Surface-modified measles vaccines encoding oligomeric, prefusion-stabilized SARS-CoV-2 spike glycoproteins boost neutralizing antibody responses to Omicron and historical variants, independent of measles seropositivity.

Serum titers of SARS-CoV-2-neutralizing antibodies (nAbs) correlate well with protection from symptomatic COVID-19 but decay rapidly in the months following vaccination or infection. In contrast, measles-protective nAb titers are lifelong after measles vaccination, possibly due to persistence of the live-attenuated virus in lymphoid tissues. We, therefore, sought to generate a live recombinant measles vaccine capable of driving high SARS-CoV-2 nAb responses. Since previous clinical testing of a live measles vaccine encoding a SARS-CoV-2 spike glycoprotein resulted in suboptimal anti-spike antibody titers, our new vectors were designed to encode prefusion-stabilized SARS-CoV-2 spike glycoproteins, trimerized via an inserted peptide domain, and displayed on a dodecahedral miniferritin scaffold. Additionally, to circumvent the blunting of vaccine efficacy by preformed anti-measles antibodies, we extensively modified the measles surface glycoproteins. Comprehensive in vivo mouse testing demonstrated the potent induction of high titer nAbs in measles-immune mice and confirmed the significant contributions to overall potency afforded by prefusion stabilization, trimerization, and miniferritin display of the SARS-CoV-2 spike glycoprotein. In animals primed and boosted with a measles virus (MeV) vaccine encoding the ancestral SARS-CoV-2 spike, high-titer nAb responses against ancestral virus strains were only weakly cross-reactive with the Omicron variant. However, in primed animals that were boosted with a MeV vaccine encoding the Omicron BA.1 spike, antibody titers to both ancestral and Omicron strains were robustly elevated, and the passive transfer of serum from these animals protected K18-ACE2 mice from infection and morbidity after exposure to BA.1 and WA1/2020 strains. Our results demonstrate that by engineering the antigen, we can develop potent measles-based vaccine candidates against SARS-CoV-2.IMPORTANCEAlthough the live-attenuated measles virus (MeV) is one of the safest and most efficacious human vaccines, a measles-vectored COVID-19 vaccine candidate expressing the SARS-CoV-2 spike failed to elicit neutralizing antibody (nAb) responses in a phase-1 clinical trial, especially in measles-immune individuals. Here, we constructed a comprehensive panel of MeV-based COVID-19 vaccine candidates using a MeV with extensive modifications on the envelope glycoproteins (MeV-MR). We show that artificial trimerization of the spike is critical for the induction of nAbs and that their magnitude can be significantly augmented when the spike protein is synchronously fused to a dodecahedral scaffold. Furthermore, preexisting measles immunity did not abolish heterologous immunity elicited by our vector. Our results highlight the importance of antigen optimization in the development of spike-based COVID-19 vaccines and therapies.

Oncolytic α-herpesvirus and myeloid-tropic cytomegalovirus cooperatively enhance systemic antitumor responses.

Oncolytic virotherapy aims to activate host antitumor immunity. In responsive tumors, intratumorally injected herpes simplex viruses (HSVs) have been shown to lyse tumor cells, resulting in local inflammation, enhanced tumor antigen presentation, and boosting of antitumor cytotoxic lymphocytes. In contrast to HSV, cytomegalovirus (CMV) is nonlytic and reprograms infected myeloid cells, limiting their antigen-presenting functions and protecting them from recognition by natural killer (NK) cells. Here, we show that when co-injected into mouse tumors with an oncolytic HSV, mouse CMV (mCMV) preferentially targeted tumor-associated myeloid cells, promoted the local release of proinflammatory cytokines, and enhanced systemic antitumor immune responses, leading to superior control of both injected and distant contralateral tumors. Deletion of mCMV genes m06, which degrades major histocompatibility complex class I (MHC class I), or m144, a viral MHC class I homolog that inhibits NK activation, was shown to diminish the antitumor activity of the HSV/mCMV combination. However, an mCMV recombinant lacking the m04 gene, which escorts MHC class I to the cell surface, showed superior HSV adjuvanticity. CMV is a potentially promising agent with which to reshape and enhance antitumor immune responses following oncolytic HSV therapy.

