine IL-2 substituted for the poliovirus VP3 and VP1 genes. The IL-2 genes were cloned into the replicon so as to maintain the translational reading frame with the remaining poliovirus proteins. Transfection of either replicon into cells resulted in the expression of replicon-encoded proteins and replication of replicon RNA. Using a procedure developed in this laboratory, we have encapsidated these replicons into authentic polio virions by passaging the replicons in the presence of a recombinant vaccinia virus, VVP1, which expresses the capsid precursor, P1, protein. Using a quantitative immunoassay, we determined that the majority of the IL-2 produced remained intracellular, with approximately 1%-2% released from the infected cells, and that the IL-2 was biologically active. The results of these studies demonstrate the utility of poliovirus replicons for expression of small bioactive molecules and are discussed with respect to future applications as immune adjuvants as well as potential new tumor therapies.

Antibody to poliovirus genome-linked protein (VPg) precipitates in vitro synthesized RNA attached to VPg-precursor polypeptide(s).

Antibody to poliovirus genome-linked protein VPg, specifically precipitated RNA synthesized in vitro by the poliovirus replicase and host factor in response to poliovirion RNA. A significant amount of the immunoprecipitated RNA was RNAase T1 resistant, sedimented at approximately 2-4 S and was shown to be largely polyuridylic acid. RNAase A digestion or alkali hydrolysis of the immunoprecipitated RNA left [32P]UMP-labeled material which comigrated on SDS-polyacrylamide gels with known VPg-precursor polypeptides. The results presented in this paper suggested that VPg was involved in the host factor-dependent, poliovirus replicase-catalyzed in vitro RNA synthesis, most probably in the form of a larger precursor protein.

Expression of poliovirus P3 proteins using a recombinant vaccinia virus results in proteolytically active 3CD precursor protein without further processing to 3Cpro and 3Dpol.

The expression of the poliovirus genome occurs by the translation of a single open reading frame to generate a long polyprotein which is subsequently processed by viral encoded proteases. The initial proteolytic cleavages result in the production of a P1 polyprotein which contains the capsid proteins, and the P2 and P3 polyproteins which contain proteins required for replication. The P3 polyprotein consists of the 3AB protein (containing the viral genome-linked protein, VPg), the viral protease, 3Cpro, and RNA polymerase, 3Dpol. To further study the expression and proteolytic processing of poliovirus P3 proteins in vivo, we have utilized recombinant vaccinia virus vectors to express nucleotides 5240-7400 containing the P3 region proteins of poliovirus. The P3 protein expressed from the recombinant vaccinia virus VV-P3 exhibited in vivo proteolytic activity as evident by processing of the polyprotein to generate the 3CD protein, consisting of a fusion between the 3Cpro and 3Dpol proteins. Further processing of the 3CD protein to 3Cpro and 3Dpol, however, was not detected in cells infected with VV-P3. Subcellular fractionation of VV-P3-infected cells demonstrated that the 3CD protein was present in both the soluble and membrane fractions. Finally, the 3CD protein expressed from VV-P3 was stable in cells co-infected with VV-P3 and poliovirus and no further processing to 3Dpol was detected. These results are discussed with regards to in vivo studies which suggest that the 3CD polyprotein is not a precursor to 3Dpol in poliovirus-infected cells.

Stability of HIV type 1 proviral genomes that contain two distinct primer-binding sites.

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNALys,3 positioned at an 18-nucleotide sequence in the RNA genome referred to as the primer-binding site (PBS). We have found that mutations within the PBS and a region upstream in U5, designated the A loop, influenced the selection of the tRNA primer used to initiate reverse transcription. Surprisingly, a proviral genome that contained a PBS and A loop complementary to tRNAPro resulted in the generation of viruses that contained two PBSs within the same genome: one of the PBSs in the virus was complementary to tRNALys,3 while the second PBS was complementary to tRNAIle, tRNAPro, or tRNALys,3. There were 14 nucleotides separating the two PBSs in the viral genome. In the current study, DNA encompassing U5 and the dual PBS complementary to the different tRNAs were amplified by PCR and exchanged for the corresponding region in an infectious HIV-1 clone, HXB2. Transfection of the different proviruses into cells resulted in the production of viruses that were infectious as determined by coculture with SupT1 cells. PCR was used to amplify the PBS regions from the different proviral DNAs followed by DNA sequencing of individual PCR clones. Proviruses containing the dual PBS complementary to tRNALys,3 and tRNAIle stably maintained the dual PBS complementary to both of these tRNAs following in vitro culture, although we noted consistent G-to-T and AA-to-GG substitutions in the 14-nucleotide region between the PBSs. The viruses derived from genomes that contained the dual PBS complementary to tRNALys,3 and tRNAPro also maintained both PBSs following in vitro culture; a single mutation was noted after in vitro culture in the 14-nucleotide region between the PBSs, which changed a consensus integration site (CA dinucleotide) prior to the PBS complementary to tRNAPro. In contrast, the proviral genomes containing the dual PBS complementary to tRNALys,3 were not stable and reverted back to a single PBS complementary to tRNALys,3. The results of our studies suggest that only the 5'-proximal PBS has been used to initiate reverse transcription. On the basis of our results, a mechanism is proposed for the generation of a dual PBS, which provides new insights into HIV-1 reverse transcription.

