SARS-CoV-2 has resulted in high levels of morbidity and mortality world-wide, and severe complications can occur in older populations. Humoral immunity induced by authorized vaccines wanes within 6 months, and frequent boosts may only offer transient protection. GRT-R910 is an investigational self-amplifying mRNA (samRNA)-based SARS-CoV-2 vaccine delivering full-length Spike and selected conserved non-Spike T cell epitopes. This study reports interim analyses for a phase I open-label dose-escalation trial evaluating GRT-R910 in previously vaccinated healthy older adults (NCT05148962). Primary endpoints of safety and tolerability were assessed. Most solicited local and systemic adverse events (AEs) following GRT-R910 dosing were mild to moderate and transient, and no treatment-related serious AEs were observed. The secondary endpoint of immunogenicity was assessed via IgG binding assays, neutralization assays, interferon-gamma ELISpot, and intracellular cytokine staining. Neutralizing antibody titers against ancestral Spike and variants of concern were boosted or induced by GRT-R910 and, contrasting to authorized vaccines, persisted through at least 6 months after the booster dose. GRT-R910 increased and/or broadened functional Spike-specific T cell responses and primed functional T cell responses to conserved non-Spike epitopes. This study is limited due to small sample size, and additional data from ongoing studies will be required to corroborate these interim findings.

The coronavirus disease 2019 (COVID-19) pandemic continues to spread globally, highlighting the urgent need for safe and effective vaccines that could be rapidly mobilized to immunize large populations. We report the preclinical development of a self-amplifying mRNA (SAM) vaccine encoding a prefusion stabilized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein and demonstrate strong cellular and humoral immune responses at low doses in mice and rhesus macaques. The homologous prime-boost vaccination regimen of SAM at 3, 10 and 30 μg induced potent neutralizing antibody (nAb) titers in rhesus macaques following two SAM vaccinations at all dose levels, with the 10 μg dose generating geometric mean titers (GMT) 48-fold greater than the GMT of a panel of SARS-CoV-2 convalescent human sera. Spike-specific T cell responses were observed with all tested vaccine regimens. SAM vaccination provided protective efficacy against SARS-CoV-2 challenge as both a homologous prime-boost and as a single boost following ChAd prime, demonstrating reduction of viral replication in both the upper and lower airways. The SAM vaccine is currently being evaluated in clinical trials as both a homologous prime-boost regimen at low doses and as a boost following heterologous prime.

Vaccines targeting neoantigens identified from common tumor driver mutations are of increasing interest as evidence of clinical benefit builds, and opportunities to combine such vaccines with immune modulators are growing. Our individualized neoantigen vaccine (GRANITE) has shown clinical benefit and strong, consistent CD8 T cell induction in patients. We have developed an analogous off-the-shelf product (SLATE) targeting shared neoantigens that offers manufacturing simplicity and faster administration to patients with shared driver mutations. A Phase 1/2 trial of a heterologous prime/boost vaccine regimen using a chimpanzee adenovirus (ChAd) prime and self amplifying mRNA (SAM) boosts (SLATE, NCT03953235) was initiated to assess safety, tolerability, and immunogenicity in patients with advanced cancers. SLATE version 1 encodes 20 unique neoantigens to various shared driver mutations (KRAS, TP53, etc.). Patients were selected if their tumors harbored one of the 20 neoantigens encoded by the vaccine cassette and an HLA Class I allele that presents that neoantigen. Administration of ChAd prime and repeated administration of 30, 100, or 300µg SAM doses were safe and well tolerated in all subjects dosed (n=26), with no evidence of increasing reactogenicity with sequential dosing. Early efficacy signals (molecular responses; one unconfirmed RECIST response) were observed in NSCLC subjects all treated with and progressed on prior anti-PD(L)1. Analysis of T cell responses pre and post immunizations by ex vivo IFNγ ELISpot did not show robust responses to KRAS neoantigens across all patients. However, objective CD8 T cell responses to KRAS antigens post vaccination were detectable after in vitro stimulation, suggesting the induction of low-level KRAS specific T cell responses in vivo. In contrast, HLA-matched responses to TP53 neoantigens encoded by the vaccine were consistently detected via ex vivo ELISpot in these same patients. These data suggests that an immunodominant T cell response to the TP53 mutations may have outcompeted the response to the less immunogenic KRAS mutations restricted and presented by the same HLA in vivo. Differential surface peptide-HLA (pHLA) density may explain these discordant findings, and subsequent targeted mass spectrometry analyses revealed detection of TP53 pHLA complexes at a higher frequency compared to KRAS mutations in single HLA-allele cell lines. Redesigned vaccine cassettes excluding the TP53 epitopes and repeating KRAS epitopes demonstrated increased immune responses (ex vivo IFNγ ELISpot) compared to cassette version 1 in HLA transgenic mice, further supporting the tumor neoantigen immunodominance hierarchy observed in humans dosed with SLATE version 1. A re-designed product (SLATE v2) focusing exclusively on KRAS mutations (G12C, G12D, G12V and Q61H) is currently being assessed in phase 2 in patients with advanced KRAS-driven tumors. Citation Format: Amy R. Rappaport, Christine D. Palmer, Annie Shen, Claudia X. Dominguez, Meghan G. Hart, Lauren D. Kraemer, Sonia Koulavouth, Martina Marrali, Jason R. Jaroslavsky, Charmaine N. Nganje, Ciaran D. Scallan, Sue-Jean Hong, Leonid Gitlin, Monica Lane, Daniel V. Catenacci, Chrisann Kyi, David P. Carbone, Hossein Borghaei, Raphael Rousseau, Andrew Ferguson, Karin Jooss. Optimization of shared neoantigen vaccine design to increase vaccine potency: From bench to bedside and back [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3578.

