Intrathecal Gene Therapy for Giant Axonal Neuropathy.

BACKGROUND: Giant axonal neuropathy is a rare, autosomal recessive, pediatric, polysymptomatic, neurodegenerative disorder caused by biallelic loss-of-function variants in GAN, the gene encoding gigaxonin. METHODS: We conducted an intrathecal dose-escalation study of scAAV9/JeT-GAN (a self-complementary adeno-associated virus-based gene therapy containing the GAN transgene) in children with giant axonal neuropathy. Safety was the primary end point. The key secondary clinical end point was at least a 95% posterior probability of slowing the rate of change (i.e., slope) in the 32-item Motor Function Measure total percent score at 1 year after treatment, as compared with the pretreatment slope. RESULTS: One of four intrathecal doses of scAAV9/JeT-GAN was administered to 14 participants - 3.5×1013 total vector genomes (vg) (in 2 participants), 1.2×1014 vg (in 4), 1.8×1014 vg (in 5), and 3.5×1014 vg (in 3). During a median observation period of 68.7 months (range, 8.6 to 90.5), of 48 serious adverse events that had occurred, 1 (fever) was possibly related to treatment; 129 of 682 adverse events were possibly related to treatment. The mean pretreatment slope in the total cohort was -7.17 percentage points per year (95% credible interval, -8.36 to -5.97). At 1 year after treatment, posterior mean changes in slope were -0.54 percentage points (95% credible interval, -7.48 to 6.28) with the 3.5×1013-vg dose, 3.23 percentage points (95% credible interval, -1.27 to 7.65) with the 1.2×1014-vg dose, 5.32 percentage points (95% credible interval, 1.07 to 9.57) with the 1.8×1014-vg dose, and 3.43 percentage points (95% credible interval, -1.89 to 8.82) with the 3.5×1014-vg dose. The corresponding posterior probabilities for slowing the slope were 44% (95% credible interval, 43 to 44); 92% (95% credible interval, 92 to 93); 99% (95% credible interval, 99 to 99), which was above the efficacy threshold; and 90% (95% credible interval, 89 to 90). Between 6 and 24 months after gene transfer, sensory-nerve action potential amplitudes increased, stopped declining, or became recordable after being absent in 6 participants but remained absent in 8. CONCLUSIONS: Intrathecal gene transfer with scAAV9/JeT-GAN for giant axonal neuropathy was associated with adverse events and resulted in a possible benefit in motor function scores and other measures at some vector doses over a year. Further studies are warranted to determine the safety and efficacy of intrathecal AAV-mediated gene therapy in this disorder. (Funded by the National Institute of Neurological Disorders and Stroke and others; ClinicalTrials.gov number, NCT02362438.).

High-dose systemic adeno-associated virus vector administration causes liver and sinusoidal endothelial cell injury.

We analyzed retrospective data from toxicology studies involving administration of high doses of adeno-associated virus expressing different therapeutic transgenes to 21 cynomolgus and 15 rhesus macaques. We also conducted prospective studies to investigate acute toxicity following high-dose systemic administration of enhanced green fluorescent protein-expressing adeno-associated virus to 10 rhesus macaques. Toxicity was characterized by transaminitis, thrombocytopenia, and alternative complement pathway activation that peaked on post-administration day 3. Although most animals recovered, some developed ascites, generalized edema, hyperbilirubinemia, and/or coagulopathy that prompted unscheduled euthanasia. Study endpoint livers from animals that recovered and from unscheduled necropsies of those that succumbed to toxicity were analyzed via hypothesis-driven histopathology and unbiased single-nucleus RNA sequencing. All liver cell types expressed high transgene transcript levels at early unscheduled timepoints that subsequently decreased. Thrombocytopenia coincided with sinusoidal platelet microthrombi and sinusoidal endothelial injury identified via immunohistology and single-nucleus RNA sequencing. Acute toxicity, sinusoidal injury, and liver platelet sequestration were similarly observed with therapeutic transgenes and enhanced green fluorescent protein at doses ≥1 × 1014 GC/kg, suggesting it was the consequence of high-dose systemic adeno-associated virus administration, not green fluorescent protein toxicity. These findings highlight a potential toxic effect of high-dose intravenous adeno-associated virus on nonhuman primate liver microvasculature.

Prednisolone and rapamycin reduce the plasma cell gene signature and may improve AAV gene therapy in cynomolgus macaques.

