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Background: The SARS-CoV-2 pandemic has affected more than 250 million people worldwide resulting in 5 million deaths. To contain the pandemic, there is a continued need for safe vaccines that provide durable protection at low and scalable doses that easily delivered. Previously, we showed that an adeno-associated virus (AAV)-based vaccine candidate (AC1) elicited high humoral and cellular immunogenicity in mice and nonhuman primates (NHP) following a single injection, which provided near-sterilizing immunity against SARS-CoV-2 in NHP. Here, we developed optimized AAVCOVID vaccine candidates for higher potency and protection against variants of concern (VOC). Methods: The promoter in AC1 vector was substituted by three different promoters to increase the expression of Spike and they were tested in mice by single IM injection. Transgene expression and anti-Spike antibody and cellular responses were determined to assess vector potency. Then, the candidate that showed higher potency (ACM1) was engineered to express the Beta (ACM-Beta) and Delta (ACM-Delta) VOC Spike. The immunogenicity provided by ACM-Beta and ACM-Delta was characterized in mice and NHP. The cross-reactivity with the Wuhan and VOC Spikes was also assessed in the animals immunized with different Spike variants. Finally, challenge and durability studies were performed in NHPs vaccinated with the new candidates. Results: Vaccination with ACM1 candidate (miniCMV promoter) resulted in 100-fold higher Spike expression and 40-fold higher antibody responses compared to the prototypic AC1 candidate in mice. When ACM1, ACM-Beta and ACM-Delta were compared in mice, we found that the immune responses against the self-transgene were not significantly different. However, cross-reactivity was different, being ACM-Delta the candidate that better cross-neutralized the different VOC. Similar results were observed in NHP: higher potency of the candidates carrying the miniCMV promoter and similar cross-reactivity profiles. Additionally, ACM-Beta showed protection against Beta SARS-CoV-2 challenge and a durability study for ACM-Delta is ongoing. Conclusion: This work shows the adaptability and versatility of AAVCOVID vaccine platform to improve potency and protect against VOC. These observations together with 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.
RESUMO
Purpose : To assess toxicity and efficacy of subretinal gene replacement in BBS10 mice. Overexpression toxicity in BBS1 mice occurred with gene therapy;BBS1 is part of the BBSome, while BBS10 is part of the BBS/CCT chaperonin complex. Methods : A knock out mouse model of Bardet Biedl Syndrome type 10 (BBS10) was developed. AAV2/5-Bbs10FLAG and AAV2/Anc80-Bbs10FLAG vectors were created. Subretinal 2 ul injections of 1E12, 2E12 or 4E12VG/ml were performed in 62 Bbs10-/-and 12 WT mice. Immunoblotting and immunohistochemistry were utilized to assess protein production and restoration of Bbs10 gene function. ERG, OCT, and visually guided swim assay (VGSA) were used to assess efficacy. Due to COVID-19, long term data was collected only for 12 mice treated with 2E9 or 4E9 AAV2/Anc80-Bbs10FLAG and 3 controls. Results : Neither AAV2/5-Bbs10FLAG nor AAV2/Anc80-Bbs10FLAG were toxic in WT or Bbs10-/-. One month after injection, FLAG was detected in treated, but not untreated Bbs10-/-eyes, documenting presence of BBS10 protein.BBS7,undetectable or barely detectable within photoreceptor cilia in untreated Bbs10 eyes,was present in photoreceptor cilia in treated Bbs10 eyes.VGSA was partially rescued via either AAV2/5 or AAV2/Anc80 treated at P30-P60 when tested in the dark at age 3.5 months(p= 1.36E005)and in both light and dark at age 7 months (p=. <0.0001 light;p= 0.0094 dark). VGSA improvement endured in AAV2/Anc80 treated mice at 9-12 months old(p=0.0113). Treated eyes had higher amplitude ERG than untreated fellow eyes at 10-11.5 months old (highest p=0.0425). OCT at 11-14 months demonstrated presence of outer nuclear layer in 5/11 treated eyes compared to 0/11 untreated fellow eyes and 0/6 untreated control eyes. 5Hz flicker response was not present at any age in untreated Bbs10 (n=18 eyes), but developed in 9 of 12 eyes treated before 4 months old. 4 of 12 treated eyes still had recordable 5 Hz at 11-14 mos, all treated with 4E9. Histology at 11 months demonstrated robust ONL, inner and outer segments with numerous cones, and normal localization of STX3 adjacent to the injection site in 2 treated eyes. Conclusions : In the Bbs10 mouse subretinal gene therapy with AAV2/Anc80-Bbs10FLAG rescues retinal phenotype . Lack of 5 Hz flicker ERG response in untreated eyes was rescued with early high titer gene therapy suggesting that BBS10 plays an early role in cone development and/or function. Human BBS10 clinical trials are needed.