A phase I oncolytic virus trial with vesicular stomatitis virus expressing human interferon beta and tyrosinase related protein 1 administered intratumorally and intravenously in uveal melanoma: safety, efficacy, and T cell responses.

Introduction: Metastatic uveal melanoma (MUM) has a poor prognosis and treatment options are limited. These patients do not typically experience durable responses to immune checkpoint inhibitors (ICIs). Oncolytic viruses (OV) represent a novel approach to immunotherapy for patients with MUM. Methods: We developed an OV with a Vesicular Stomatitis Virus (VSV) vector modified to express interferon-beta (IFN-ß) and Tyrosinase Related Protein 1 (TYRP1) (VSV-IFNß-TYRP1), and conducted a Phase 1 clinical trial with a 3 + 3 design in patients with MUM. VSV-IFNß-TYRP1 was injected into a liver metastasis, then administered on the same day as a single intravenous (IV) infusion. The primary objective was safety. Efficacy was a secondary objective. Results: 12 patients with previously treated MUM were enrolled. Median follow up was 19.1 months. 4 dose levels (DLs) were evaluated. One patient at DL4 experienced dose limiting toxicities (DLTs), including decreased platelet count (grade 3), increased aspartate aminotransferase (AST), and cytokine release syndrome (CRS). 4 patients had stable disease (SD) and 8 patients had progressive disease (PD). Interferon gamma (IFNγ) ELIspot data showed that more patients developed a T cell response to virus encoded TYRP1 at higher DLs, and a subset of patients also had a response to other melanoma antigens, including gp100, suggesting epitope spreading. 3 of the patients who responded to additional melanoma antigens were next treated with ICIs, and 2 of these patients experienced durable responses. Discussion: Our study found that VSV-IFNß -TYRP1 can be safely administered via intratumoral (IT) and IV routes in a previously treated population of patients with MUM. Although there were no clear objective radiographic responses to VSV-IFNß-TYRP1, dose-dependent immunogenicity to TYRP1 and other melanoma antigens was seen.

Preclinical safety assessment of MV-s-NAP, a novel oncolytic measles virus strain armed with an H . pylori immunostimulatory bacterial transgene.

Despite recent therapeutic advances, metastatic breast cancer (MBC) remains incurable. Engineered measles virus (MV) constructs based on the attenuated MV Edmonston vaccine platform have demonstrated significant oncolytic activity against solid tumors. The Helicobacter pylori neutrophil-activating protein (NAP) is responsible for the robust inflammatory reaction in gastroduodenal mucosa during bacterial infection. NAP attracts and activates immune cells at the site of infection, inducing expression of pro-inflammatory mediators. We engineered an MV strain to express the secretory form of NAP (MV-s-NAP) and showed that it exhibits anti-tumor and immunostimulatory activity in human breast cancer xenograft models. In this study, we utilized a measles-infection-permissive mouse model (transgenic IFNAR KO-CD46Ge) to evaluate the biodistribution and safety of MV-s-NAP. The primary objective was to identify potential toxic side effects and confirm the safety of the proposed clinical doses of MV-s-NAP prior to a phase I clinical trial of intratumoral administration of MV-s-NAP in patients with MBC. Both subcutaneous delivery (corresponding to the clinical trial intratumoral administration route) and intravenous (worst case scenario) delivery of MV-s-NAP were well tolerated: no significant clinical, laboratory or histologic toxicity was observed. This outcome supports the safety of MV-s-NAP for oncolytic virotherapy of MBC. The first-in-human clinical trial of MV-s-NAP in patients with MBC (ClinicalTrials.gov: NCT04521764) was subsequently activated.

Boosting of SARS-CoV-2 immunity in nonhuman primates using an oral rhabdoviral vaccine.