Adeno-associated virus (AAV) vector gene therapy is a promising approach to treat rare genetic diseases; however, an ongoing challenge is how to best modulate host immunity to improve transduction efficiency and therapeutic outcomes. This report presents two studies characterizing multiple prophylactic immunosuppression regimens in male cynomolgus macaques receiving an AAVrh10 gene therapy vector expressing human coagulation factor VIII (hFVIII). In study 1, no immunosuppression was compared with prednisolone, rapamycin (or sirolimus), rapamycin and cyclosporin A in combination, and cyclosporin A and azathioprine in combination. Prednisolone alone demonstrated higher mean peripheral blood hFVIII expression; however, this was not sustained upon taper. Anti-capsid and anti-hFVIII antibody responses were robust, and vector genomes and transgene mRNA levels were similar to no immunosuppression at necropsy. Study 2 compared no immunosuppression with prednisolone alone or in combination with rapamycin or methotrexate. The prednisolone/rapamycin group demonstrated an increase in mean hFVIII expression and a mean delay in anti-capsid IgG development until after rapamycin taper. Additionally, a significant reduction in the plasma cell gene signature was observed with prednisolone/rapamycin, suggesting that rapamycin's tolerogenic effects may include plasma cell differentiation blockade. Immunosuppression with prednisolone and rapamycin in combination could improve therapeutic outcomes in AAV vector gene therapy.

Phase I study of liver depot gene therapy in late-onset Pompe disease.

Gene therapy with an adeno-associated virus serotype 8 (AAV8) vector (AAV8-LSPhGAA) could eliminate the need for enzyme replacement therapy (ERT) by creating a liver depot for acid α-glucosidase (GAA) production. We report initial safety and bioactivity of the first dose (1.6 × 1012 vector genomes/kg) cohort (n = 3) in a 52-week open-label, single-dose, dose-escalation study (NCT03533673) in patients with late-onset Pompe disease (LOPD). Subjects discontinued biweekly ERT after week 26 based on the detection of elevated serum GAA activity and the absence of clinically significant declines per protocol. Prednisone (60 mg/day) was administered as immunoprophylaxis through week 4, followed by an 11-week taper. All subjects demonstrated sustained serum GAA activities from 101% to 235% of baseline trough activity 2 weeks following the preceding ERT dose. There were no treatment-related serious adverse events. No subject had anti-capsid T cell responses that decreased transgene expression. Muscle biopsy at week 24 revealed unchanged muscle glycogen content in two of three subjects. At week 52, muscle GAA activity for the cohort was significantly increased (p < 0.05). Overall, these initial data support the safety and bioactivity of AAV8-LSPhGAA, the safety of withdrawing ERT, successful immunoprophylaxis, and justify continued clinical development of AAV8-LSPhGAA therapy in Pompe disease.

Engineering and expression of a RhoA peptide against respiratory syncytial virus infection in plants.

MAIN CONCLUSION : A RhoA-derived peptide fused to carrier molecules from plants showed enhanced biological activity of in vitro assays against respiratory syncytial virus compared to the RhoA peptide alone or the synthetic RhoA peptide. A RhoA-derived peptide has been reported for over a decade as a potential inhibitor of respiratory syncytial virus (RSV) infection both in vitro and in vivo and is anticipated to be a promising alternative to monoclonal antibody-based therapy against RSV infection. However, there are several challenges to furthering development of this antiviral peptide, including improvement in the peptide's bioavailability, development of an efficient delivery system and identification of a cost-effective production platform. In this study, we have engineered a RhoA peptide as a genetic fusion to two carrier molecules, either lichenase (LicKM) or the coat protein (CP) of Alfalfa mosaic virus. These constructs were introduced into Nicotiana benthamiana plants using a tobacco mosaic virus-based expression vector and targets purified. The results demonstrated that the RhoA peptide fusion proteins were efficiently expressed in N. benthamiana plants, and that two of the resulting fusion proteins, RhoA-LicKM and RhoA2-FL-d25CP, inhibited RSV growth in vitro by 50 and 80 %, respectively. These data indicate the feasibility of transient expression of this biologically active antiviral RhoA peptide in plants and the advantage of using a carrier molecule to enhance target expression and efficacy.

Stabilization of HAC1 influenza vaccine by spray drying: formulation development and process scale-up.