Checkpoint inhibitor (CPI) therapies provide limited benefit to patients with tumors of low immune reactivity. T cell-inducing vaccines hold promise to exert long-lasting disease control in combination with CPI therapy. Safety, tolerability and recommended phase 2 dose (RP2D) of an individualized, heterologous chimpanzee adenovirus (ChAd68) and self-amplifying mRNA (samRNA)-based neoantigen vaccine in combination with nivolumab and ipilimumab were assessed as primary endpoints in an ongoing phase 1/2 study in patients with advanced metastatic solid tumors (NCT03639714). The individualized vaccine regimen was safe and well tolerated, with no dose-limiting toxicities. Treatment-related adverse events (TRAEs) >10% included pyrexia, fatigue, musculoskeletal and injection site pain and diarrhea. Serious TRAEs included one count each of pyrexia, duodenitis, increased transaminases and hyperthyroidism. The RP2D was 10 12 viral particles (VP) ChAd68 and 30 µg samRNA. Secondary endpoints included immunogenicity, feasibility of manufacturing and overall survival (OS). Vaccine manufacturing was feasible, with vaccination inducing long-lasting neoantigen-specific CD8 T cell responses. Several patients with microsatellite-stable colorectal cancer (MSS-CRC) had improved OS. Exploratory biomarker analyses showed decreased circulating tumor DNA (ctDNA) in patients with prolonged OS. Although small study size limits statistical and translational analyses, the increased OS observed in MSS-CRC warrants further exploration in larger randomized studies.

The immunogenicity and efficacy of RNA-based vaccine platforms has been abundantly shown through their application in prophylactic SARS-CoV2 vaccines. Contrasting to mRNA based vectors, self amplifying mRNA platforms may offer dose-sparing and superior induction of T cell responses, and may also trigger distinct innate immune pathways, which may exert adjuvanting or inhibiting effects on vaccine-induced immunity. Optimal dosing for a novel self-amplifying mRNA (SAM) in a heterologous prime-boost vaccination approach consisting of Chimpanzee Adenovirus (ChAd) prime and SAM boosts was evaluated in two first-in-human phase 1/2 clinical trials assessing personalized neoantigen vaccines in patients with metastatic cancer (NCT03639714, NCT03953235). SAM vaccine dose escalation was performed to assess safety, tolerability, and immunogenicity, including administration of up to 8 SAM doses at 30, 100, or 300µg following a fixed dose of ChAd (1012 vp) over the course of a year. SAM was safe and well tolerated at all 3 dose levels, with no evidence of increasing reactogenicity with sequential doses. However, while immune monitoring via IFNγ ELISpot revealed that the 30µg SAM dose boosted T cell responses induced by the ChAd prime, the 100µg and 300µg SAM doses resulted in maintenance of T cell levels, without a clear T cell boost, suggesting a non-linear and likely bell-shaped dose-response curve to SAM in humans. Follow-up studies in non-human primates (NHPs) using a model antigen revealed dose-dependent increases in serum IFNa levels following administration of increasing SAM doses. Similarly, while multiple inflammatory cytokines were transiently increased following both ChAd and SAM administration in patients, serum IFNa levels were only increased 24h post SAM administration and correlated positively with SAM dose. Increased IFNa levels post SAM dosing suggested activation of mRNA-sensing innate immune pathways that may reduce the amplification of, and/or antigen expression by, the SAM vector and thus blunt T cell boosting at higher SAM doses. In addition, analysis of T cell responses in patients and NHPs showed increased boosting of T cell responses with longer intervals. These data lead to a reduction of the SAM dose to 30µg and adjusting SAM dosing intervals to 8 weeks in the Phase 2 portion of these clinical studies. Multiple patients have been dosed with the adjusted vaccine regimen, and preliminary data suggest robust boosting of ChAd-induced neoantigen-specific T cell responses with the selected SAM dosing regimen and the 30µg dose. We anticipate that this translational approach of adjusting clinical vaccine regimens based on strong translational immune data will increase the potency of our heterologous neoantigen vaccine, and subsequently provide more durable clinical benefit to patients with cancer. Citation Format: Christine D. Palmer, Amy R. Rappaport, Meghan G. Hart, Lauren D. Kraemer, Sonia Kounlavouth, Martina Marrali, Jason R. Jaroslavsky, Charmaine N. Nganje, Annie Shen, Gregory R. Boucher, Melissa A. Kachura, Ciaran D. Scallan, Sue-Jean Hong, Leonid Gitlin, Alexander I. Spira, Chrisann Kyi, Daniel V. Catenacci, Raphael Rousseau, Andrew Ferguson, Karin Jooss. Lower doses of self-amplifying mRNA drive superior neoantigen-specific CD8 T cell responses in cancer patients versus high doses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4159.