An orally active vaccine capable of boosting SARS-CoV-2 immune responses in previously infected or vaccinated individuals would help efforts to achieve and sustain herd immunity. Unlike mRNA-loaded lipid nanoparticles and recombinant replication-defective adenoviruses, replicating vesicular stomatitis viruses with SARS-CoV-2 spike glycoproteins (VSV-SARS2) were poorly immunogenic after intramuscular administration in clinical trials. Here, by G protein trans-complementation, we generated VSV-SARS2(+G) virions with expanded target cell tropism. Compared to parental VSV-SARS2, G-supplemented viruses were orally active in virus-naive and vaccine-primed cynomolgus macaques, powerfully boosting SARS-CoV-2 neutralizing antibody titers. Clinical testing of this oral VSV-SARS2(+G) vaccine is planned.

Clinical activity of single-dose systemic oncolytic VSV virotherapy in patients with relapsed refractory T-cell lymphoma.

Clinical success with intravenous (IV) oncolytic virotherapy (OV) has to-date been anecdotal. We conducted a phase 1 clinical trial of systemic OV and investigated the mechanisms of action in responding patients. A single IV dose of vesicular stomatitis virus (VSV) interferon-ß (IFN-ß) with sodium iodide symporter (NIS) was administered to patients with relapsed/refractory hematologic malignancies to determine safety and efficacy across 4 dose levels (DLs). Correlative studies were undertaken to evaluate viremia, virus shedding, virus replication, and immune responses. Fifteen patients received VSV-IFNß-NIS. Three patients were treated at DL1 through DL3 (0.05, 0.17, and 0.5 × 1011 TCID50), and 6 were treated at DL4 (1.7 × 1011 TCID50) with no dose-limiting toxicities. Three of 7 patients with T-cell lymphoma (TCL) had responses: a 3-month partial response (PR) at DL2, a 6-month PR, and a complete response (CR) ongoing at 20 months at DL4. Viremia peaked at the end of infusion, g was detected. Plasma IFN-ß, a biomarker of VSV-IFNß-NIS replication, peaked between 4 hours and 48 hours after infusion. The patient with CR had robust viral replication with increased plasma cell-free DNA, high peak IFN-ß of 18 213 pg/mL, a strong anti-VSV neutralizing antibody response, and increased numbers of tumor reactive T-cells. VSV-IFNß-NIS as a single agent was effective in patients with TCL, resulting in durable disease remissions in heavily pretreated patients. Correlative analyses suggest that responses may be due to a combination of direct oncolytic tumor destruction and immune-mediated tumor control. This trial is registered at www.clinicaltrials.gov as #NCT03017820.

Oncolytic virotherapy - Forging its place in the immunomodulatory paradigm for Multiple Myeloma.

The treatment focus for multiple myeloma (MM) has recently pivoted towards immune modulating strategies, with T-cell redirection therapies currently at the forefront of drug development. Yet, despite this revolution in treatment, MM remains without a sustainable cure. At the same time, tremendous advancement has been made in recombinant and gene editing techniques for oncolytic viruses (OV), which have increased their tumor specificity, improved safety, and enhanced the oncolytic and immunostimulatory potential. These breakthrough developments in oncolytic virotherapy have opened new avenues for OVs to be used in combination with other immune-based therapies such as checkpoint inhibitors, chimeric antigen receptor T-cells (CAR-T) and bispecific T-cell engagers. In this review, the authors place the spotlight on systemic oncolytic virotherapy as an adaptable immunotherapeutic for MM, highlight the unique mechanism of OVs in activating the immune-suppressive marrow microenvironment, and lastly showcase the OV platforms and the promising combination strategies in the pipeline for MM.

Development of a Clinically Relevant Reporter for Chimeric Antigen Receptor T-cell Expansion, Trafficking, and Toxicity.

