Recombinant Particles Research Articles

Self-Replicating Alphaviruses: From Pathogens to Therapeutic Agents

Alphaviruses are known for being model viruses for studying cellular functions related to viral infections but also for causing epidemics in different parts of the world. More recently, alphavirus-based expression systems have demonstrated efficacy as vaccines against infectious diseases and as therapeutic applications for different cancers. Point mutations in the non-structural alphaviral replicase genes have generated enhanced transgene expression and created temperature-sensitive expression vectors. The recently engineered trans-amplifying RNA system can provide higher translational efficiency and eliminate interference with cellular translation. The self-replicating feature of alphaviruses has provided the advantage of extremely high transgene expression of vaccine-related antigens and therapeutic anti-tumor and immunostimulatory genes, which has also permitted significantly reduced doses for prophylactic and therapeutic applications, potentially reducing adverse events. Furthermore, alphaviruses have shown favorable flexibility as they can be delivered as recombinant viral particles, RNA replicons, or DNA-replicon-based plasmids. In the context of infectious diseases, robust immune responses against the surface proteins of target agents have been observed along with protection against challenges with lethal doses of infectious agents in rodents and primates. Similarly, the expression of anti-tumor genes and immunostimulatory genes from alphavirus vectors has provided tumor growth inhibition, tumor regression, and cures in animal cancer models. Moreover, protection against tumor challenges has been observed. In clinical settings, patient benefits have been reported. Alphaviruses have also been considered for the treatment of neurological disorders due to their neurotrophic preference.

Characterization of bi-segmented and tri-segmented recombinant Pichinde virus particles.

Mammarenaviruses include several highly virulent pathogens (e.g., Lassa virus) capable of causing severe hemorrhagic fever diseases for which there are no approved vaccines and limited treatment options. Mammarenaviruses are enveloped, bi-segmented ambisense RNA viruses. There is limited knowledge about cellular proteins incorporated into progeny virion particles and their potential biological roles in viral infection. Pichinde virus (PICV) is a prototypic arenavirus used to characterize mammarenavirus replication and pathogenesis. We have developed a recombinant PICV with a tri-segmented RNA genome as a viral vector platform. Whether the tri-segmented virion differs from the wild-type bi-segmented one in viral particle morphology and protein composition has not been addressed. In this study, recombinant PICV (rPICV) virions with a bi-segmented (rP18bi) and a tri-segmented (rP18tri) genome were purified by density-gradient ultracentrifugation and analyzed by cryo-electron microscopy and mass spectrometry. Both virion types are pleomorphic with spherical morphology and have no significant difference in size despite rP18tri having denser particles. Both virion types also contain similar sets of cellular proteins. Among the highly enriched virion-associated cellular proteins are components of the endosomal sorting complex required for transport pathway and vesicle trafficking, such as ALIX, Tsg101, VPS, CHMP, and Ras-associated binding proteins, which have known functions in virus assembly and budding. Other enriched cellular proteins include peripheral and transmembrane proteins, chaperone proteins, and ribosomal proteins; their biological roles in viral infection warrant further analysis. Our study provides important insights into mammarenavirus particle formation and aids in the future development of viral vectors and antiviral discovery.IMPORTANCEMammarenaviruses, such as Lassa virus, are enveloped RNA viruses that can cause severe hemorrhagic fever diseases (Lassa fever) with no approved vaccine and limited therapeutic options. Cellular proteins incorporated into progeny virion particles and their biological roles in mammarenavirus infection have not been well characterized. Pichinde virus (PICV) is a prototypic mammarenavirus used as a surrogate model for Lassa fever. We used cryo-electron microscopy and proteomic analysis to characterize the morphology and protein contents of the purified PICV particles that package either two (bi-segmented) or three (tri-segmented) genomic RNA segments. Our results demonstrate a similar virion morphology but different particle density for the bi- and tri-segmented viral particles and reveal major virion-associated cellular proteins. This study provides important insights into the virus-host interactions that can be used for antiviral development and optimizing arenavirus-based vaccine vectors.

Recombinant adenoviruses expressing HPV16/18 E7 upregulate the HDAC6 and DNMT3B genes in C33A cells.

