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Abstract The rapid development and deployment of vaccines following the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been estimated to have saved millions of lives. Despite their immense success, there remains a need for next-generation vaccination approaches for SARS-CoV-2 and future emerging coronaviruses and other respiratory viruses. Here we utilized a Newcastle Disease virus (NDV) vectored vaccine expressing the ancestral SARS-CoV-2 spike protein in a pre-fusion stabilized chimeric conformation (NDV-PFS). When delivered intranasally, NDV-PFS protected both Syrian hamsters and K18 mice against Delta and Omicron SARS-CoV-2 variants of concern. Additionally, intranasal vaccination induced robust, durable protection that was extended to 6 months post-vaccination. Overall, our data provide evidence that NDV-vectored vaccines represent a viable next-generation mucosal vaccination approach.

Abstract Background The advancement of AAV vectors into clinical testing has accelerated rapidly over the past two decades. While many of the AAV vectors being utilized in clinical trials are derived from natural serotypes, engineered serotypes are progressing toward clinical translation due to their enhanced tissue tropism and immune evasive properties. However, novel AAV vectors require formulation and stability testing to determine optimal storage conditions prior to their use in a clinical setting. Results Here, we evaluated the thermal stability of AAV6.2FF, a rationally engineered capsid with strong tropism for lung and muscle, in two different buffer formulations; phosphate buffered saline (PBS), or PBS supplemented with 0.001% non-ionic surfactant Pluronic F68 (PF-68). Aliquots of AAV6.2FF vector encoding the firefly luciferase reporter gene (AAV6.2FF-ffLuc) were incubated at temperatures ranging from -20°C to 55°C for varying periods of time and the impact on infectivity and particle integrity evaluated. Additionally, the impact of several rounds of freeze-thaw treatments on the infectivity of AAV6.2FF was investigated. Vector infectivity was measured by quantifying firefly luciferase expression in HEK 293 cells and AAV particle integrity was measured by qPCR quantification of encapsidated viral DNA. Conclusions Our data demonstrate that formulating AAV6.2FF in PBS containing 0.001% PF-68 leads to increased stability and particle integrity at temperatures between -20℃ to 21℃ and protection against the destructive effects of freeze-thaw. Finally, AAV6.2FF-GFP formulated in PBS supplemented with 0.001% PF-68 displayed higher transduction efficiency in vivo in murine lung epithelial cells following intranasal administration than vector buffered in PBS alone further demonstrating the beneficial properties of PF-68.

IntroductionTumor microenvironments are immunosuppressive due to progressive accumulation of mutations in cancer cells that can drive expression of a range of inhibitory ligands and cytokines, and recruitment of immunomodulatory cells, including myeloid-derived suppressor cells (MDSC), tumor-associated macrophages, and regulatory T cells (Tregs).MethodsTo reverse this immunosuppression, we engineered mesogenic Newcastle disease virus (NDV) to express immunological checkpoint inhibitors anti-cytotoxic T lymphocyte antigen-4 and soluble programmed death protein-1.ResultsIntratumoral administration of recombinant NDV (rNDV) to mice bearing intradermal B16-F10 melanomas or subcutaneous CT26LacZ colon carcinomas led to significant changes in the tumor-infiltrating lymphocyte profiles. Vectorizing immunological checkpoint inhibitors in NDV increased activation of intratumoral natural killer cells and cytotoxic T cells and decreased Tregs and MDSCs, suggesting induction of a pro-inflammatory state with greater infiltration of activated CD8+ T cells. These notable changes translated to higher ratios of activated effector/suppressor tumor-infiltrating lymphocytes in both cancer models, which is a promising prognostic marker. Whereas all rNDV-treated groups showed evidence of tumor regression and increased survival in the CT26LacZ and B16-F10, only treatment with NDV expressing immunological checkpoint blockades led to complete responses compared to tumors treated with NDV only.DiscussionThese data demonstrated that NDV expressing immunological checkpoint inhibitors could reverse the immunosuppressive state of tumor microenvironments and enhance tumor-specific T cell responses.