Although chimeric antigen receptor T (CART)-cell therapy has been successful in treating certain hematologic malignancies, wider adoption of CART-cell therapy is limited because of minimal activity in solid tumors and development of life-threatening toxicities, including cytokine release syndrome (CRS). There is a lack of a robust, clinically relevant imaging platform to monitor in vivo expansion and trafficking to tumor sites. To address this, we utilized the sodium iodide symporter (NIS) as a platform to image and track CART cells. We engineered CD19-directed and B-cell maturation antigen (BCMA)-directed CART cells to express NIS (NIS+CART19 and NIS+BCMA-CART, respectively) and tested the sensitivity of 18F-TFB-PET to detect trafficking and expansion in systemic and localized tumor models and in a CART-cell toxicity model. NIS+CART19 and NIS+BCMA-CART cells were generated through dual transduction with two vectors and demonstrated exclusive 125I uptake in vitro. 18F-TFB-PET detected NIS+CART cells in vivo to a sensitivity level of 40,000 cells. 18F-TFB-PET confirmed NIS+BCMA-CART-cell trafficking to the tumor sites in localized and systemic tumor models. In a xenograft model for CART-cell toxicity, 18F-TFB-PET revealed significant systemic uptake, correlating with CART-cell in vivo expansion, cytokine production, and development of CRS-associated clinical symptoms. NIS provides a sensitive, clinically applicable platform for CART-cell imaging with PET scan. 18F-TFB-PET detected CART-cell trafficking to tumor sites and in vivo expansion, correlating with the development of clinical and laboratory markers of CRS. These studies demonstrate a noninvasive, clinically relevant method to assess CART-cell functions in vivo.

IMMUNO-COV v2.0: Development and Validation of a High-Throughput Clinical Assay for Measuring SARS-CoV-2-Neutralizing Antibody Titers.

Neutralizing antibodies are key determinants of protection from future infection, yet well-validated high-throughput assays for measuring titers of SARS-CoV-2-neutralizing antibodies are not generally available. Here, we describe the development and validation of IMMUNO-COV v2.0, a scalable surrogate virus assay, which titrates antibodies that block infection of Vero-ACE2 cells by a luciferase-encoding vesicular stomatitis virus displaying SARS-CoV-2 spike glycoproteins (VSV-SARS2-Fluc). Antibody titers, calculated using a standard curve consisting of stepped concentrations of SARS-CoV-2 spike monoclonal antibody, correlated closely (P < 0.0001) with titers obtained from a gold standard 50% plaque-reduction neutralization test (PRNT50%) performed using a clinical isolate of SARS-CoV-2. IMMUNO-COV v2.0 was comprehensively validated using data acquired from 242 assay runs performed over 7 days by five analysts, utilizing two separate virus lots, and 176 blood samples. Assay performance was acceptable for clinical use in human serum and plasma based on parameters including linearity, dynamic range, limit of blank and limit of detection, dilutional linearity and parallelism, precision, clinical agreement, matrix equivalence, clinical specificity and sensitivity, and robustness. Sufficient VSV-SARS2-Fluc virus reagent has been banked to test 5 million clinical samples. Notably, a significant drop in IMMUNO-COV v2.0 neutralizing antibody titers was observed over a 6-month period in people recovered from SARS-CoV-2 infection. Together, our results demonstrate the feasibility and utility of IMMUNO-COV v2.0 for measuring SARS-CoV-2-neutralizing antibodies in vaccinated individuals and those recovering from natural infections. Such monitoring can be used to better understand what levels of neutralizing antibodies are required for protection from SARS-CoV-2 and what booster dosing schedules are needed to sustain vaccine-induced immunity. IMPORTANCE Since its emergence at the end of 2019, SARS-CoV-2, the causative agent of COVID-19, has caused over 100 million infections and 2.4 million deaths worldwide. Recently, countries have begun administering approved COVID-19 vaccines, which elicit strong immune responses and prevent disease in most vaccinated individuals. A key component of the protective immune response is the production of neutralizing antibodies capable of preventing future SARS-CoV-2 infection. Yet, fundamental questions remain regarding the longevity of neutralizing antibody responses following infection or vaccination and the level of neutralizing antibodies required to confer protection. Our work is significant as it describes the development and validation of a scalable clinical assay that measures SARS-CoV-2-neutraling antibody titers. We have critical virus reagent to test over 5 million samples, making our assay well suited for widespread monitoring of SARS-CoV-2-neutralizing antibodies, which can in turn be used to inform vaccine dosing schedules and answer fundamental questions regarding SARS-CoV-2 immunity.