High-risk human papillomavirus (HPV) is a carcinogenic virus associated with nearly all cases of cervical cancer, as well as an increasing number of anal and oral cancers. The two carcinogenic proteins of HPV, E6 and E7, can immortalize keratinocytes and are essential for HPV-related cellular transformation. Currently, the global regulatory effects of these oncogenic proteins on the host proteome are not fully understood, and further exploration of the functions and carcinogenic mechanisms of E6 and E7 proteins is needed. We used a previously established platform in our laboratory for constructing recombinant adenoviral plasmids expressing the HPV16 E7 gene to further construct recombinant virus particles expressing HPV16/18 E6, E7, and both E6 and E7 genes. These recombinant viruses were used to infect C33A cells to achieve sustained expression of the HPV16/18 E6/E7 genes. Subsequently, total RNA was extracted and RNA-Seq technology was employed for transcriptome sequencing to identify differentially expressed genes associated with HPV infection in cervical cancer. RNA-Seq analysis revealed that overexpression of the HPV16/18 E6/E7 genes upregulated GP6, CD36, HDAC6, ESPL1, and DNMT3B among the differentially expressed genes (DEGs) associated with cervical cancer. Spearman correlation analysis revealed a statistically significant correlation between the HDAC6 and DNMT3B genes and key pathways, including DNA replication, tumor proliferation signature, G2M checkpoint, p53 pathways, and PI3K/AKT/mTOR signaling pathways. Further, qRT-PCR and Western blot analyses indicated that both HPV16/18 E7 can upregulate the expression of HDAC6 and DNMT3B, genes associated with HPV infection-related cervical cancer. The successful expression of HPV16/18 E6/E7 in cells indicates that the recombinant viruses retain the replication and infection capabilities of Ad4. Furthermore, the recombinant viruses expressing HPV16/18 E7 can upregulate the HDAC6 and DNMT3B genes involved in cervical cancer pathways, thereby influencing the cell cycle. Additionally, HDAC6 and DNMT3B are emerging as important therapeutic targets for cancer. This study lays the foundation for further exploration of the oncogenic mechanisms of HPV E6/E7 and may provide new directions for the treatment of HPV-related cancers.

3D‐Printed and Recombinant Spider Silk Particle Reinforced Collagen Composite Scaffolds for Soft Tissue Engineering

AbstractCollagen is one main component of the extracellular matrix (ECM) in natural tissues and is, therefore, well suited as a biomaterial for tissue engineering. In this study, a method is presented to 3D‐bioprint collagen into a precipitation bath comprising recombinantly produced spider silk protein eADF4(C16) yielding a composite with excellent mechanical properties. The spider silk precipitation bath induced assembly of the collagen into fibrils, and subsequent addition of potassium phosphate buffer lead to the formation of silk particles and stabilization of the collagen fibrils. The produced collagen‐silk composite scaffolds show an internal structure of homogeneously distributed and interacting collagen fibrils and spider silk particles with significantly better mechanical properties compared to plain collagen scaffolds. Further, enzymatic degradation assays of the scaffolds over a 7‐day period show higher stability of the collagen‐silk scaffolds compared to plain collagen scaffolds in the presence of wound proteases. Using the spider silk variant eADF4(C16‐RGD) further increases compressive stress and elastic modulus compared to that of the unmodified variant. Finally, it is shown that the unique collagen‐spider silk composite scaffolds comprising the cell‐binding domains of collagen and the RGD sequence in the spider silk variant represent a promising material for soft tissue regeneration.

Characterization of residual microRNAs in AAV vector batches produced in HEK293 mammalian cells and Sf9 insect cells

With more than 130 clinical trials and 8 approved gene therapy products, adeno-associated virus (AAV) stands as one of the most popular vehicles to deliver therapeutic DNA invivo. One critical quality attribute analyzed in AAV batches is the presence of residual DNA, as it could pose genotoxic risks or induce immune responses. Surprisingly, the presence of small cell-derived RNAs, such as microRNAs (miRNAs), has not been investigated previously. In this study, we examined the presence of miRNAs in purified AAV batches produced in mammalian or in insect cells. Our findings revealed that miRNAs were present in all batches, regardless of the production cell line or capsid serotype (2 and 8). Quantitative assays indicated that miRNAs were co-purified with the recombinant AAV particles in a proportion correlated with their abundance in the production cells. The level of residual miRNAs was reduced via an immunoaffinity chromatography purification process including a tangential flow filtration step or by RNase treatment, suggesting that most miRNA contaminants are likely non-encapsidated. In summary, we demonstrate, for the first time, that miRNAs are co-purified with AAV particles. Further investigations are required to determine whether these miRNAs could interfere with the safety or efficacy of AAV-mediated gene therapy.