Immunotherapies revive host immune responses against tumors by stimulating innate and adaptive immune effector cells with antitumor functions. Thus, detailed studies of immunological cell phenotypes and functions within the tumor microenvironment (TME) following immunotherapy treatments is critical to identifying the determinants of therapeutic success, optimizing treatment regimens, and driving curative outcomes. Oncolytic viruses such as Orf virus (OrfV) are multifunctional biologics that preferentially infect and kill cancer cells while simultaneously causing inflammation that drives anticancer immune responses. Here, we describe the immunological impact of OrfV on the ascites TME in a preclinical model of advanced-stage epithelial ovarian cancer. OrfV promoted the infiltration of several immune effector cells with increased expression of activation markers and effector cytokines into the ascites TME, which correlated with reduced ascites tumor burden and improved survival. The kinetics of the immune response and change in tumor burden following OrfV therapy revealed an optimal re-administration time to sustain antitumor immunity, further extending survival. The data presented highlight the importance of investigating immune response kinetics following immunotherapy and demonstrate that detailed kinetic profiling of immune responses can reveal novel insights into mechanisms of action that can guide the development of more effective therapies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged following an outbreak of unexplained viral illness in China in late 2019. Since then, it has spread globally causing a pandemic that has resulted in millions of deaths and has had enormous economic and social consequences. The emergence of SARS-CoV-2 saw the rapid and widespread development of a number of vaccine candidates worldwide, and this never-before-seen pace of vaccine development led to several candidates progressing immediately through clinical trials. Many countries have now approved vaccines for emergency use, with large-scale vaccination programs ongoing. Despite these successes, there remains a need for ongoing pre-clinical and clinical development of vaccine candidates against SARS-CoV-2, as well as vaccines that can elicit strong mucosal immune responses. Here, we report on the efficacy of a Newcastle disease virus-vectored vaccine candidate expressing SARS-CoV-2 spike protein (NDV-FLS) administered to cynomolgus macaques. Macaques given two doses of the vaccine via respiratory immunization developed robust immune responses and had reduced viral RNA levels in nasal swabs and in the lower airway. Our data indicate that NDV-FLS administered mucosally provides significant protection against SARS-CoV-2 infection, resulting in reduced viral burden and disease manifestation, and should be considered as a viable candidate for clinical development.

Here, Parzych et al describe the development of a DNA-delivered SARS-CoV-2 mAb cocktail displaying synergistic RBD binding that confers broad neutralizing activity against variants of concern and prophylactic efficacy in multiple small animal models.

Filoviruses cause severe hemorrhagic fever with case fatality rates as high as 90%. Filovirus-specific monoclonal antibodies (mAbs) confer protection in nonhuman primates as late as 5 days after challenge, and FDA-approved mAbs REGN-EB3 and mAb114 have demonstrated efficacy against Ebola virus (EBOV) infection in humans. Vectorized antibody expression mediated by adeno-associated virus (AAV) can generate protective and sustained concentrations of therapeutic mAbs in animal models for a variety of infectious diseases, including EBOV. Here we demonstrate that AAV6.2FF-mediated expression of murine IgG2a EBOV mAbs, 2G4 and 5D2, protects from mouse-adapted (MA)-EBOV infection with none of the surviving mice developing anti-VP40 antibodies above background. Protective serum concentrations of AAV6.2FF-2G4/AAV6.2FF-5D2 did not alter endogenous antibody responses to heterologous virus infection. AAV-mediated expression of EBOV mAbs 100 and 114, and pan-ebolavirus mAbs, FVM04, ADI-15878, and CA45, as human IgG1 antibodies conferred protection against MA-EBOV at low serum concentrations, with minimum protective serum levels as low as 2 μg/mL. Vectorized expression of murine IgG2a or human IgG1 mAbs led to sustained expression in the serum of mice for >400 days or for the lifetime of the animal, respectively. AAV6.2FF-mediated mAb expression offers an alternative to recombinant antibody administration in scenarios where long-term protection is preferable to passive immunization.