A brief review of reporter gene imaging in oncolytic virotherapy and gene therapy.

Reporter gene imaging (RGI) can accelerate development timelines for gene and viral therapies by facilitating rapid and noninvasive in vivo studies to determine the biodistribution, magnitude, and durability of viral gene expression and/or virus infection. Functional molecular imaging systems used for this purpose can be divided broadly into deep-tissue and optical modalities. Deep-tissue modalities, which can be used in animals of any size as well as in human subjects, encompass single photon emission computed tomography (SPECT), positron emission tomography (PET), and functional/molecular magnetic resonance imaging (f/mMRI). Optical modalities encompass fluorescence, bioluminescence, Cerenkov luminescence, and photoacoustic imaging and are suitable only for small animal imaging. Here we discuss the mechanisms of action and relative merits of currently available reporter gene systems, highlighting the strengths and weaknesses of deep tissue versus optical imaging systems and the hardware/reagents that are used for data capture and processing. In light of recent technological advances, falling costs of imaging instruments, better availability of novel radioactive and optical tracers, and a growing realization that RGI can give invaluable insights across the entire in vivo translational spectrum, the approach is becoming increasingly essential to facilitate the competitive development of new virus- and gene-based drugs.

Improved Noninvasive In Vivo Tracking of AAV-9 Gene Therapy Using the Perchlorate-Resistant Sodium Iodide Symporter from Minke Whale.

The sodium iodide symporter (NIS) is widely used as a reporter gene to noninvasively monitor the biodistribution and durability of vector-mediated gene expression via gamma scintigraphy, single-photon emission computed tomography (SPECT), and positron-emission tomography (PET). However, the approach is limited by background signal due to radiotracer uptake by endogenous NIS-expressing tissues. In this study, using the SPECT tracer pertechnetate (99mTcO4) and the PET tracer tetrafluoroborate (B18F4), in combination with the NIS inhibitor perchlorate, we compared the transport properties of human NIS and minke whale (Balaenoptera acutorostrata scammoni) NIS in vitro and in vivo. Based on its relative resistance to perchlorate, the NIS protein from minke whale appeared to be the superior candidate reporter gene. SPECT and PET imaging studies in nude mice challenged with NIS-encoding adeno-associated virus (AAV)-9 vectors confirmed that minke whale NIS, in contrast to human and endogenous mouse NIS, continues to function as a reliable reporter even when background radiotracer uptake by endogenous NIS is blocked by perchlorate.

Retargeted and Stealth-Modified Oncolytic Measles Viruses for Systemic Cancer Therapy in Measles Immune Patients.

Measles viruses (MV) are rapidly inactivated by anti-measles neutralizing antibodies, which has limited their clinical performance as oncolytic agents. Here, by substituting the H and F surface glycoproteins of MV with those from the homologous canine distemper virus (CDV) and engineering the CDV H attachment protein to target EGFR or CD38, we generated a fully retargeted MV capable of resisting neutralization by measles-immune human serum. The resultant recombinant MVs encoding retargeted CDV envelope glycoproteins had similar growth kinetics as the control MV, showed the expected engineered receptor specificities for cell entry, intercellular fusion, and target cell killing, and were blind to native CDV receptors. In contrast to the control MV, recombinant MVs incorporating CDV F and H glycoproteins retained full infectivity when exposed to high concentrations of pooled measles-immune human serum. Comparing viruses bearing MV or CDV glycoproteins in the SKOV3ip.1 model, only the virus bearing an EGFR-retargeted CDV envelope glycoprotein complex was capable of limiting tumor growth and extending the survival in measles immune mice. MV, "stealthed" and retargeted using engineered CDV surface glycoproteins, may be a promising platform to advance for systemic cancer therapy in measles immune patients.

Oncolytic Virus with Attributes of Vesicular Stomatitis Virus and Measles Virus in Hepatobiliary and Pancreatic Cancers.

