ABSTRACT
Introduction: Older age is an important risk factor for severe COVID-19 disease. Understanding the biological mechanisms that link aging to the pathogenesis of COVID-19 is essential for developing of therapeutic strategies. We hypothesized that cell senescence, a basic aging process that plays a pivotal role in lung diseases, is involved in the pathogenesis of COVID-19 including the development of long-lasting lung alterations. Methods: To evaluate the impact of SARS-CoV-2 infection on cell senescence, we 1) analyzed publicly available datasets of scRNA-seq performed in BALF cells from patients with moderate or severe/critical COVID-19;2) investigated lung samples from cynomolgus macaques infected with 106 pfu of a SARS-CoV-2 clinical isolate. Two macaques were sacrificed at 4 days post-infection (dpi.) and two others at 30 dpi. Results: In BALF obtained within 10 days after symptom onset, the expression of several senescence markers, i.e., CDKN2A, CDKN1A (encoding p21), uPAR, CXCL8, IGFBP3, and GDF15 was significantly increased in epithelial cells in BALF from patients with severe COVID-19, suggesting that lung-cell senescence induction was contemporary of viral detection. Next, we investigated macaques at 4 and 30 dpi, corresponding respectively to the viral load peak and to the absence of detectable viral RNA in BALF (1). Immunohistochemical analysis revealed numerous SARS-CoV-2 antigen-stained cells, also co-stained for senescence markers p16- and p21. The lungs at 30 dpi no longer contained the consolidated parenchymal areas seen at 4 dpi but showed extensive lung parenchyma remodelling, with thickening of the alveoli and pulmonary vessel walls and abundant extracellular matrix deposits as assessed by collagen staining. These lesions were accompanied with massive accumulation of p16- and p21-positive cells, mostly pneumocytes II and ECs. Of note, p16 staining of most ECs was seen in pulmonary vessels, notably those occluded by thrombosis and showing intraluminal vWF staining. Cells stained for p16 were also stained for the DNA damage markers γ-H2AX protein and p53-binding protein 1. Conclusions: Our data constitute the first evidence of temporal and topographic relations between senescent-cell accumulation and pulmonary lesions induced by SARS-CoV-2 infection.
ABSTRACT
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.
ABSTRACT
Background: A1 and HP serum levels are associated with, spread, severity and recovery of SI.1 Low A1 level measured 10 years prior to SI exposure, is also a clinical risk factor (fragility) for SI.2 In patients (Pts) it is difficult to assess the real number of days post infection (Dpi). The aim of this pilot study was to compare the A1 and HP dynamic changes during the first 4 weeks after SI in a nonhuman primate model, the macaque (NHP), that recapitulates mild COVID- 19 symptoms, in 2 ancillary experimental SI,3 vs Pts of an updated prospective observational study.1 Methods: NHP had 3-5 years of age, 2 female rhesus and 2 cynomolgus, and originating from Mauritian and Chinese AAALAC certified breeding centers, respectively, infected intranasally and intratracheally with 2×107 plaque-forming units of the clinical isolate hCoV-19/France/lDF0372/2020. Viral loads in nasopharyngeal, tracheal and rectal fluids were analyzed using an RT-qPCR assay, (Roche). Pts were those included in the observational study of SI in the GHPS hospital (intermediate severity 3to5 WHO grades), the Dpi being defined since the first symptom. A1 and HP were measured in all 283 Pts and in a subset of 58 with at least 3 repeated samples, a population (n=7482) representative of French population was used as Controls. Results: (Figure): In NHP, A1 had no significant changes from Dpi7 to Dpi26 (Panel A), contrarily to Pts who had lower values than Controls (blue box) (Repeated ANOVA Dunnets test) P<.001) before, during SI, and after recovery in all or repeated samples (Panel B). In NHP, HP was peaking at 7Dpi, close to the peak of nasal viral loads, still elevated at 20Dpi (Panel C);CRP in the same HP was peaking later at 13Dpi, and still high at 20Dpi.3 In Pts, HP changes were similar with a peak at 14Dpi and returned to normal values at 60Dpi (Panel D). Conclusion: These results validate in NHP the HP changes observed in hospitalized patients with intermediate severity SI. HP is an earlier marker of SI than CRP, and ApoA1 changes should be evaluated in NHP with shorter Dpi.