Adeno-associated virus (AAV) vectors are among the most widely used delivery vehicles for in vivo gene therapy as they mediate robust and sustained transgene expression with limited toxicity. However, a significant impediment to the broad clinical success of AAV-based therapies is the widespread presence of pre-existing humoral immunity to AAVs in the human population. This immunity arises from the circulation of non-pathogenic endemic human AAV serotypes. One possible solution is to use non-human AAV capsids to pseudotype transgene-containing AAV vector genomes of interest. Due to the low probability of human exposure to animal AAVs, pre-existing immunity to animal-derived AAV capsids should be low. Here, we characterize two novel AAV capsid sequences: one derived from porcine colon tissue and the other from a caprine adenovirus stock. Both AAV capsids proved to be effective transducers of HeLa and HEK293T cells in vitro. In vivo, both capsids were able to transduce the murine nose, lung, and liver after either intranasal or intraperitoneal administration. In addition, we demonstrate that the porcine AAV capsid likely arose from multiple recombination events involving human- and animal-derived AAV sequences. We hypothesize that recurrent recombination events with similar and distantly related AAV sequences represent an effective mechanism for enhancing the fitness of wildtype AAV populations.

Poxviruses have been used extensively as vaccine vectors for human and veterinary medicine and have recently entered the clinical realm as immunotherapies for cancer. We present a comprehensive method for producing high-quality lots of the poxvirus Parapoxvirus ovis (OrfV) for use in preclinical models of vaccinology and cancer therapy. OrfV is produced using a permissive sheep skin-derived cell line and is released from infected cells by repeated freeze-thaw combined with sonication. We present two methods for isolation and purification of bulk virus. Isolated virus is concentrated to high titer using polyethylene glycol to produce the final in vivo-grade product. We also describe methods for quantifying OrfV infectious virions and determining genomic copy number to evaluate virus stocks. The methods herein will provide researchers with the ability to produce high-quality, high-titer OrfV for use in preclinical studies, and support the translation of OrfV-derived technologies into the clinic.

Novel immunotherapies continue to be developed and tested for application against a plethora of diseases. The clinical translation of immunotherapies requires an understanding of their mechanisms. The contributions of antibodies in driving long-term responses following immunotherapies continue to be revealed given their diverse effector functions. Developing an in-depth understanding of the role of antibodies in treatment efficacy is required to optimize immunotherapies and improve the chance of successfully translating them into the clinic. However, analyses of antibody responses can be challenging in the context of antigen-agnostic immunotherapies, particularly in the context of cancers that lack pre-defined target antigens. As such, robust methods are needed to evaluate the capacity of a given immunotherapy to induce beneficial antibody responses, and to identify any therapy-limiting antibodies. We previously developed a comprehensive method for detecting antibody responses induced by antigen-agnostic immunotherapies for application in pre-clinical models of vaccinology and cancer therapy. Here, we extend this method to a high-throughput, flow cytometry-based assay able to identify and quantify isotype-specific virus- and tumor-associated antibody responses induced by immunotherapies using small sample volumes with rapid speed and high sensitivity. This method provides a valuable and flexible protocol for investigating antibody responses induced by immunotherapies, which researchers can use to expand their analyses and optimize their own treatment regimens.

Monoclonal antibodies (mAbs) are important treatment modalities for preventing and treating infectious diseases, especially for those lacking prophylactic vaccines or effective therapies. Recent advances in mAb gene cloning from naturally infected or immunized individuals has led to the development of highly potent human mAbs against a wide range of human and animal pathogens. While effective, the serum half-lives of mAbs are quite variable, with single administrations usually resulting in short-term protection, requiring repeated doses to maintain therapeutic concentrations for extended periods of time. Moreover, due to their limited time in circulation, mAb therapies are rarely given prophylactically; instead, they are generally administered therapeutically after the onset of symptoms, thus preventing mortality, but not morbidity. Adeno-associated virus (AAV) vectors have an established record of high-efficiency in vivo gene transfer in a variety of animal models and humans. When delivered to post-mitotic tissues such as skeletal muscle, brain, and heart, or to organs in which cells turn over slowly, such as the liver and lungs, AAV vector genomes assume the form of episomal concatemers that direct transgene expression, often for the lifetime of the cell. Based on these attributes, many research groups have explored AAV-vectored delivery of highly potent mAb genes as a strategy to enable long-term expression of therapeutic mAbs directly in vivo following intramuscular or intranasal administration. However, clinical trials in humans and studies in nonhuman primates (NHPs) indicate that while AAVs are a powerful and promising platform for vectored immunoprophylaxis (VIP), further optimization is needed to decrease anti-drug antibody (ADA) and anti-capsid antibody responses, ultimately leading to increased serum transgene expression levels and improved therapeutic efficacy. The following review will summarize the current landscape of AAV VIP in NHP models, with an emphasis on vector and transgene design as well as general delivery system optimization. In addition, major obstacles to AAV VIP, along with implications for clinical translation, will be discussed.