Leveraging biotin-based proximity labeling to identify cellular factors governing early alphaherpesvirus infection.

Neurotropic alphaherpesviruses, including herpes simplex virus type 1 and pseudorabies virus, establish a lifelong presence within the peripheral nervous system of their mammalian hosts. Upon entering cells, two conserved tegument proteins, pUL36 and pUL37, traffic DNA-containing capsids to nuclei. These proteins support long-distance retrograde axonal transport and invasion of the nervous system in vivo. To better understand how pUL36 and pUL37 function, recombinant viral particles carrying BioID2 fused to these proteins were produced to biotinylate cellular proteins in their proximity (<10 nm) during infection. Eighty-six high-confidence host proteins were identified by mass spectrometry and subsequently targeted by CRISPR-Cas9 gene editing to assess their contributions to early infection. Proteins were identified that both supported and antagonized infection in immortalized human epithelial cells. The latter included zyxin, a protein that localizes to focal adhesions and regulates actin cytoskeletal dynamics. Zyxin knockout cells were hyper-permissive to infection and could be rescued with even modest expression of GFP-zyxin. These results provide a resource for studies of the virus-cell interface and identify zyxin as a novel deterrent to alphaherpesvirus infection.IMPORTANCENeuroinvasive alphaherpesviruses are highly prevalent with many members found across mammals [e.g., herpes simplex virus type 1 (HSV-1) in humans and pseudorabies virus in pigs]. HSV-1 causes a range of clinical manifestations from cold sores to blindness and encephalitis. There are no vaccines or curative therapies available for HSV-1. A fundamental feature of these viruses is their establishment of lifelong infection of the nervous system in their respective hosts. This outcome is possible due to a potent neuroinvasive property that is coordinated by two proteins: pUL36 and pUL37. In this study, we explore the cellular protein network in proximity to pUL36 and pUL37 during infection and examine the impact of knocking down the expression of these proteins upon infection.

Insights into the Pathogenesis and Development of Recombinant Japanese Encephalitis Virus Genotype 3 as a Vaccine.

Japanese encephalitis virus (JEV), a flavivirus transmitted by mosquitoes, has caused epidemics and severe neurological diseases in Asian countries. In this study, we developed a cDNA infectious clone, pBAC JYJEV3, of the JEV genotype 3 strain (EF571853.1) using a bacterial artificial chromosome (BAC) vector. The constructed infectious clone was transfected into Vero cells, where it exhibited infectivity and induced cytopathic effects akin to those of the parent virus. Confocal microscopy confirmed the expression of the JEV envelope protein. Comparative analysis of growth kinetics revealed similar replication dynamics between the parental and recombinant viruses, with peak titers observed 72 h post-infection (hpi). Furthermore, plaque assays demonstrated comparable plaque sizes and morphologies between the viruses. Cryo-electron microscopy confirmed the production of recombinant virus particles with a morphology identical to that of the parent virus. Immunization studies in mice using inactivated parental and recombinant viruses revealed robust IgG responses, with neutralizing antibody production increasing over time. These results showcase the successful generation and characterization of a recombinant JEV3 virus and provide a platform for further investigations into JEV pathogenesis and vaccine development.

Falsa positivización del antígeno de superficie del virus de la Hepatitis B (HBsAg) tras vacunación.