The coronavirus disease 2019 (COVID-19) pandemic continues to spread globally, highlighting the urgent need for safe and effective vaccines that could be rapidly mobilized to immunize large populations. We report the preclinical development of a self-amplifying mRNA (SAM) vaccine encoding a prefusion stabilized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein and demonstrate potent cellular and humoral immune responses at low doses in mice and rhesus macaques. The homologous prime-boost vaccination regimen of SAM at 3, 10 and 30 μg induced potent neutralizing antibody titers in rhesus macaques following two SAM vaccinations at all dose levels, with the 10 μg dose generating geometric mean titers (GMT) 48-fold greater than the GMT of a panel of SARS-CoV-2 convalescent human sera. Spike-specific T cell responses were observed at all dose levels. SAM vaccination provided protective efficacy against SARS-CoV-2 challenge as both a homologous prime-boost and as a single boost following ChAd prime, demonstrating reduction of viral replication in both the upper and lower airways. Protection was most effective with a SAM prime-boost vaccination regimen at 10 and 30 μg and with a ChAd/SAM heterologous prime-boost regimen. The SAM vaccine is currently being evaluated in clinical trials as both a homologous prime-boost regimen at low doses and as a boost following heterologous prime.

Abstract Tumor-specific neoantigens (TSNAs) are present in a majority of tumor types and are key targets for T cells released by immune checkpoint blockade therapy. Given that TSNAs are non-self antigens, they are particularly attractive cancer vaccine targets. We have developed a potent heterologous prime/boost immunization approach to deliver predicted TSNAs to patients, which is comprised of a replication incompetent chimpanzee adenoviral vector (ChAdV) for the prime vaccination and a self-replicating, synthetic viral vector (srRNA) for repeated boost vaccinations. The ChAdV vector is similar in design to other adenoviral vectors that have demonstrated induction of high titer, polyfunctional and durable CD4 and CD8 T-cell responses against non-self antigens that protected humans against infections. The srRNA vector is based on Venezuelan equine encephalitis virus (VEE), where sequences encoding the structural proteins of VEE were deleted and replaced by a TSNA expression cassette. For delivery in vivo, the srRNA is formulated with a lipid nanoparticle (LNP), which facilitates efficient cellular uptake of the RNA and enhances antigen expression as well as the resulting immune response. We demonstrate that the srRNA vector effectively replicates in vitro and in vivo resulting in durable and high levels of antigen expression. To characterize the vectors in pre-clinical animal models, a prototypical expression cassette that encodes multiple mouse MHC class I tumor antigens as well as Mamu-A01 restricted class I antigens for monitoring immune responses in mice and non-human primates (NHPs) was introduced into both vector systems. We demonstrate that immunization of mice with either vector results in strong antigen-specific CD8 T-cell responses against the encoded murine epitopes. The heterologous prime/boost approach provided a statistically significant survival advantage to tumor bearing mice when compared to untreated mice. The potency of the immunization platform was also assessed in Indian rhesus macaques and the platform demonstrated a quick onset of T-cell responses 1 week post ChAdV prime vaccination with peak T-cell responses against all delivered Mamu-A01 restricted class I antigens at 2-3 weeks. Such ChAdV primed T-cell responses were effectively boosted by the LNP formulated srRNA vector. Co-administration of anti-CTLA4 with the vaccine demonstrated enhanced vaccine induced immune response. These data demonstrate that the heterologous prime/boost platform effectively programs robust T-cell immunity toward encoded non-self antigens in NHPs, which is a highly predictive model of vaccine responses in humans. This vaccine platform is targeted for entry into clinical trials in mid 2018. Citation Format: Wade Blair, Gijsbert Grotenbreg, Ciaran Scallan, Amy Rappaport, Renee Greer, Leonid Gitlin, Kieu Lam, James Heyes, Anne Van Abbema, Hadley Hanson, Petra Schreiner, karin Jooss. A novel heterologous prime boost vaccine system drives tumor specific and potent CD8 T cell responses for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 724.