PURPOSE: Stable vaccines with long shelf lives and reduced dependency on the cold chain are ideal for stockpiling and rapid deployment during public emergencies, including pandemics. Spray drying is a low-cost process that has potential to produce vaccines stable at a wide range of temperatures. Our aim was to develop a stable formulation of a recombinant H1N1 influenza hemagglutinin vaccine candidate and take it to pilot-scale spray-drying production. METHODS: Eight formulations containing different excipients were produced and assayed for antigen stability, powder characteristics, and immunogenicity after storage at a range of temperatures, resulting in the identification of four promising candidates. A pilot-scale spray-drying process was then developed for further testing of one formulation. RESULTS: The pilot-scale process was used to reproducibly manufacture three batches of the selected formulation with yields >90%. All batches had stable physical properties and in vitro potency for 6 months at temperatures from -20°C to +50°C. Formulations stored for 3 months elicited immunogenic responses in mice equivalent to a frozen lot of bulk vaccine used as a stability control. CONCLUSIONS: This study demonstrates the feasibility of stabilizing subunit vaccines using a spray-drying process and the suitability of the process for manufacturing a candidate product.

Neonatal Fc Receptor Inhibition Enables Adeno-Associated Virus Gene Therapy Despite Pre-Existing Humoral Immunity.

Advances in adeno-associated virus (AAV)-based gene therapy are transforming our ability to treat rare genetic disorders and address other unmet medical needs. However, the natural prevalence of anti-AAV neutralizing antibodies (NAbs) in humans currently limits the population who can benefit from AAV-based gene therapies. Neonatal Fc receptor (FcRn) plays an essential role in the long half-life of IgG, a key NAb. Researchers have developed several FcRn-inhibiting monoclonal antibodies to treat autoimmune diseases, as inhibiting the interaction between FcRn and IgG Fc can reduce circulating IgG levels to 20-30% of the baseline. We evaluated the utility of one such monoclonal antibody, M281, to reduce pre-existing NAb levels and to permit gene delivery to the liver and heart via systemic AAV gene therapy in mice and nonhuman primates. M281 successfully reduced NAb titers along with total IgG levels; it also enhanced gene delivery to the liver and other organs after intravenous administration of AAV in NAb-positive animals. These results indicate that mitigating pre-existing humoral immunity via disruption of the FcRn-IgG interaction may make AAV-based gene therapies effective in NAb-positive patients.

Engineering of a plant-produced virus-like particle to improve the display of the Plasmodium falciparum Pfs25 antigen and transmission-blocking activity of the vaccine candidate.

Malaria kills around 409,000 people a year, mostly children under the age of five. Malaria transmission-blocking vaccines work to reduce malaria prevalence in a community and have the potential to be part of a multifaceted approach required to eliminate the parasites causing the disease. Pfs25 is a leading malaria transmission-blocking antigen and has been successfully produced in a plant expression system as both a subunit vaccine and as a virus-like particle. This study demonstrates an improved version of the virus-like particle antigen display molecule by eliminating known protease sites from the prior A85 variant. This re-engineered molecule, termed B29, displays three times the number of Pfs25 antigens per virus-like particle compared to the original Pfs25 virus-like particle. An improved purification scheme was also developed, resulting in a substantially higher yield and improved purity. The molecule was evaluated in a mouse model and found to induce improved transmission-blocking activity at lower doses and longer durations than the original molecule.

Immune transgene-dependent myocarditis in macaques after systemic administration of adeno-associated virus expressing human acid alpha-glucosidase.

Immune responses to human non-self transgenes can present challenges in preclinical studies of adeno-associated virus (AAV) gene therapy candidates in nonhuman primates. Although anti-transgene immune responses are usually mild and non-adverse, they can confound pharmacological readouts and complicate translation of results between species. We developed a gene therapy candidate for Pompe disease consisting of AAVhu68, a clade F AAV closely related to AAV9, that expresses an engineered human acid-alpha glucosidase (hGAA) tagged with an insulin-like growth factor 2 variant (vIGF2) peptide for enhanced cell uptake. Rhesus macaques were administered an intravenous dose of 1x1013 genome copies (GC)/kg, 5x1013 GC/kg, or 1 x 1014 GC/kg of AAVhu68.vIGF2.hGAA. Some unusually severe adaptive immune responses to hGAA presented, albeit with a high degree of variability between animals. Anti-hGAA responses ranged from absent to severe cytotoxic T-cell-mediated myocarditis with elevated troponin I levels. Cardiac toxicity was not dose dependent and affected five out of eleven animals. Upon further investigation, we identified an association between toxicity and a major histocompatibility complex class I haplotype (Mamu-A002.01) in three of these animals. An immunodominant peptide located in the C-terminal region of hGAA was subsequently identified via enzyme-linked immunospot epitope mapping. Another notable observation in this preclinical safety study cohort pertained to the achievement of robust and safe gene transfer upon intravenous administration of 5x1013 GC/kg in one animal with a low pre-existing neutralizing anti-capsid antibodies titer (1:20). Collectively, these findings may have significant implications for gene therapy inclusion criteria.

