Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models

The ongoing SARS-CoV-2 pandemic is controlled but not halted by public health measures and mass vaccination strategies which have exclusively relied on intramuscular vaccines. Intranasal vaccines can prime or recruit to the respiratory epithelium mucosal immune cells capable of preventing infection. Here we report a comprehensive series of studies on this concept using various mouse models, including HLA class II-humanized transgenic strains. We found that a single intranasal (i.n.) dose of serotype-5 adenoviral vectors expressing either the receptor binding domain (Ad5-RBD) or the complete ectodomain (Ad5-S) of the SARS-CoV-2 spike protein was effective in inducing i) serum and bronchoalveolar lavage (BAL) anti-spike IgA and IgG, ii) robust SARS-CoV-2-neutralizing activity in the serum and BAL, iii) rigorous spike-directed T helper 1 cell/cytotoxic T cell immunity, and iv) protection of mice from a challenge with the SARS-CoV-2 beta variant. Intramuscular (i.m.) Ad5-RBD or Ad5-S administration did not induce serum or BAL IgA, and resulted in lower neutralizing titers in the serum. Moreover, prior immunity induced by an intramuscular mRNA vaccine could be potently enhanced and modulated towards a mucosal IgA response by an i.n. Ad5-S booster. Notably, Ad5 DNA was found in the liver or spleen after i.m. but not i.n. administration, indicating a lack of systemic spread of the vaccine vector, which has been associated with a risk of thrombotic thrombocytopenia. Unlike in otherwise genetically identical HLA-DQ6 mice, in HLA-DQ8 mice Ad5-RBD vaccine was inferior to Ad5-S, suggesting that the RBD fragment does not contain a sufficient collection of helper-T cell epitopes to constitute an optimal vaccine antigen. Our data add to previous promising preclinical results on intranasal SARS-CoV-2 vaccination and support the potential of this approach to elicit mucosal immunity for preventing transmission of SARS-CoV-2

Quantification of myocardial infarct area based on TRAFFn relaxation time maps:comparison with cardiovascular magnetic resonance late gadolinium enhancement, T1ρ and T2 in vivo

Abstract Background: Two days after myocardial infarction (MI), the infarct consists mostly on necrotic tissue, and the myocardium is transformed through granulation tissue to scar in two weeks after the onset of ischemia in mice. In the current work, we determined and optimized cardiovascular magnetic resonance (CMR) methods for the detection of MI size during the scar formation without contrast agents in mice. Methods: We characterized MI and remote areas with rotating frame relaxation time mapping including relaxation along fictitious field in nth rotating frame (RAFFn), T1ρ and T2 relaxation time mappings at 1, 3, 7, and 21 days after MI. These results were compared to late gadolinium enhancement (LGE) and Sirius Red-stained histology sections, which were obtained at day 21 after MI. Results: All relaxation time maps showed significant differences in relaxation time between the MI and remote area. Areas of increased signal intensities after gadolinium injection and areas with increased TRAFF2 relaxation time were highly correlated with the MI area determined from Sirius Red-stained histology sections (LGE: R² = 0.92, P < 0.01, TRAFF2: R² = 0.95, P < 0.001). Infarct area determined based on T1ρ relaxation time correlated highly with Sirius Red histology sections (R² = 0.97, P < 0.01). The smallest overestimation of the LGE-defined MI area was obtained for TRAFF2 (5.6 ± 4.2%) while for T1ρ overestimation percentage was > 9% depending on T1ρ pulse power. Conclusion: T1ρ and TRAFF2 relaxation time maps can be used to determine accurately MI area at various time points in the mouse heart. Determination of MI size based on TRAFF2 relaxation time maps could be performed without contrast agents, unlike LGE, and with lower specific absorption rate compared to on-resonance T1ρ relaxation time mapping

Lymphatic insufficiency leads to distinct myocardial infarct content assessed by magnetic resonance TRAFFn, T1ρ and T₂ relaxation times

Abstract The role of cardiac lymphatics in the pathogenesis of myocardial infarction (MI) is unclear. Lymphatic system regulates cardiac physiological processes such as edema and tissue fluid balance, which affect MI pathogenesis. Recently, MI and fibrosis have been assessed using endogenous contrast in magnetic resonance imaging (MRI) based on the relaxation along a fictitious field with rank n (RAFFn). We extended the RAFFn applications to evaluate the effects of lymphatic insufficiency on MI with comparison to longitudinal rotating frame (T1ρ) and T₂ relaxation times. MI was induced in transgenic (TG) mice expressing soluble decoy VEGF receptor 3 that reduces lymphatic vessel formation and their wild-type (WT) control littermates for comparison. The RAFFn relaxation times with rank 2 (TRAFF2), and rank 4 (TRAFF4), T1ρ and T₂ were acquired at time points 0, 3, 7, 21 and 42 days after the MI at 9.4 T. Infarct sizes were determined based on TRAFF2, TRAFF4, T1ρ and T₂ relaxation time maps. The area of differences (AOD) was calculated based on the MI areas determined on T₂ and TRAFF2, TRAFF4 or T1ρ relaxation time maps. Hematoxylin–eosin and Sirius red stained histology sections were prepared to confirm MI locations and sizes. MI was detected as increased TRAFF2, TRAFF4, T1ρ and T₂ relaxation times. Infarct sizes were similar on all relaxation time maps during the experimental period. Significantly larger AOD values were found together with increased AOD values in the TG group compared to the WT group. Histology confirmed these findings. The lymphatic deficiency was found to increase cardiac edema in MI. The combination of TRAFF2 (or TRAFF4) and T₂ characterizes MI and edema in the myocardium in both lymphatic insufficiency and normal mice without any contrast agents