Nodamura Virus (NoV) is a nodavirus originally isolated from insects that can replicate in a wide variety of hosts, including mammals. Because of their simplicity and ability to replicate in many diverse hosts, NoV, and the Nodaviridae in general, provide a unique window into the evolution of viruses and host-virus interactions. Here we show that the C-terminus of the viral polymerase exhibits extreme structural and evolutionary flexibility. Indeed, fewer than 10 positively charged residues from the 110 amino acid-long C-terminal region of protein A are required to support RNA1 replication. Strikingly, this region can be replaced by completely unrelated protein sequences, yet still produce a functional replicase. Structure predictions, as well as evolutionary and mutational analyses, indicate that the C-terminal region is structurally disordered and evolves faster than the rest of the viral proteome. Thus, the function of an intrinsically unstructured protein region can be independent of most of its primary sequence, conferring both functional robustness and sequence plasticity on the protein. Our results provide an experimental explanation for rapid evolution of unstructured regions, which enables an effective exploration of the sequence space, and likely function space, available to the virus.

Herein, we present a straightforward surface modification technique for PDMS‐based microfluidic devices. The method takes advantage of the high reactivity of concentrated sulfuric acid to enhance the surface properties of PDMS bulk material. This results in alteration of the surface morphology and chemical composition that is in‐depth characterized by ATR‐FTIR, EDX, SEM, and XPS. In comparison to untreated PDMS, modified substrates exhibit a significantly reduced diffusive uptake of small organic molecules while retaining its low electroosmotic properties. This was demonstrated by exposing the channels of a microfluidic device to concentrated rhodamine B solution followed by fluorescence microscopy. The surface modification procedure was used to improve chip‐based electrophoretic separations. Separation efficiencies of FITC‐labeled amines/amino acids obtained in treated and untreated PDMS‐devices as well as in glass chips were compared. We obtained higher efficiencies in H2SO4 treated PDMS chips compared to untreated ones but lower efficiencies than those obtained in commercial microfluidic glass devices.

Real-time chemical reaction monitoring in microfluidic environments is demonstrated using luminescent chemical sensors integrated in PDMS/glass-based microscale reactors. A fabrication procedure is presented that allows for straightforward integration of thin polymer layers with optical sensing functionality in microchannels of glass-PDMS chips of only 150 μm width and of 10 to 35 μm height. Sensor layers consisting of polystyrene and an oxygen-sensitive platinum porphyrin probe with film thicknesses of about 0.5 to 4 μm were generated by combining spin coating and abrasion techniques. Optimal coating procedures were developed and evaluated. The chip-integrated sensor layers were calibrated and investigated with respect to stability, reproducibility and response times. These microchips allowed observation of dissolved oxygen concentration in the range of 0 to over 40 mg L(-1) with a detection limit of 368 μg L(-1). The sensor layers were then used for observation of a model reaction, the oxidation of sulphite to sulphate in a microfluidic chemical reactor and could observe sulphite concentrations of less than 200 μM. Real-time on-line monitoring of this chemical reaction was realized at a fluorescence microscope setup with 405 nm LED excitation and CCD camera detection.

We present a fast and versatile method to produce functional micro free-flow electrophoresis chips. Microfluidic structures were generated between two glass slides applying multistep liquid-phase lithography, omitting troublesome bonding steps or cost-intensive master structures. Utilizing a novel spacer-less approach with the photodefinable polymer polyethyleneglycol dimethacrylate (PEG-DA), microfluidic devices with hydrophilic channels of only 25 μm in height were generated. The microfluidic chips feature ion-permeable segregation walls between the electrode channels and the separation bed and hydrophilic surfaces. The performance of the chip is demonstrated by free-flow electrophoretic separation of fluorescent xanthene dyes and fluorescently labeled amino acids.