Integrated vector genomes may contribute to long-term expression in primate liver after AAV administration.

The development of liver-based adeno-associated virus (AAV) gene therapies is facing concerns about limited efficiency and durability of transgene expression. We evaluated nonhuman primates following intravenous dosing of AAV8 and AAVrh10 vectors for over 2 years to better define the mechanism(s) of transduction that affect performance. High transduction of non-immunogenic transgenes was achieved, although expression declined over the first 90 days to reach a lower but stable steady state. More than 10% of hepatocytes contained single nuclear domains of vector DNA that persisted despite the loss of transgene expression. Greater reductions in vector DNA and RNA were observed with immunogenic transgenes. Genomic integration of vector sequences, including complex concatemeric structures, were detected in 1 out of 100 cells at broadly distributed loci that were not in proximity to genes associated with hepatocellular carcinoma. Our studies suggest that AAV-mediated transgene expression in primate hepatocytes occurs in two phases: high but short-lived expression from episomal genomes, followed by much lower but stable expression, likely from integrated vectors.

Intravenous immunoglobulin prevents peripheral liver transduction of intrathecally delivered AAV vectors.

Gene therapy using neurotropic adeno-associated virus vectors represents an emerging solution for genetic disorders affecting the central nervous system. The first approved central nervous system-targeting adeno-associated virus gene therapy, Zolgensma®, for treating spinal muscular atrophy is administered intravenously at high doses that cause liver-associated adverse events in 20%-30% of patients. Intrathecal routes of vector administration, such as the intra-cisterna magna route, provide efficient gene transduction to central nervous system cells while reducing off-target liver transduction. However, significant levels of liver transduction often occur upon intra-cisterna magna vector delivery in preclinical studies. Using vectors expressing monoclonal antibody transgenes, we examined whether passive transfer of adeno-associated virus-neutralizing antibodies as intravenous immunoglobulin before intrathecal adeno-associated virus delivery improved the safety of viral gene therapy targeting the central nervous system in mice and nonhuman primates. We used intracerebroventricular and intra-cisterna magna routes for vector administration to mice and nonhuman primates, respectively, and evaluated transgene expression and vector genome distribution. Our data indicate that pretreatment with intravenous immunoglobulin significantly reduced gene transduction to the liver and other peripheral organs but not to the central nervous system in both species. With further refinement, this method may improve the safety of adeno-associated virus-based, central nervous system-targeting gene therapies in clinical settings.

Erratum: Intravenous immunoglobulin prevents peripheral liver transduction of intrathecally delivered AAV vectors.

[This corrects the article DOI: 10.1016/j.omtm.2022.09.017.].

Prednisolone reduces the interferon response to AAV in cynomolgus macaques and may increase liver gene expression.

Ornithine transcarbamylase deficiency is a rare X-linked genetic urea cycle disorder leading to episodes of acute hyperammonemia, adverse cognitive and neurological effects, hospitalizations, and in some cases death. DTX301, a non-replicating, recombinant self-complimentary adeno-associated virus vector serotype 8 (scAAV8)-encoding human ornithine transcarbamylase, is a promising gene therapy for ornithine transcarbamylase deficiency; however, the impact of sex and prophylactic immunosuppression on ornithine transcarbamylase gene therapy outcomes is not well characterized. This study sought to describe the impact of sex and immunosuppression in adult, sexually mature female and male cynomolgus macaques through day 140 after DTX301 administration. Four study groups (n = 3/group) were included: male non-immunosuppressed; male immunosuppressed; female non-immunosuppressed; and female immunosuppressed. DTX301 was well tolerated with and without immunosuppression; no notable differences were observed between female and male groups across outcome measures. Prednisolone-treated animals exhibited a trend toward greater vector genome and transgene expression, although the differences were not statistically significant. The hepatic interferon gene signature was significantly decreased in prednisolone-treated animals, and a significant inverse relationship was observed between interferon gene signature levels and hepatic vector DNA and transgene RNA. These observations were not sustained upon immunosuppression withdrawal. Further studies may determine whether the observed effect can be prolonged.

Safety and immunogenicity of a plant-derived recombinant protective antigen (rPA)-based vaccine against Bacillus anthracis: A Phase 1 dose-escalation study in healthy adults.