Recombinant vesicular stomatitis virus (VSV)-fusion and hemagglutinin (FH) was developed by substituting the promiscuous VSV-G glycoprotein (G) gene in the backbone of VSV with genes encoding for the measles virus envelope proteins F and H. Hybrid VSV-FH exhibited a multifaceted mechanism of cancer-cell killing and improved neurotolerability over parental VSV in preclinical studies. In this study, we evaluated VSV-FH in vitro and in vivo in models of hepatobiliary and pancreatic cancers. Our results indicate that high intrahepatic doses of VSV-FH did not result in any significant toxicity and were well tolerated by transgenic mice expressing the measles virus receptor CD46. Furthermore, a single intratumoral treatment with VSV-FH yielded improved survival and complete tumor regressions in a proportion of mice in the Hep3B hepatocellular carcinoma model but not in mice xenografted with BxPC-3 pancreatic cancer cells. Our preliminary findings indicate that VSV-FH can induce potent oncolysis in hepatocellular and pancreatic cancer cell lines with concordant results in vivo in hepatocellular cancer and discordant in pancreatic cancer without the VSV-mediated toxic effects previously observed in laboratory animals. Further study of VSV-FH as an oncolytic virotherapy is warranted in hepatocellular carcinoma and pancreatic cancer to understand broader applicability and mechanisms of sensitivity and resistance.

CRISPR/Cas9-mediated introduction of the sodium/iodide symporter gene enables noninvasive in vivo tracking of induced pluripotent stem cell-derived cardiomyocytes.

Techniques that enable longitudinal tracking of cell fate after myocardial delivery are imperative for optimizing the efficacy of cell-based cardiac therapies. However, these approaches have been underutilized in preclinical models and clinical trials, and there is considerable demand for site-specific strategies achieving long-term expression of reporter genes compatible with safe noninvasive imaging. In this study, the rhesus sodium/iodide symporter (NIS) gene was incorporated into rhesus macaque induced pluripotent stem cells (RhiPSCs) via CRISPR/Cas9. Cardiomyocytes derived from NIS-RhiPSCs (NIS-RhiPSC-CMs) exhibited overall similar morphological and electrophysiological characteristics compared to parental control RhiPSC-CMs at baseline and with exposure to physiological levels of sodium iodide. Mice were injected intramyocardially with 2 million NIS-RhiPSC-CMs immediately following myocardial infarction, and serial positron emission tomography/computed tomography was performed with 18 F-tetrafluoroborate to monitor transplanted cells in vivo. NIS-RhiPSC-CMs could be detected until study conclusion at 8 to 10 weeks postinjection. This NIS-based molecular imaging platform, with optimal safety and sensitivity characteristics, is primed for translation into large-animal preclinical models and clinical trials.

Development and validation of IMMUNO-COV™: a high-throughput clinical assay for detecting antibodies that neutralize SARS-CoV-2.

We here describe the development and validation of IMMUNO-COV™, a high-throughput clinical test to quantitatively measure SARS-CoV-2-neutralizing antibodies, the specific subset of anti-SARS-CoV-2 antibodies that block viral infection. The test measures the capacity of serum or purified antibodies to neutralize a recombinant Vesicular Stomatitis Virus (VSV) encoding the SARS-CoV-2 spike glycoprotein. This recombinant virus (VSV-SARS-CoV-2-S-Δ19CT) induces fusion in Vero cell monolayers, which is detected as luciferase signal using a dual split protein (DSP) reporter system. VSV-SARS-CoV-2-S-Δ19CT infection was blocked by monoclonal α-SARS-CoV-2-spike antibodies and by plasma or serum from SARS-CoV-2 convalescing individuals. The assay exhibited 100% specificity in validation tests, and across all tests zero false positives were detected. In blinded analyses of 230 serum samples, only two unexpected results were observed based on available clinical data. We observed a perfect correlation between results from our assay and 80 samples that were also assayed using a commercially available ELISA. To quantify the magnitude of the anti-viral response, we generated a calibration curve by adding stepped concentrations of α-SARS-CoV-2-spike monoclonal antibody to pooled SARS-CoV-2 seronegative serum. Using the calibration curve and a single optimal 1:100 serum test dilution, we reliably measured neutralizing antibody levels in each test sample. Virus neutralization units (VNUs) calculated from the assay correlated closely (p < 0.0001) with PRNT EC50 values determined by plaque reduction neutralization test against a clinical isolate of SARS-CoV-2. Taken together, these results demonstrate that the IMMUNO-COV™ assay accurately quantitates SARS-CoV-2 neutralizing antibodies in human sera and therefore is a potentially valuable addition to the currently available serological tests. The assay can provide vital information for comparing immune responses to the various SARS-CoV-2 vaccines that are currently in development, or for evaluating donor eligibility in convalescent plasma therapy studies.

