A Novel CRISPR-Engineered, Stem Cell-Derived Cellular Vaccine

COVID-19 has forced rapid clinical translation of novel vaccine technologies, principally mRNA vaccines, that have resulted in meaningful efficacy and adequate safety in response to the global pandemic. Notwithstanding this success, there remains an opportunity for innovation in vaccine technology to address current limitations and meet the challenges of inevitable future pandemics. We describe a universal vaccine cell (UVC) rationally designed to mimic the natural physiologic immunity induced post viral infection of host cells. Induced pluripotent stem cells were CRISPR engineered to delete MHC-I expression and simultaneously overexpress a NK Ligand adjuvant to increase rapid cellular apoptosis which was hypothesized to enhance viral antigen presentation in the resulting immune microenvironment leading to a protective immune response. Cells were further engineered to express the parental variant WA1/2020 SARS-CoV-2 spike protein as a representative viral antigen prior to irradiation and cryopreservation. The cellular vaccine was then used to immunize non-human primates in a standard 2-dose, IM injected prime + boost vaccination with 1e8 cells per 1 ml dose resulting in robust neutralizing antibody responses (1e3 nAb titers) with decreasing levels at 6 months duration. Similar titers generated in this established NHP model have translated into protective human neutralizing antibody levels in SARS-Cov-2 vaccinated individuals. Animals vaccinated with WA1/2020 spike antigens were subsequently challenged with 1.0 x 105 TCID50 infectious Delta (B.1.617.2) SARS-CoV-2 in a heterologous challenge which resulted in an approximately 3-log order decrease in viral RNA load in the lungs. These heterologous viral challenge results reflect the ongoing real-world experience of original variant WA1/2020 spike antigen vaccinated populations exposed to rapidly emerging variants like Delta and now Omicron. This cellular vaccine is designed to be a rapidly scalable cell line with a modular poly-antigenic payload to allow for practical, large-scale clinical manufacturing and use in an evolving viral variant environment. Human clinical translation of the UVC is being actively explored for this and potential future pandemics.

577. COVI-VAC™, a Live Attenuated COVID-19 Vaccine, Provides Single Dose Protection Against Heterologous Challenge with SARS-CoV-2 Beta (B.1.351) in the Syrian Golden Hamster Model

Background Although multiple COVID-19 vaccines are currently in use, emergence of novel SARS-CoV-2 variants with reduced neutralization raises concern of future vaccine escape. COVI-VAC™ is a live attenuated SARS-CoV-2 strain based on WA/1 being developed as an intranasal COVID-19 vaccine. COVI-VAC is attenuated through removal of the furin cleavage site and introduction of 283 silent, deoptimizing mutations that maintain viral amino acid sequence but slow viral replication in vivo by up to 5 logs. Notably, COVI-VAC presents all viral antigens in their native conformation and is not limited to spike. COVI-VAC demonstrated attenuation, immunogenicity and single dose protection in both the Syrian golden hamster and non-human primate models and currently in Phase 1 clinical trials. In this study, we evaluated efficacy of COVI-VAC against challenge with the Beta/B.1.351 variant in Syrian golden hamsters. Methods Syrian golden hamsters, 7-10 weeks of age were, vaccinated intranasally with 8.25x104 PFU COVI-VAC (n=28) or vehicle control (n=16). Twenty seven days post-vaccination, animals were challenged intranasally with 3x104 PFU of wildtype (WT) SARS-CoV-2 Beta. Animals were weighed daily. Further analysis is being conducted with serum and key tissues from pre and post challenge timepoints to include neutralizing antibody, biodistribution (subgenomic qPCR) and histopathology. Results COVI-VAC prevented weight loss following challenge with the heterologous variant of SARS-CoV-2, B.1.351/Beta (Figure). Results of additional analyses will be available before the IDWeek meeting. Change in Weight following SARS-CoV-2 Beta Challenge Conclusion COVI-VAC is protective against heterologous challenge with SARS-CoV-2 Beta. By presenting all viral antigens, COVI-VAC may be less affected by viral evolution than spike-based vaccines. Disclosures Anna Kushnir, PHD, Codagenix Inc (Employee) Steffen Mueller, PhD, Codagenix Inc (Board Member, Employee, Shareholder) Sybil Tasker, MD, MPH, FIDSA, Codagenix Inc (Employee, Shareholder) J. Robert Coleman, PhD, Codagenix Inc. (Board Member, Employee, Shareholder)