BACKGROUND: The potential use of Bacillus anthracis as a bioterrorism weapon requires a safe and effective vaccine that can be immediately distributed for mass vaccination. Protective antigen (PA), a principal component of virulence factors edema toxin and lethal toxin of B. anthracis, has been the topic of extensive research. Previously, full-length PA (PA83) was manufactured using a transient plant-based expression system. Immunization with this PA83 antigen formulated with Alhydrogel® adjuvant elicited strong neutralizing immune responses in mice and rabbits and protected 100% of rabbits from a lethal aerosolized B. anthracis challenge. This Phase 1 study evaluates this vaccine's safety and immunogenicity in healthy human volunteers. METHODS: This first-in-human, single-blind, Phase 1 study was performed at a single center to investigate the safety, reactogenicity, and immunogenicity of the plant-derived PA83-FhCMB vaccine at four escalating dose levels (12.5, 25, 50 or 100 µg) with Alhydrogel® in healthy adults 18-49 years of age (inclusive). Recipients received three doses of vaccine intramuscularly at 28-day intervals. Safety was evaluated on days 3, 7, and 14 following vaccination. Immunogenicity was assessed using an enzyme-linked immunosorbent assay (ELISA) and a toxin neutralizing antibody (TNA) assay on days 0, 14, 28, 56, 84, and 180. RESULTS: All four-dose ranges were safe and immunogenic, with no related serious adverse events observed. Peak ELISA Geometric Mean Concentration (GMC) and TNA ED50 Geometric Mean Titer (GMT) were noted at Day 84, 1 month after the final dose, with the most robust response detected in the highest dose group. Antibody responses decreased by Day 180 across all dose groups. Long-term immunogenicity data beyond six months was not collected. CONCLUSIONS: This is the first study demonstrating a plant-derived subunit anthrax vaccine's safety and immunogenicity in healthy adults. The results support further clinical investigation of the PA83-FhCMB vaccine. ClinicalTrials.gov identifier. NCT02239172.

A non-glycosylated, plant-produced human monoclonal antibody against anthrax protective antigen protects mice and non-human primates from B. anthracis spore challenge.

The health and economic burden of infectious diseases in general and bioterrorism in particular necessitate the development of medical countermeasures. One proven approach to reduce the disease burden and spread of pathogen is treatment with monoclonal antibodies (mAb). mAbs can prevent or reduce severity of the disease by variety of mechanisms, including neutralizing pathogen growth, limiting its spread from infected to adjacent cells, or by inhibiting biological activity of toxins, such as anthrax lethal toxin. Here, we report the production of glycosylated (pp-mAb (PA) ) and non-glycosylated (pp-mAb (PANG) ) versions of a plant-derived mAb directed against protective antigen (PA) of Bacillus anthracis in Nicotiana benthamiana plants using agroinfiltration. Both forms of the antibody were able to neutralize anthrax lethal toxin activity in vitro and protect mice against an intraperitoneal challenge with spores of B. anthracis Sterne strain. A single 180 µg intraperitoneal dose of pp-mAb (PA) or pp-mAb (PANG) provided 90% and 100% survival, respectively. When tested in non-human primates, pp-mAb (PANG) was demonstrated to be superior to pp-mAb (PA) in that it had a significantly longer terminal half-life and conferred 100% protection against a lethal dose of aerosolized anthrax spore challenge after a single 5 mg/kg intravenous dose compared to a 40% survival rate conferred by pp-mAb (PA) . This study demonstrates the potential of a plant-produced non-glycosylated antibody as a useful tool for the treatment of inhalation anthrax.

Plant-based rapid production of recombinant subunit hemagglutinin vaccines targeting H1N1 and H5N1 influenza.

In 2009, a novel H1N1 swine influenza virus was isolated from infected humans in Mexico and the United States, and rapidly spread around the world. Another virus, a highly pathogenic avian influenza virus of the H5N1 subtype, identified by the World Health Organization as a potential pandemic threat in 1997, continues to be a significant risk. While vaccination is the preferred strategy for the prevention and control of influenza infections, the traditional egg-based approach to producing influenza vaccines does not provide sufficient capacity and adequate speed to satisfy global needs to combat newly emerging strains, seasonal or potentially pandemic. Significant efforts are underway to develop and implement new cell substrates with improved efficiency for influenza vaccine development and manufacturing. In recent years, plants have been used to produce recombinant proteins including subunit vaccines and antibodies. The main advantages of using plant systems for the production of vaccine antigens against influenza are their independence from pathogenic viruses, and cost and time efficiency. Here, we describe the large-scale production of recombinant hemagglutinin proteins from A/California/04/09 (H1N1) and A/Indonesia/05/05 (H5N1) strains of influenza virus in Nicotiana benthamiana plants, and their immunogenicity (serum hemagglutination inhibition and virus neutralizing antibodies), and safety in animal models. These results support the testing of these candidate vaccines in human volunteers and also the utility of our plant expression system for large-scale recombinant influenza vaccine production.