Neutrophils have conflicting roles in the context of cancers, where they have been associated with contributing to both anti-tumor and pro-tumor responses. Their functional heterogenicity is plastic and can be manipulated by environmental stimuli, which has fueled an area of research investigating therapeutic strategies targeting neutrophils. Dendritic cell (DC)-based cancer vaccination is an immunotherapy that has exhibited clinical promise but has shown limited clinical efficacy. Enhancing our understanding of the communications occurring during DC cancer vaccination can uncover opportunities for enhancing the DC vaccine platform. There have been observed communications between neutrophils and DCs during natural immune responses. However, their crosstalk has been poorly studied in the context of DC vaccination. Here, we review the dual functionality of neutrophils in the context of cancers, describe the crosstalk between neutrophils and DCs during immune responses, and discuss their implications in DC cancer vaccination. This discussion will focus on how neutrophil extracellular traps can influence immune responses in the tumor microenvironment and what roles they may play in promoting or hindering DC vaccine-induced anti-tumor efficacy.

Pseudomonas aeruginosa is a bacterial pathogen of global concern and is responsible for 10–15% of nosocomial infections worldwide. This opportunistic bacterial pathogen is known to cause serious complications in immunocompromised patients and is notably the leading cause of morbidity and mortality in patients suffering from cystic fibrosis. Currently, the only line of defense against P. aeruginosa infections is antibiotic treatment. Due to the acquired and adaptive resistance mechanisms of this pathogen, the prevalence of multidrug resistant P. aeruginosa strains has increased, presenting a major problem in healthcare settings. To date, there are no approved licensed vaccines to protect against P. aeruginosa infections, prompting the urgent need alternative treatment options. An alternative to traditional vaccines is vectored immunoprophylaxis (VIP), which utilizes a safe and effective adeno-associated virus (AAV) gene therapy vector to produce sustained levels of therapeutic monoclonal antibodies (mAbs) in vivo from a single intramuscular injection. In this review, we will provide an overview of P. aeruginosa biology and key mechanisms of pathogenesis, discuss current and emerging treatment strategies for P. aeruginosa infections and highlight AAV-VIP as a promising novel therapeutic platform.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19) that has caused a pandemic with millions of human infections. There continues to be a pressing need to develop potential therapies and vaccines to inhibit SARS-CoV-2 infection to mitigate the ongoing pandemic. Epidemiological data from the current pandemic indicates that there may be sex-dependent differences in disease outcomes. To investigate these differences, we proposed to use common small animal species that are frequently used to model disease with viruses. However, common laboratory strains of mice are not readily infected by SARS-CoV-2 because of differences in the angiotensin-converting enzyme 2 (ACE2), the cellular receptor for the virus. To overcome this limitation, we transduced common laboratory accessible strains of mice of different sexes and age groups with a novel a triple AAV6 mutant, termed AAV6.2FF, encoding either human ACE2 or luciferase via intranasal administration to promote expression in the lung and nasal turbinates. Infection of AAV-hACE2-transduced mice with SARS-CoV-2 resulted in high viral titers in the lungs and nasal turbinates, establishment of an IgM and IgG antibody response, and modulation of lung and nasal turbinate cytokine profiles. There were insignificant differences in infection characteristics between age groups and sex-related differences; however, there were significant strain-related differences between BALB/c vs. C57BL/6 mice. We show that AAV-hACE2-transduced mice are a useful for determining immune responses and for potential evaluation of SARS-CoV-2 vaccines and antiviral therapies, and this study serves as a model for the utility of this approach to rapidly develop small-animal models for emerging viruses.