Preliminary evaluation of Brazilian green propolis as coadjuvant of a subunit vaccine against lethal leptospirosis

Vaccination would be preferred to leptospirosis control measures. However, current vaccines are heat killed whole-cell bacterins that generate serovar specific protection and several side effects. Modern molecular assays have revealed antigens that may replace traditional whole-cell vaccines. Among these, LigB protein is surface-exposed outer membrane protein of virulent leptospires and therefore potential target of a protective immune response. Some unsuccessful attempts at using these antigens as vaccines have been reported. However, we believe that immune modulation through alternative adjuvants and co-adjuvants may overcome previous setbacks. In this light, our study aimed to evaluate the protective immune response in hamsters vaccinated with 40 µg of rLigBNI using oil adjuvant (OA), with or without green propolis (GP) as co-adjuvant. Upon a challenge, all groups immunized with rLigBNI, coupled or not with GP, were highly immunogenic and revealed statistically significant (p<0.05) protection of hamsters from lethal leptospirosis. Additional studies are being carried out to assess the optimum dose, protection against heterologous challenge, and vaccine dynamics.

Trained Immunity Confers Prolonged Protection From Listeriosis

Trained immunity refers to the ability of the innate immune system exposed to a first challenge to provide an enhanced response to a secondary homologous or heterologous challenge. We reported that training induced with β-glucan one week before infection confers protection against a broad-spectrum of lethal bacterial infections. Whether this protection persists over time is unknown. To tackle this question, we analyzed the immune status and the response to Listeria monocytogenes (L. monocytogenes) of mice trained 9 weeks before analysis. The induction of trained immunity increased bone marrow myelopoiesis and blood counts of Ly6Chigh inflammatory monocytes and polymorphonuclear neutrophils (PMNs). Ex vivo, whole blood, PMNs and monocytes from trained mice produced increased levels of cytokines in response to microbial products and limited the growth of L. monocytogenes. In vivo, following challenge with L. monocytogenes, peripheral blood leukocytes were massively depleted in control mice but largely preserved in trained mice. PMNs were reduced also in the spleen from control mice, and increased in the spleen of trained mice. In transwell experiments, PMNs from trained mice showed increased spontaneous migration and CXCL2/MIP2α-induced chemotaxis, suggesting that training promotes the migration of PMNs in peripheral organs targeted by L. monocytogenes. Trained PMNs and monocytes had higher glycolytic activity and mitochondrial respiration than control cells when exposed to L. monocytogenes. Bacterial burden and dissemination in blood, spleen and liver as well as systemic cytokines and inflammation (multiplex bead assay and bioluminescence imaging) were reduced in trained mice. In full agreement with these results, mice trained 9 weeks before infection were powerfully protected from lethal listeriosis. Altogether, these data suggest that training increases the generation and the antimicrobial activity of PMNs and monocytes, which may confer prolonged protection from lethal bacterial infection.

A SARS-CoV-2 spike ferritin nanoparticle vaccine protects against heterologous challenge with B.1.1.7 and B.1.351 virus variants in Syrian golden hamsters

The emergence of SARS-CoV-2 variants of concern (VOC) requires adequate coverage of vaccine protection. We evaluated whether a spike ferritin nanoparticle vaccine (SpFN), adjuvanted with the Army Liposomal Formulation QS21 (ALFQ), conferred protection against the B.1.1.7 and B.1.351 VOCs in Syrian golden hamsters. SpFN-ALFQ was administered as either single or double-vaccination (0 and 4 week) regimens, using a high (10 μg) or low (0.2 μg) immunogen dose. Animals were intranasally challenged at week 11. Binding antibody responses were comparable between high- and low-dose groups. Neutralizing antibody titers were equivalent against WA1, B.1.1.7, and B.1.351 variants following two high dose two vaccinations. SpFN-ALFQ vaccination protected against SARS-CoV-2-induced disease and viral replication following intranasal B.1.1.7 or B.1.351 challenge, as evidenced by reduced weight loss, lung pathology, and lung and nasal turbinate viral burden. These data support the development of SpFN-ALFQ as a broadly protective, next-generation SARS-CoV-2 vaccine.