An influenza N1 neuraminidase-specific monoclonal antibody with broad neuraminidase inhibition activity against H5N1 HPAI viruses.

H5N1 avian influenza continues to be a potential pandemic threat. Several vaccine candidates based on potentially pandemic influenza strains and antiviral drugs have been tested in preclinical and clinical studies. The data obtained so far have shown some promise, but have also revealed some shortcomings with both of these approaches. We have identified and characterized an H5N1 neuraminidasespecific monoclonal antibody which specifically inhibits N1 neuraminidase activity of highly pathogenic avian influenza (HPAI) strains from clades 1 and 2. We have also shown the protective efficacy of this antibody in animal challenge models using homologous virus. Specific and effective inhibition of N1 NA could make this mAb a useful therapeutic tool in the treatment of human infection, in particular with oseltamivirand zanamivir-resistant strains of HPAI.

Antibodies to plant-produced Plasmodium falciparum sexual stage protein Pfs25 exhibit transmission blocking activity.

Malaria is a serious and sometimes fatal mosquito-borne disease caused by a protozoan parasite. Each year, it is estimated that over one million people are killed by malaria, yet the disease is preventable and treatable. Developing vaccines against the parasite is a critical component in the fight against malaria and these vaccines can target different stages of the pathogen's life cycle. We are targeting sexual stage proteins of P. falciparum which are found on the surface of the parasite reproductive cells present in the mosquito gut. Antibodies against these proteins block the progression of the parasite's life cycle in the mosquito, and thus block transmission to the next human host. Transmission blocking vaccines are essential to the malaria eradication program to ease the disease burden at the population level. We have successfully produced multiple versions of the Pfs25 antigen in a plant virus-based transient expression system and have evaluated these vaccine candidates in an animal model. The targets are expressed in plants at a high level, are soluble and most importantly, generate strong transmission blocking activity as determined by a standard membrane feeding assay. These data demonstrate the feasibility of expressing Plasmodium antigens in a plant-based system for the economic production of a transmission blocking vaccine against malaria.

Immunogenicity of an AAV-based, room-temperature stable, single dose COVID-19 vaccine in mice and non-human primates.

The SARS-CoV-2 pandemic has affected more than 70 million people worldwide and resulted in over 1.5 million deaths. A broad deployment of effective immunization campaigns to achieve population immunity at global scale will depend on the biological and logistical attributes of the vaccine. Here, two adeno-associated viral (AAV)-based vaccine candidates demonstrate potent immunogenicity in mouse and nonhuman primates following a single injection. Peak neutralizing antibody titers remain sustained at 5 months and are complemented by functional memory T-cells responses. The AAVrh32.33 capsid of the AAVCOVID vaccine is an engineered AAV to which no relevant pre-existing immunity exists in humans. Moreover, the vaccine is stable at room temperature for at least one month and is produced at high yields using established commercial manufacturing processes in the gene therapy industry. Thus, this methodology holds as a very promising single dose, thermostable vaccine platform well-suited to address emerging pathogens on a global scale.

An AAV-based, room-temperature-stable, single-dose COVID-19 vaccine provides durable immunogenicity and protection in non-human primates.

The SARS-CoV-2 pandemic has affected more than 185 million people worldwide resulting in over 4 million deaths. To contain the pandemic, there is a continued need for safe vaccines that provide durable protection at low and scalable doses and can be deployed easily. Here, AAVCOVID-1, an adeno-associated viral (AAV), spike-gene-based vaccine candidate demonstrates potent immunogenicity in mouse and non-human primates following a single injection and confers complete protection from SARS-CoV-2 challenge in macaques. Peak neutralizing antibody titers are sustained at 1 year and complemented by functional memory T cell responses. The AAVCOVID vector has no relevant pre-existing immunity in humans and does not elicit cross-reactivity to common AAVs used in gene therapy. Vector genome persistence and expression wanes following injection. The single low-dose requirement, high-yield manufacturability, and 1-month stability for storage at room temperature may make this technology well suited to support effective immunization campaigns for emerging pathogens on a global scale.