Summary: The pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19). Worldwide efforts are being made to develop vaccines to mitigate this pandemic. We engineered two recombinant Newcastle disease virus (NDV) vectors expressing either the full-length SARS-CoV-2 spike protein (NDV-FLS) or a version with a 19 amino acid deletion at the carboxy terminus (NDV-Δ19S). Hamsters receiving two doses (prime-boost) of NDV-FLS developed a robust SARS-CoV-2-neutralizing antibody response, with elimination of infectious virus in the lungs and minimal lung pathology at five days post-challenge. Single-dose vaccination with NDV-FLS significantly reduced SARS-CoV-2 replication in the lungs but only mildly decreased lung inflammation. NDV-Δ19S-treated hamsters had a moderate decrease in SARS-CoV-2 titers in lungs and presented with severe microscopic lesions, suggesting that truncation of the spike protein was a less effective strategy. In summary, NDV-vectored vaccines represent a viable option for protection against COVID-19.

Epithelial ovarian cancer is the deadliest gynecological malignancy. The lack of effective treatments highlights the need for novel therapeutic interventions. The aim of this study was to investigate whether sustained adeno-associated virus (AAV) vector-mediated expression of vascular normalizing agents 3TSR and Fc3TSR and the antiangiogenic monoclonal antibody, Bevacizumab, with or without oncolytic virus treatment would improve survival in an orthotopic syngeneic mouse model of epithelial ovarian carcinoma. AAV vectors were administered 40 days post-tumor implantation and combined with oncolytic avian orthoavulavirus-1 (AOaV-1) 20 days later, at the peak of AAV-transgene expression, to ascertain whether survival could be extended. Flow cytometry conducted on blood samples, taken at an acute time point post-AOaV-1 administration (36 h), revealed a significant increase in activated NK cells in the blood of all mice that received AOaV-1. T cell analysis revealed a significant increase in CD8+ tumor specific T cells in the blood of AAV-Bevacizumab+AOaV-1 treated mice compared to control mice 10 days post AOaV-1 administration. Immunohistochemical staining of primary tumors harvested from a subset of mice euthanized 90 days post tumor implantation, when mice typically have large primary tumors, secondary peritoneal lesions, and extensive ascites fluid production, revealed that AAV-3TSR, AAV-Fc3TSR+AOaV-1, or AAV-Bevacizumab+AOaV-1 treated mice had significantly more tumor-infiltrating CD8+ T cells than PBS controls. Despite AAV-mediated transgene expression waning faster in tumor-bearing mice than in non-tumor bearing mice, all three of the AAV therapies significantly extended survival compared to control mice; with AAV-Bevacizumab performing the best in this model. However, combining AAV therapies with a single dose of AOaV-1 did not lead to significant extensions in survival compared to AAV therapies on their own, suggesting that additional doses of AOaV-1 may be required to improve efficacy in this model. These results suggest that vectorizing anti-angiogenic and vascular normalizing agents is a viable therapeutic option that warrants further investigation, including optimizing combination therapies.

The ongoing COVID-19 pandemic drew global attention to infectious diseases, attracting numerous resources for development of pandemic preparedness plans and vaccine platforms—technologies with robust manufacturing processes that can quickly be pivoted to target emerging diseases. Newcastle Disease Virus (NDV) has been studied as a viral vector for human and veterinary vaccines, but its production relies heavily on embryonated chicken eggs, with very few studies producing NDV in cell culture. Here, NDV is produced in suspension Vero cells, and analytical assays (TCID50 and ddPCR) are developed to quantify infectious and total viral titer. NDV-GFP and NDV-FLS (SARS-CoV-2 full-length spike protein) constructs were adapted to replicate in Vero and HEK293 suspension cultures using serum-free media, while fine-tuning parameters such as MOI, temperature, and trypsin concentration. Shake flask productions with Vero cells resulted in infectious titers of 1.07 × 108 TCID50/mL for NDV-GFP and 1.33 × 108 TCID50/mL for NDV-FLS. Production in 1 L batch bioreactors also resulted in high titers in culture supernatants, reaching 2.37 × 108 TCID50/mL for NDV-GFP and 3.16 × 107 TCID50/mL for NDV-FLS. This shows effective NDV production in cell culture, building the basis for a scalable vectored-vaccine manufacturing process that can be applied to different targets.