Intranasal Immunization with the Influenza A Virus Encoding Truncated NS1 Protein Protects Mice from Heterologous Challenge by Restraining the Inflammatory Response in the Lungs

Influenza viruses with an impaired NS1 protein are unable to antagonize the innate immune system and, therefore, are highly immunogenic because of the self-adjuvating effect. Hence, NS1-mutated viruses are considered promising candidates for the development of live-attenuated influenza vaccines and viral vectors for intranasal administration. We investigated whether the immunogenic advantage of the virus expressing only the N-terminal half of the NS1 protein (124 a.a.) can be translated into the induction of protective immunity against a heterologous influenza virus in mice. We found that immunization with either the wild-type A/PR/8/34 (H1N1) influenza strain (A/PR8/NSfull) or its NS1-shortened counterpart (A/PR8/NS124) did not prevent the viral replication in the lungs after the challenge with the A/Aichi/2/68 (H3N2) virus. However, mice immunized with the NS1-shortened virus were better protected from lethality after the challenge with the heterologous virus. Besides showing the enhanced influenza-specific CD8+ T-cellular response in the lungs, immunization with the A/PR8/NS124 virus resulted in reduced concentrations of proinflammatory cytokines and the lower extent of leukocyte infiltration in the lungs after the challenge compared to A/PR8/NSfull or the control group. The data show that intranasal immunization with the NS1-truncated virus may better induce not only effector T-cells but also certain immunoregulatory mechanisms, reducing the severity of the innate immune response after the heterologous challenge.

A CCHFV DNA vaccine protects against heterologous challenge and establishes GP38 as immunorelevant in mice

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that causes severe hemorrhagic fever disease in humans. Currently, no licensed CCHF vaccines exist, and the protective epitopes remain unclear. Previously, we tested a DNA vaccine expressing the M-segment glycoprotein precursor gene of the laboratory CCHFV strain IbAr 10200 (CCHFV-M10200). CCHFV-M10200 provided >60% protection against homologous CCHFV-IbAr 10200 challenge in mice. Here, we report that increasing the dose of CCHFV-M10200 provides complete protection from homologous CCHFV challenge in mice, and significant (80%) protection from challenge with the clinically relevant heterologous strain CCHFV-Afg09-2990. We also report complete protection from CCHFV-Afg09-2990 challenge following vaccination with a CCHFV-Afg09-2990 M-segment DNA vaccine (CCHFV-MAfg09). Finally, we show that the non-structural M-segment protein, GP38, influences CCHF vaccine immunogenicity and provides significant protection from homologous CCHFV challenge. Our results demonstrate that M-segment DNA vaccines elicit protective CCHF immunity and further illustrate the immunorelevance of GP38.

Impact of Pre-Existing Immunity on Live Attenuated Influenza Vaccine-Induced Cross-Protective Immunity

The efficacy of the intranasally (i.n.) delivered live attenuated influenza vaccine (LAIV) is variable and, in some seasons, suboptimal. In this study, we report that LAIV exhibits cross-protective efficacy in mice, potentially associated with cellular immunity as opposed to antigen-specific antibody responses. However, pre-exposure to the intramuscularly (i.m.) delivered inactivated influenza vaccine (IIV) severely impaired LAIV-induced cross-protection against heterologous challenge, potentially by inhibiting replication of LAIV. Our findings suggest that pre-existing immunity afforded by IIV suppresses cross-protective T cell immunogenicity of LAIV.

Schistosoma haematobium Extracellular Vesicle Proteins Confer Protection in a Heterologous Model of Schistosomiasis

Helminth parasites release extracellular vesicles which interact with the surrounding host tissues, mediating host–parasite communication and other fundamental processes of parasitism. As such, vesicle proteins present attractive targets for the development of novel intervention strategies to control these parasites and the diseases they cause. Herein, we describe the first proteomic analysis by LC-MS/MS of two types of extracellular vesicles (exosome-like, 120 k pellet vesicles and microvesicle-like, 15 k pellet vesicles) from adult Schistosoma haematobium worms. A total of 57 and 330 proteins were identified in the 120 k pellet vesicles and larger 15 k pellet vesicles, respectively, and some of the most abundant molecules included homologues of known helminth vaccine and diagnostic candidates such as Sm-TSP2, Sm23, glutathione S-transferase, saponins and aminopeptidases. Tetraspanins were highly represented in the analysis and found in both vesicle types. Vaccination of mice with recombinant versions of three of these tetraspanins induced protection in a heterologous challenge (S. mansoni) model of infection, resulting in significant reductions (averaged across two independent trials) in liver (47%, 38% and 41%) and intestinal (47%, 45% and 41%) egg burdens. These findings offer insight into the mechanisms by which anti-tetraspanin antibodies confer protection and highlight the potential that extracellular vesicle surface proteins offer as anti-helminth vaccines.