Oncolytic measles virus therapy enhances tumor antigen-specific T-cell responses in patients with multiple myeloma.

Oncolytic virus therapy leads to immunogenic death of virus-infected tumor cells and this has been shown in preclinical models to enhance the cytotoxic T-lymphocyte response against tumor-associated antigens (TAAs), leading to killing of uninfected tumor cells. To investigate whether oncolytic virotherapy can increase immune responses to tumor antigens in human subjects, we studied T-cell responses against a panel of known myeloma TAAs using PBMC samples obtained from ten myeloma patients before and after systemic administration of an oncolytic measles virus encoding sodium iodide symporter (MV-NIS). Despite their prior exposures to multiple immunosuppressive antimyeloma treatment regimens, T-cell responses to some of the TAAs were detectable even before measles virotherapy. Measurable baseline T-cell responses against MAGE-C1 and hTERT were present. Furthermore, MV-NIS treatment significantly (P < 0.05) increased T-cell responses against MAGE-C1 and MAGE-A3. Interestingly, one patient who achieved complete remission after MV-NIS therapy had strong baseline T-cell responses both to measles virus proteins and to eight of the ten tested TAAs. Our data demonstrate that oncolytic virotherapy can function as an antigen agnostic vaccine, increasing cytotoxic T-lymphocyte responses against TAAs in patients with multiple myeloma, providing a basis for continued exploration of this modality in combination with immune checkpoint blockade.

Mapping of Ion and Substrate Binding Sites in Human Sodium Iodide Symporter (hNIS).

The human sodium iodide symporter (hNIS) is a theranostic reporter gene which concentrates several clinically approved SPECT and PET radiotracers and plays an essential role for the synthesis of thyroid hormones as an iodide transporter in the thyroid gland. Development of hNIS mutants which could enhance translocation of the desired imaging ions is currently underway. Unfortunately, it is hindered by lack of understanding of the 3D organization of hNIS and its relation to anion transport. There are no known crystal structures of hNIS in any of its conformational states. Homology modeling can be very effective in such situations; however, the low sequence identity between hNIS and relevant secondary transporters with available experimental structures makes the choice of a template and the generation of 3D models nontrivial. Here, we report a combined application of homology modeling and molecular dynamics refining of the hNIS structure in its semioccluded state. The modeling was based on templates from the LeuT-fold protein family and was done with emphasis on the refinement of the substrate-ion binding pocket. The consensus model developed in this work is compared to available biophysical and biochemical experimental data for a number of different LeuT-fold proteins. Some functionally important residues contributing to the formation of putative binding sites and permeation pathways for the cotransported Na+ ions and I- substrate were identified. The model predictions were experimentally tested by generation of mutant versions of hNIS and measurement of relative (to WT hNIS) 125I- uptake of 35 hNIS variants.

Enhanced noninvasive imaging of oncology models using the NIS reporter gene and bioluminescence imaging.