Epigenetic Upregulation of Endogenous VEGF-A Reduces Myocardial Infarct Size in Mice

<div><p>“Epigenetherapy” alters epigenetic status of the targeted chromatin and modifies expression of the endogenous therapeutic gene. In this study we used lentiviral <i>in vivo</i> delivery of small hairpin RNA (shRNA) into hearts in a murine infarction model. shRNA complementary to the promoter of vascular endothelial growth factor (VEGF-A) was able to upregulate endogenous VEGF-A expression. Histological and multiphoton microscope analysis confirmed the therapeutic effect in the transduced hearts. Magnetic resonance imaging (MRI) showed <i>in vivo</i> that the infarct size was significantly reduced in the treatment group 14 days after the epigenetherapy. Importantly, we show that promoter-targeted shRNA upregulates all isoforms of endogenous VEGF-A and that an intact hairpin structure is required for the shRNA activity. In conclusion, regulation of gene expression at the promoter level is a promising new treatment strategy for myocardial infarction and also potentially useful for the upregulation of other endogenous genes.</p></div

ELISA assay of myocardial infarction samples and analysis for single-stranded vectors for a mechanistical view.

<p>(a) ELISA analysis of VEGF-A protein from transduced hearts, (b) ELISA assay from growth medium of C166 cells transduced with LV-451 and corresponding single stranded vectors using MOI 10, 7 days time point. (c) RT-PCR analysis of VEGF-A mRNA levels. C166 cells were transduced with LV-451 and corresponding single stranded vectors using MOI 10, 11 days time point. (d) qChIP assay of C166 cells using antibodies against H3K4me2. Cells were transduced with LV-451 and corresponding single stranded vectors using MOI 10, 11 days timepoint. All results are shown as mean ± SD.</p

Intracellular distribution of LV-451 expressed RNA and VEGF-A mRNA in transduced cells.

<p>C166 cells were subjected to RNA-FISH analysis with LV-451 or VEGF mRNA probes. (a) Confocal microscopy images of LV-451 transduced (MOI 10) cells 72 h post transduction. Distribution of LV-451 RNA (green) and VEGF-A mRNA (red) probe binding induced signals is shown. Nuclei were visualized with DAPI (grey). Scale bars, 5 µm. (b) Quantification of LV-451 RNA or VEGF-A mRNA RNA-FISH signal spots detected in LV-451 transduced (MOI 4, 40, 200) cells at 72 h post transduction and in nontransduced control cells. The amount of signal was calculated in the nucleus (white), the cytosol (grey) and whole cell (black). Error bars = SD. (c) Nucleus size in response to LV transduction. CTRL sample is nontransduced C166 cells and LV-451 is C166 cells transduced with LV-451 vector.</p

Multiphoton microscopy and histology analysis of myocardial infarction animals.

<p>(a) Multiphoton laser scanning microscopy (MPLSM) analysis of GFP expression in transduced mouse heart, (b) Immunohistological analysis of GFP expression in mouse heart, (c) antibody omitted control, (d and k) Massons Trichrome staining from mouse heart transduced with VEGF-A upregulating LV-451 and shRNA control, respectively, (e and l) insert from infarcted area of d and k, respectively, (h and o) insert from infarct borderzone (f, i, m, p) alpha-SMA staining of smooth muscle cells, arrows point to arteriols formed, (g, j, n, q) CD-31 staining of endothelial cells. Scale bars (a) 100 µm, (d and k) 2000 µm, (e, f, g, h, i, j, l, m, n, o, p, q) 200 µm.</p

Analysis for mechanism of action for promoter targeted shRNAs.

<p>(a) RT-PCR analysis for different VEGF-A isoforms. The expression levels for different isoforms were studied using primers specific to each isoform. Total VEGF-A protein level was measured with ELISA. (b) Reversing DNA methylation with 5-Azacytidine treatment induces responses in MS1 cells but erases responses in C166 cells. Cells were treated with 1 µM 5-Azacytidine, transduced with different vectors on day 3 and samples were collected on day 8. qRT-PCR analysis of VEGF-A and B-actin mRNA levels in MS1 cells and C166 cells. (c) qChIP assay in MS1 cells using antibody against H3K27me3. (d) The VEGF-A gene promoter in C166 cells was also analyzed for basal DNA methylation levels without 5-Azacytidine treatment using MeDIP. Cells were transduced with different vectors using MOI 10, 10 days timepoint. (e) RT-PCR analysis of VEGF-A mRNA levels after C166 cells were transfected with siRNA oligos. Results are calculated in reference to housekeeping gene ACTB and control oligo. (f) CBP-CREB interaction inhibitor (7.5 µM) abolishes the upregulation of VEGF-A by LV-451 in C166 cells. For all results, mean ± SD shown.</p