Introduction: The chronic hemodialysis population has a greater exposure and risk to the hepatitis B virus, which is why immunoprophylaxis using the double dose scheme (40mcg) on days 0-30-60-180, with application of this regimen for a second extra time in nonresponders, is common practice in this population. Clinical case: This is a 67-year-old male patient of Afro-Ecuatorian ethnicity who started hemodialysis (HD) in August 2023 for chronic kidney disease (CKD) secondary to diabetic nephropathy. When starting HD, he had negative serology for hepatitis and negativity for HBsAg. For this reason, he received his first vaccination dose on November 29, 2023, and on December 15, he had HBsAg positivity with values of 4.14 S/CO (≥1 positive), with Ac-ANTI-HBSAG levels &lt; 2 IU/L. Management: Given the findings, it was decided to isolate the patient with control of liver function, which is normal, and a new test of HBsAg levels in 15 days, which were subsequently negative. Conclusion: In this case, we want to remember the false politicization of HBsAg after vaccination in hemodialysis patients, a situation associated with the vaccine being composed of live attenuated viruses or recombinant particles of surface proteins.

The development and application of pseudoviruses: assessment of SARS-CoV-2 pseudoviruses

Although most Coronavirus disease (COVID-19) patients can recover fully, the disease remains a significant cause of morbidity and mortality. In addition to the consequences of acute infection, a proportion of the population experiences long-term adverse effects associated with SARS-CoV-2. Therefore, it is still critical to comprehend the virus’s characteristics and how it interacts with its host to develop effective drugs and vaccines against COVID-19. SARS-CoV-2 pseudovirus, a replication-deficient recombinant glycoprotein chimeric viral particle, enables investigations of highly pathogenic viruses to be conducted without the constraint of high-level biosafety facilities, considerably advancing virology and being extensively employed in the study of SARS-CoV-2. This review summarizes three methods of establishing SARS-CoV-2 pseudovirus and current knowledge in vaccine development, neutralizing antibody research, and antiviral drug screening, as well as recent progress in virus entry mechanism and susceptible cell screening. We also discuss the potential advantages and disadvantages.

Enrichment and separation of recombinant adeno-associated virus particles by aqueous two-phase extraction

The adeno-associated virus (AAV) has been widely employed as a popular gene therapy vector. However, current methods for its separation and purification exhibit numerous limitations. In this study, polyethylene glycol (PEG)/salt aqueous two-phase system (ATPS) was utilized as an efficient and environmentally-benign approach to separate AAV. The separation efficiency of AAV particles from other cellular components was investigated using various ATPS compositions. The effects of PEG molecular weight, types of phase-forming salts, and concentrations of different components were tested and compared. The optimal separation conditions were determined to be 17 % (w/w) PEG 600, 16 % (w/w) sodium citrate, and 15 % (w/w) crude cell lysate. The AAV particles were predominantly enriched in the interphase, while the impurities were observed in the top and bottom phases. High AAV recovery (>99 %) and efficient impurity removal (>95 %) were achieved. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transmission electron microscopy were used to characterize and verify the separated AAV particles. The results indicated that aqueous two-phase extraction possesses a robust capability for the enrichment and separation of AAV particles directly from crude cell lysates, and it shows promising potential for the separation of other viral particles.

Retardation of Aβ42 fibril formation by apolipoprotein A-I and recombinant HDL particles

The double nucleation mechanism of amyloid β (Aβ) peptide aggregation is retained from buffer to cerebrospinal fluid (CSF) but with reduced rate of all microscopic processes. Here, we used a bottom-up approach to identify retarding factors in CSF. We investigated the Aβ42 fibril formation as a function of time in the absence and presence of apolipoprotein A-I (ApoA-I), recombinant high-density lipoprotein (rHDL) particles, or lipid vesicles. A retardation was observed in the presence of ApoA-I or rHDL particles, most pronounced with ApoA-I, but not with lipid vesicles. Global kinetic analysis implies that rHDL interferes with secondary nucleation. The effect of ApoA-I could best be described as an interference with secondary and to a smaller extent primary nucleation. Using surface plasmon resonance and microfluidics diffusional sizing analyses, we find that both rHDL and ApoA-I interact with Aβ42 fibrils but not Aβ42 monomer, thus the effect on kinetics seems to involve interference with the catalytic surface for secondary nucleation. The Aβ42 fibrils were imaged using cryogenic-electron microscopy and found to be longer when formed in the presence of ApoA-I or rHDL, compared to formation in buffer. A retarding effect, as observed in CSF, could be replicated using a simpler system, from key components present in CSF but purified from a CSF-free host. However, the effect of CSF is stronger implying the presence of additional retarding factors.