Noninvasive bioluminescence imaging (BLI) of luciferase-expressing tumor cells has advanced pre-clinical evaluation of cancer therapies. Yet despite its successes, BLI is limited by poor spatial resolution and signal penetration, making it unusable for deep tissue or large animal imaging and preventing precise anatomical localization or signal quantification. To refine pre-clinical BLI methods and circumvent these limitations, we compared and ultimately combined BLI with tomographic, quantitative imaging of the sodium iodide symporter (NIS). To this end, we generated tumor cell lines expressing luciferase, NIS, or both reporters, and established tumor models in mice. BLI provided sensitive early detection of tumors and relatively easy monitoring of disease progression. However, spatial resolution was poor, and as the tumors grew, deep thoracic tumor signals were massked by overwhelming surface signals from superficial tumors. In contrast, NIS-expressing tumors were readily distinguished and precisely localized at all tissue depths by positron emission tomography (PET) or single photon emission computed tomography (SPECT) imaging. Furthermore, radiotracer uptake for each tumor could be quantitated noninvasively. Ultimately, combining BLI and NIS imaging represented a significant enhancement over traditional BLI, providing more information about tumor size and location. This combined imaging approach should facilitate comprehensive evaluation of tumor responses to given therapies.

Systemic Safety of a Recombinant AAV8 Vector for Human Cocaine Hydrolase Gene Therapy: A Good Laboratory Practice Preclinical Study in Mice.

Cocaine addiction continues to impose major burdens on affected individuals and broader society but is highly resistant to medical treatment or psychotherapy. This study was undertaken with the goal of Food and Drug Administration (FDA) permission for a first-in-human clinical trial of a gene therapy for treatment-seeking cocaine users to become and remain abstinent. The approach was based on intravenous administration of AAV8-hCocH, an adeno-associated viral vector encoding a modified plasma enzyme that metabolizes cocaine into harmless by-products. To assess systemic safety, we conducted "Good Laboratory Practice" (GLP) studies in cocaine-experienced and cocaine-naive mice at doses of 5E12 and 5E13 vector genomes/kg. Results showed total lack of viral vector-related adverse effects in all tests performed. Instead, mice given one injection of AAV8-hCocH and regular daily injections of cocaine had far less tissue pathology than cocaine-injected mice with no vector treatment. Biodistribution analysis showed the vector located almost exclusively in the liver. These results indicate that a liver-directed AAV8-hCocH gene transfer at reasonable dosage is safe, well tolerated, and effective. Thus, gene transfer therapy emerges as a radically new approach to treat compulsive cocaine abuse. In fact, based on these positive findings, the FDA recently accepted our latest request for investigational new drug application (IND 18579).

Oncolytic Viruses: Priming Time for Cancer Immunotherapy.

New immuno-oncology therapies are improving cancer treatments beyond the former standard of care, as evidenced by the recent and continuing clinical approvals for immunotherapies in a broad range of indications. However, a majority of patients (particularly those with immunologically cold tumors) still do not benefit, highlighting the need for rational combination approaches. Oncolytic viruses (OV) both directly kill tumor cells and inflame the tumor microenvironment. While OV spread can be limited by the generation of antiviral immune responses, the initial local tumor cell killing can reverse the immunosuppressive tumor microenvironment, resulting in more effective release of tumor-associated antigens (TAAs), cross-presentation, and antitumoral effector T cell recruitment. Moreover, many OVs can be engineered to express immunomodulatory genes. Rational combination approaches to cancer immunotherapy include the use of OVs in combination with immune checkpoint inhibitors (ICIs) or adoptive T cell therapy (ACT) to promote sustained antitumoral immune responses. OV combinations have additive or synergistic efficacy in preclinical tumor models with ICIs or ACT. Several preclinical studies have confirmed systemic reactivation and proliferation of adoptively transferred antitumoral T cells in conjunction with oncolytic OVs (expressing cytokines or TAAs) resulting from the specific tumor cell killing and immunostimulation of the tumor microenvironment which leads to increased tumor trafficking, activity, and survival. Recent clinical trials combining OVs with ICIs have shown additive effects in melanoma. Additional clinical data in an expanded range of patient indications are eagerly awaited. The relative timings of OV and ICI combination remains under-studied and is an area for continued exploration. Studies systematically exploring the effects of systemic ICIs prior to, concomitantly with, or following OV therapy will aid in the future design of clinical trials to enhance efficacy and increase patient response rates.