Journal: bioRxiv

Article Title: Cross-Protection Induced by Highly Conserved Human B, CD4 +, and CD8 + T Cell Epitopes-Based Coronavirus Vaccine Against Severe Infection, Disease, and Death Caused by Multiple SARS-CoV-2 Variants of Concern

doi: 10.1101/2023.05.24.541850

Figure Lengend Snippet: (A) Experimental plan showing screening process of COVID-19 patients ( n = 210) into Asymptomatic and Symptomatic categories based on clinical parameters. Blood and nasopharyngeal swabs were collected from all the subjects and a qRT-PCR assay was performed. Six novel nonsynonymous mutations (Δ69–70, Δ242–244, N501Y, E484K, L452R, and T478K) were used to identify the haplotypes unique to different SARS-CoV-2 variants of concern (Omicron (B.1.1.529 (BA.1)), Omicron (B.1.1.529 (BA.2)), Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Epsilon (B.1.427/B.1.429)) and variants of interest (Eta (B.1.525), R.1, Zeta (P.2), Iota (B.1.526) and B.1.2/501Y or B.1.1.165). (B) ELISpot images and bar diagrams showing average frequencies of IFN-γ producing cell spots from immune cells from PBMCs (1 × 10 6 cells per well) of COVID-19 infected with highly pathogenic SARS-CoV-2 variants of concern Beta (B.1.351) ( left panel ) and Omicron (B.1.1.529) ( right panel ). Cells were stimulated for 48 hours with 10mM of 16 immunodominant CD8 + T cell peptides derived from SARS-CoV-2 structural (Spike, Envelope, Membrane) and nonstructural (orf1ab, ORF6, ORF7b, ORF8a, ORF10) proteins. (C) ELISpot images and bar diagrams showing average frequencies of IFN-γ producing cell spots from immune cells from PBMCs (1 × 10 6 cells per well) of COVID-19 infected with SARS-CoV-2 variants of concern Alpha (B.1.1.7) ( left panel ) and Omicron (B.1.1.529) ( right panel ). Cells were stimulated for 48 hours with 10mM of 6 immunodominant CD4 + T cell peptides derived from SARS-CoV-2 structural (Spike, Membrane, Nucleocapsid) and nonstructural (ORF1a, ORF6, ORF8a) proteins. The bar diagrams show the average/mean numbers (± SD) of IFN-γ-spot forming cells (SFCs) after CD8 + T cell peptide-stimulation PBMCs of Asymptomatic and Symptomatic COVID-19 patients. Dotted lines represent an arbitrary threshold set to evaluate the relative magnitude of the response. A strong response is defined for mean SFCs > 25 per 1 × 10 6 stimulated PBMCs. Results were considered statistically significant at P < 0.05.

Article Snippet: The blocking solution was removed and 40μl/well of anti-SARS Nucleocapsid antibody (Novus Biologicals NB100–56576) at 1:1000 in 5% non-fat dry milk/PBS was added.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunospot, Infection, Derivative Assay, Membrane

Journal: bioRxiv

Article Title: Cross-Protection Induced by Highly Conserved Human B, CD4 +, and CD8 + T Cell Epitopes-Based Coronavirus Vaccine Against Severe Infection, Disease, and Death Caused by Multiple SARS-CoV-2 Variants of Concern

doi: 10.1101/2023.05.24.541850

Figure Lengend Snippet: (A) Experimental scheme of vaccination and challenge triple transgenic HLA-A*02:01/HLA-DRB1*01:01-hACE-2 mice. Triple transgenic HLA-A*02:01/HLA-DRB1*01:01-hACE-2 mice (7–8-week-old, n = 60) were immunized subcutaneously on Days 0 and 14 with a multi-epitope pan-Coronavirus vaccine consisting of a pool of conserved B, CD4 + T cell and CD8 + T cell human epitope peptides. The pool of peptides comprised 25μg of each of the 16 CD8 + T cell peptides, 6 CD4 + T cell peptides, and 7 B-cell peptides. The final composition of peptides was mixed with 25μg of CpG and 25μg of Alum. Mock-vaccinated mice were used as controls ( Mock ). Fourteen days following the second immunization, mice were intranasally challenged with each of the six different SARS-CoV-2 variants of concern (WA/USA2020, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529)). Vaccinated and mock-vaccinated mice were followed 14 days post-challenge for COVID-like symptoms, weight loss, survival, and virus replication. (B) Percent weight change recorded daily for 14 days p.i. in vaccinated and mock-vaccinated mice following the challenge with each of the six different SARS-CoV-2 variants. (C) Kaplan-Meir survival plots for vaccinated and mock-vaccinated mice following the challenge with each of the six different SARS-CoV-2 variants. (D) Virus replication in vaccinated and mock-vaccinated mice following the challenge with each of the six different SARS-CoV-2 variants detected in throat swabs on Days 2, 4, 6, 8, 10, and 14, The indicated P values are calculated using the unpaired t -test, comparing results obtained in vaccinated VERSUS mock-vaccinated mice.