A replicative recombinant HPV16 E7 expression virus upregulates CD36 in C33A cells.

In past decades, the role of high-risk HPV (HR-HPV) infection in cancer pathogenesis has been extensively studied. The viral E7 protein expressed in pre-malignant cells has been identified as an ideal target for immunological intervention. However, the cultivation of HPV in vitro remains a significant challenge, as well as the lack of methods for expressing the HPV E7 protein and generating replication-competent recombinant viral particles, which posed a major obstacle to further exploration of the function and carcinogenic mechanisms of the E7 oncoprotein. Therefore, it is imperative to investigate novel methodologies to construct replication-competent recombinant viral particles that express the HPV E7 protein to facilitate the study of its function. We initiated the construction of recombinant viral particles by utilizing the ccdB-Kan forward/reverse screening system in conjunction with the Red/ExoCET recombinant system. We followed the infection of C33A cells with the obtained recombinant virus to enable the continuous expression of HPV16 E7. Afterwards, the total RNA was extracted and performed transcriptome sequencing using RNA-Seq technology to identify differentially expressed genes associated with HPV-induced oncogenicity. We successfully established replicative recombinant viral particles expressing HPV16 E7 stably and continuously. The C33A cells were infected with recombinant viral particles to achieve overexpression of the E7 protein. Subsequently, RNA-Seq analysis was conducted to assess the changes in host cell gene expression. The results revealed an upregulation of the CD36 gene, which is associated with the HPV-induced oncogenic pathways, including PI3K-Akt and p53 signaling pathway. qRT-PCR analysis further identified that the upregulation of the CD36 gene due to the expression of HPV16 E7. The successful expression of HPV16 E7 in cells demonstrates that the replicated recombinant virus retains the replication and infection abilities of Ad4, while also upregulating the CD36 gene involved in the PI3K-Akt signaling and p53 pathways, thereby promoting cell proliferation. The outcome of this study provides a novel perspective and serves as a solid foundation for further exploration of HPV-related carcinogenesis and the development of replicative HPV recombinant vaccines capable of inducing protective immunity against HPV.

Recombinant Virus Quantification Using Single-Cell Droplet Digital PCR: A Method for Infectious Titer Quantification.

The quantification of viruses is necessary for both research and clinical applications. The methods available for RNA virus quantification possess several drawbacks, including sensitivity to inhibitors and the necessity of a standard curve generation. The main purpose of this study was to develop and validate a method for the quantification of recombinant, replication-deficient Semliki Forest virus (SFV) vectors using droplet digital PCR (ddPCR). This technique demonstrated stability and reproducibility using various sets of primers that targeted inserted transgenes, as well as the nsP1 and nsP4 genes of the SFV genome. Furthermore, the genome titers in the mixture of two types of replication-deficient recombinant virus particles were successfully measured after optimizing the annealing/extension temperature and virus:virus ratios. To measure the infectious units, we developed a single-cell ddPCR, adding the whole infected cells to the droplet PCR mixture. Cell distribution in the droplets was investigated, and β-actin primers were used to normalize the quantification. As a result, the number of infected cells and the virus infectious units were quantified. Potentially, the proposed single-cell ddPCR approach could be used to quantify infected cells for clinical applications.

Optimal Conditions for In Vitro Assembly of Respiratory Syncytial Virus Nucleocapsid-like Particles.

The nucleocapsids (NCs) of the respiratory syncytial virus (RSV) can display multiple morphologies in vivo, including spherical, asymmetric, and filamentous conformations. Obtaining homogeneous ring-like oligomers in vitro is significant since they structurally represent one turn of the characteristic RSV NC helical filament. Here, we analyzed and optimized conditions for forming homogenous, recombinant nucleocapsid-like particles (NCLPs) of RSV in vitro. We examined the effects of modifying the integrated RNA length and sequence, altering incubation time, and varying buffer parameters, including salt concentration and pH, on ring-like NCLPs assembly using negative stain electron microscopy (EM) imaging. We showed that high-quality, homogeneous particles are assembled when incubating short, adenine-rich RNA sequences with RNA-free N associated with P (N0P). Further, we reported that a co-incubation duration greater than 3 days, a NaCl concentration between 100 mM and 200 mM, and a pH between 7 and 8 are optimal for N-RNA ring assembly with polyadenine RNA sequences. We believe assembling high-quality, homogeneous NCLPs in vitro will allow for further analysis of RSV RNA synthesis. This work may also lend insights into obtaining high-resolution nucleocapsid homogeneous structures for in vitro analysis of antiviral drug candidates against RSV and related viruses.