Microfluidic chips applied to the investigation of chirality allow reaction, separation and analysis of minuscule amounts of enantiomeric molecules. Chiral chip technology is employed in fields as diverse as pharmaceutical high throughput screening and deep space exploration missions.

The early host response to pathogens is mediated by several distinct pattern recognition receptors. Cytoplasmic RNA helicases including RIG-I and MDA5 have been shown to respond to viral RNA by inducing interferon (IFN) production. Previous in vitro studies have demonstrated a direct role for MDA5 in the response to members of the Picornaviridae, Flaviviridae and Caliciviridae virus families ((+) ssRNA viruses) but not to Paramyxoviridae or Orthomyxoviridae ((−) ssRNA viruses). Contrary to these findings, we now show that MDA5 responds critically to infections caused by Paramyxoviridae in vivo. Using an established model of natural Sendai virus (SeV) infection, we demonstrate that MDA5−/− mice exhibit increased morbidity and mortality as well as severe histopathological changes in the lower airways in response to SeV. Moreover, analysis of viral propagation in the lungs of MDA5−/− mice reveals enhanced replication and a distinct distribution involving the interstitium. Though the levels of antiviral cytokines were comparable early during SeV infection, type I, II, and III IFN mRNA expression profiles were significantly decreased in MDA5−/− mice by day 5 post infection. Taken together, these findings indicate that MDA5 is indispensable for sustained expression of IFN in response to paramyxovirus infection and provide the first evidence of MDA5-dependent containment of in vivo infections caused by (−) sense RNA viruses.

We present a simple method for fast and precise replication of microfluidic master structures for moulding or soft embossing by double casting of microstructured masters with polydimethylsiloxane (PDMS). The significant achievement is a simple approach to inverse a given microstructure multiple times by means of PDMS-based soft lithography utilising hydroxypropylmethylcellulose (HPMC) as non texturing release agent. A series of PDMS copies have been generated from different silicon layouts with excellent reproducibility and precision, even submicron structures were well reproduced. The replicas were successfully applied in hot embossing and soft lithography of microfluic devices. Hence, we believe this technique is ideally suited for the economic replication of precious master structures (master sharing) commonly used in soft lithography and hot embossing.

Defective interfering (DI) particles are byproducts of virus replication that potently enhance dendritic cell (DC) maturation by virus infection. DI particles have been reported for many different viruses and are strong inducers of type I IFNs. The cellular mechanisms involved in the response to DI particles are not known. In this study, we show that 1) DI particles are recognized by more than one viral sensor independently of TLRs and type I IFN signaling; 2) The helicase MDA5 participates in the detection of DI genomes as MDA5-deficient DCs respond inefficiently to Sendai virus stocks containing DI particles; 3) DI particles stimulate the expression of IRF3-responsive genes by a uniquely potent mechanism when compared with other prototypic viral stimulus; and 4) the efficient detection of DI particles overcomes virus immune antagonism. These data highlight the outstanding adjuvant capacity of DI particles in stimulating mouse and human DCs. They also offer biological relevance to the previously reported inhibition of MDA5 by different paramyxovirus V proteins. The unique mechanism by which DI particles trigger the maturation of DCs represents a novel strategy that could be further exploited for the development of potent adjuvant molecules.

Noroviruses are important human pathogens responsible for most cases of viral epidemic gastroenteritis worldwide. Murine norovirus-1 (MNV-1) is one of several murine noroviruses isolated from research mouse facilities and has been used as a model of human norovirus infection. MNV-1 infection has been shown to require components of innate and adaptive immunity for clearance; however, the initial host protein that recognizes MNV-1 infection is unknown. Because noroviruses are RNA viruses, we investigated whether MDA5 and TLR3, cellular sensors that recognize dsRNA, are important for the host response to MNV-1. We demonstrate that MDA5(-/-) dendritic cells(DC) have a defect in cytokine response to MNV-1. In addition, MNV-1 replicates to higher levels in MDA5(-/-) DCs as well as in MDA5(-/-) mice in vivo. Interestingly, TLR3(-/-) DCs do not have a defect in vitro, but TLR3(-/-) mice have a slight increase in viral titers. This is the first demonstration of an innate immune sensor for norovirus and shows that MDA5 is required for the control of MNV-1 infection. Knowledge of the host response to MNV-1 may provide keys for prevention and treatment of the human disease.