Genetic evidence of the interaction between tRNA(Lys,3) and U5 facilitating efficient initiation of reverse transcription by human immunodeficiency virus type 1.

Previous studies using in vitro chemical and enzyme methods demonstrated that, in addition to the primer-binding site (PBS), two regions upstream of the PBS in U5 of HIV-1 RNA interact with tRNA(Lys,3) during the initiation of reverse transcription. One region corresponds to nucleotides 167-172 of U5, which are complementary to the anticodon region of tRNA(Lys,3); a second region corresponds to nucleotides 142-145 of U5, which interacts with nucleotides 43-46 of tRNA(Lys,3). To study the importance of these viral RNA-tRNA interactions in reverse transcription and viral replication, we mutated the two corresponding regions in the infectious HIV-1 proviral DNA (HXB2). Changing nucleotides 167-172 from GAAAAU to CCACAA (which is complementary to the anticodon of tRNA(His)) or changing nucleotides 142-144 from CCC to GGG did not affect protein expression or production of virus from transfected proviral DNAs. Analysis of these viruses revealed that, although all were infectious, the initial replication was delayed compared with wild-type virus. Using an endogenous reverse transcription-PCR assay, we found that the initiation of the reverse transcription in the mutant viruses was less efficient than that for the wild-type virus. Analysis of the proviral DNA sequences after 2 months of in vitro culture revealed that most progeny viruses derived from the mutant that contained the CCACAA motif had acquired nucleotide substitutions within and surrounding the CCACAA nucleotides. All the viruses recovered from the mutant that originally contained the GGG nucleotides reverted back to contain the wild-type CCC sequence. The majority of the proviral clones derived from virus containing the double mutations had gained additional mutations within the CCACAA and GGG motifs. The replication of the mutant viruses was now similar to that of the wild type. The results of these studies demonstrate that interactions between the tRNA and U5 are important for generation of an optimized initiation complex required for efficient reverse transcription.

Characterization of the expression and immunogenicity of poliovirus replicons that encode simian immunodeficiency virus SIVmac239 Gag or envelope SU proteins.

The effectiveness of the poliovirus vaccines to induce both systemic and mucosal immunity has prompted the development of this virus as a vector in which to express foreign proteins. Our laboratory has previously reported on the construction and characterization of poliovirus genomes that encode HIV-1 proteins (Porter DC, et al.: J Virol 1996;70:2643-2649). To develop this system further, we have constructed poliovirus genomes, referred to as replicons, which encode the SIVmac239 Gag or Env SU in place of the poliovirus capsid gene (P1). Since the replicons do not encode capsid proteins, they are encapsidated into poliovirus by passage with a recombinant vaccinia virus, VVP1, which provides the poliovirus capsid proteins in trans. Using this system, we have derived stocks of the encapsidated replicons which encode the SIVmac239 or Env SU protein. Infection of cells with the replicon that encodes SIVmac239 Gag resulted in the expression of a 55-kDa protein that was released from the infected cells. Analysis of the sedimentation of the released proteins by sucrose density gradient centrifugation revealed that the protein was released from the cell in the form of a virus-like particle. Infection of cells with the replicons encoding the SIVmac239 Env SU resulted in the expression of a 63-kDa protein, corresponding to the molecular mass predicted for the nonglycosylated SIVmac239 SU protein. A second protein with a molecular mass greater than 160 kDa was also immunoprecipitated. After enzymatic deglycosylation, this protein migrated at a molecular mass consistent with that for an Env SU dimer. Analysis of the medium from cells infected with the replicon encoding SIVmac239 Env SU revealed the presence of a protein of molecular mass 85-90 kDa, possibly representing a fragment of the SIVmac239 or Env SU protein. To determine the immunogenicity of the replicons encoding SIVmac239 Gag or Env SU, transgenic mice that express the human receptor for poliovirus, and are thus susceptible to poliovirus, were immunized via the intramuscular route. A serum antibody response to SIV envelope was detected following booster immunization, establishing that the encapsidated replicon was immunogenic. Finally, we demonstrate that the replicons have the capacity to infect peripheral blood mononuclear monocytes/macrophages, suggesting that this cell is a possible target for in vivo infection. The results of our studies, then, lend further support for the development and application of recombinant poliovirus replicons in a vaccine strategy.