Delivering Base Editors In Vivo by Adeno-Associated Virus Vectors.

CRISPR base editors are genome-modifying proteins capable of creating single-base substitutions in DNA but without the requirement for a DNA double-strand break. Given their ability to precisely edit DNA, they hold tremendous therapeutic potential. Here, we describe procedures for delivering base editors in vivo via adeno-associated virus (AAV) vectors, a promising engineered gene delivery vehicle capable of transducing a range of cell types and tissues. We provide step by step protocols for (i) designing and validating base editing systems, (ii) packaging base editors into recombinant AAV vector particles, (iii) delivering AAV to the central nervous system via intrathecal injection, and (iv) quantifying base editing frequencies by next-generation sequencing.

Alphaviruses in cancer immunotherapy.

Alphaviruses have frequently been engineered for cancer therapy, cancer immunotherapy, and cancer vaccine development. As members of self-replicating RNA viruses, alphaviruses provide high levels of transgene expression through efficient self-amplifying of their RNA genome in host cells. Alphavirus vectors can be used as recombinant viral particles or oncolytic viruses. Alternatively, either naked or nanoparticle-encapsulated RNA and DNA replicons can be utilized. In the context of cancer prevention and treatment, antitumor, cytotoxic and suicide genes have been expressed from alphavirus vectors to provide tumor regression and tumor eradication. Moreover, immunostimulatory genes such as cytokines and chemokines have been used for cancer immunotherapy approaches. Expression of tumor antigens has been applied for cancer vaccine development. Alphavirus vectors has demonstrated tumor regression and even cure in various preclinical animal models. Immunization has elicited strong immune responses and showed protection against challenges with tumor cells in animal models. Several clinical trials have confirmed good safety and tolerability of alphaviruses in cancer patients although therapeutic efficacy will still require optimization.

Therapeutic Applications for Oncolytic Self-Replicating RNA Viruses.

Self-replicating RNA viruses have become attractive delivery vehicles for therapeutic applications. They are easy to handle, can be rapidly produced in large quantities, and can be delivered as recombinant viral particles, naked or nanoparticle-encapsulated RNA, or plasmid DNA-based vectors. The self-replication of RNA in infected host cells provides the means for generating much higher transgene expression levels and the possibility to apply substantially reduced amounts of RNA to achieve similar expression levels or immune responses compared to conventional synthetic mRNA. Alphaviruses and flaviviruses, possessing a single-stranded RNA genome of positive polarity, as well as measles viruses and rhabdoviruses with a negative-stranded RNA genome, have frequently been utilized for therapeutic applications. Both naturally and engineered oncolytic self-replicating RNA viruses providing specific replication in tumor cells have been evaluated for cancer therapy. Therapeutic efficacy has been demonstrated in animal models. Furthermore, the safe application of oncolytic viruses has been confirmed in clinical trials. Multiple myeloma patients treated with an oncolytic measles virus (MV-NIS) resulted in increased T-cell responses against the measles virus and several tumor-associated antigen responses and complete remission in one patient. Furthermore, MV-CEA administration to patients with ovarian cancer resulted in a stable disease and more than doubled the median overall survival.

Chicken telomerase reverse transcriptase promotes the tumorigenicity of avian leukosis virus subgroup J by regulating the Wnt/β-catenin signaling pathway

This research aimed to analyze the regulatory effect of chicken telomerase reverse transcriptase (chTERT) on the Wnt/β-catenin signaling pathway and its effect on the tumorigenicity of avian leukosis virus subgroup J (ALV-J) through in vivo experiments. The chTERT eukaryotic expression plasmid and its recombinant lentivirus particles were constructed for in vivo transfection of chTERT to analyze the effect of chTERT continuously overexpressed in chickens on the tumorigenicity of ALV-J. During 156 days of the artificial ALV-J tumor-inducing process, 7 solid tumors developed in 3 chickens in the chTERT-overexpression group (n = 26*2) and no tumors developed in the control group (n = 26*2). Another 18 tumors induced by ALV-J were confirmed and collected from breeding poultry farms. And we confirmed that chTERT was significantly highly expressed in ALV-J tumors. The ELISA data suggested that the protein levels of β-catenin and c-Myc in the chicken plasma of the chTERT-overexpressing group with ALV-J infected were consistently and significantly higher than those of the control group. Compared with that of the tumor-adjacent tissues, the activity of the Wnt/β-catenin signaling pathway and expression of the c-Myc was significantly increased in ALV-J tumors. And the percentage of apoptosis in ALV-J tumors significantly lower than that in tumor-adjacent tissues. Immunohistochemistry, Western blot and RT-qPCR suggested that the replication level of ALV-J in tumors was significantly higher than that in tumor-adjacent tissues. This study suggests that chTERT plays a critical role in the tumorigenicity of ALV-J by enhancing the Wnt/β-catenin signaling pathway, which will contribute to further elucidating the tumor-inducing mechanism of ALV-J.

Concentrating all helper protein functions on a single entity allows rescue of recombinant measles virus by transfection of just two plasmids.

The generation of recombinant measles virus (MeV) from manipulated genomes on plasmid DNA is quite a complex and inefficient process. As a member of the order Mononegavirales its single-stranded ssRNA genome in negative sense orientation is not infectious, but requires co-availability of the viral RNA-dependent RNA polymerase L, the polymerase co-factor phosphoprotein P, and the nucleocapsid protein N in defined relative amounts to establish infectious centres in transfected cell cultures that release replication-competent recombinant MeV particles. For this so-called rescue, different rescue systems were developed that rely on at least four different components. In this work, we establish a functional MeV rescue system just being composed of two components: the plasmid encoding the (modified) viral genome, and a one-helper-plasmid bundling all helper functions. In contrast to a rescue-system for Newcastle Disease Virus, another paramyxovirus, co-expression of all helper proteins by the same promoter failed. Instead, adaptation of the strength of the respective promoters to drive each helper gene´s expression to the relative expression found in MeV-infected cells or other rescue systems, which indeed adjusted respective mRNA and protein expression, yielded success, albeit not yet to the same efficacy as the four-component system. Thereby, our study paves the way for the development of easier and, after further optimization, more efficient rescue systems to generate recombinant MeV for e.g. the application as a vaccine platform or oncolytic virus, for example.

EXTH-24. FILAMENTOUS PHAGE AS A NANOTHERANOSTIC FOR THE DETECTION, IMAGING, AND TREATMENT OF GLIOMAS

Abstract Current standard of care treatment for gliomas includes maximal safe surgical resection followed by concurrent chemoradiation, however, the diffuse infiltrative nature of gliomas present certain challenges for neurosurgeons to clearly visualize tumor tissue under conventional white light during microsurgical resection. Fluorescence-guided surgical techniques have been recently introduced to help surgeons better visualize tumor tissue under the operating microscope. Fluorescent dyes conjugated to nanoscale materials that are functionalized with glioma-targeting peptide sequences, such as the 39 amino acid chlorotoxin (CTX) peptide sequence, have demonstrated clinical efficacy in recognizing microscopic tumor cells during surgeries. M13 bacteriophage (phage) are versatile, genetically tunable nanocarriers that have been recently adapted for use as theranostic platforms. Applying p3 capsid CTX fusion with a unique “inho” circular single-stranded DNA (cssDNA) gene packaging system, we produced miniature CTX inho (CTX-inho phage particles with a minimum length of 50 nm that can target intracranial orthotopic patient-derived glioblastoma tumors in the brains of mice. Systemically-administered indocyanine green-conjugated CTX-inho phage accumulated in brain tumors, facilitating short-wave infrared detection. Furthermore, we show that our inho phage can carry cssDNA that are transcriptionally active when delivered to GBM22 glioma cells in vitro. The ability to modulate the capsid display, surface loading, phage length, and cssDNA gene content makes the recombinant M13 phage particle an ideal nanotheranostic platform for neuro-oncological applications.