Article Snippet: The blocking solution was removed and 40μl/well of anti-SARS Nucleocapsid antibody (Novus Biologicals NB100–56576) at 1:1000 in 5% non-fat dry milk/PBS was added.

Techniques: Transgenic Assay, Virus

Journal: bioRxiv

Article Title: Cross-Protection Induced by Highly Conserved Human B, CD4 +, and CD8 + T Cell Epitopes-Based Coronavirus Vaccine Against Severe Infection, Disease, and Death Caused by Multiple SARS-CoV-2 Variants of Concern

doi: 10.1101/2023.05.24.541850

Figure Lengend Snippet: (A) Representative images of hematoxylin and Eosin (H & E) staining of the lungs harvested on day 14 p.i. from vaccinated ( left panels ) and mock-vaccinated ( right panels ) mice. (B) Representative immunohistochemistry (IHC) sections of the lungs were harvested on Day 14 p.i. from vaccinated ( left panels ) and mock-vaccinated ( right panels ) mice and stained with SARS-CoV-2 Nucleocapsid antibody. Black arrows point to the antibody staining. Fluorescence microscopy images showing infiltration of CD8 + T cells (C) and of CD4 + T cells (D) in the lungs from vaccinated ( left panels ) and mock-vaccinated ( right panels ) mice. Lung sections were co-stained using DAPI ( blue ) and mAb specific to CD8 + T cells ( Pink ) (magnification, 20x). The white arrows point to CD8 + and CD4 + T cells infiltrating the infected lungs.

Article Snippet: The blocking solution was removed and 40μl/well of anti-SARS Nucleocapsid antibody (Novus Biologicals NB100–56576) at 1:1000 in 5% non-fat dry milk/PBS was added.

Techniques: Staining, Immunohistochemistry, Fluorescence, Microscopy, Infection

Journal: bioRxiv

Article Title: Cross-Protection Induced by Highly Conserved Human B, CD4 +, and CD8 + T Cell Epitopes-Based Coronavirus Vaccine Against Severe Infection, Disease, and Death Caused by Multiple SARS-CoV-2 Variants of Concern

doi: 10.1101/2023.05.24.541850

Figure Lengend Snippet: FACS plots and bar graphs showing the (A) expression of CD8 + T cell function markers, (B) CD4 + T cell function associated markers, (C) CD8 + T effector memory response (CD44 + CD62L − ), and CD8 + T resident memory (CD103 + CD69 + ) response, and (D) CD4 + T effector memory response (CD44 + CD62L − ), and CD4 + resident memory (CD103 + CD69 + ) response in the lung of vaccinated and mock-vaccinated groups of mice infected with multiple SARS-CoV-2 variants. Bars represent means ± SEM. Data were analyzed by student’s t -test. Results were considered statistically significant at P < 0.05.

Article Snippet: The blocking solution was removed and 40μl/well of anti-SARS Nucleocapsid antibody (Novus Biologicals NB100–56576) at 1:1000 in 5% non-fat dry milk/PBS was added.

Techniques: Expressing, Cell Function Assay, Infection

Journal: bioRxiv

Article Title: Cross-Protection Induced by Highly Conserved Human B, CD4 +, and CD8 + T Cell Epitopes-Based Coronavirus Vaccine Against Severe Infection, Disease, and Death Caused by Multiple SARS-CoV-2 Variants of Concern

doi: 10.1101/2023.05.24.541850

Figure Lengend Snippet: ELISpot images and bar diagrams showing average frequencies of IFN-γ producing cell spots from mononuclear cells from lung tissue (1 × 10 6 cells per well) of vaccinated and mock-vaccinated mice challenged with ( A ) WA/USA2020, ( B ) Alpha (B.1.1.7), ( C ) Beta (B.1.351), ( D ) Gamma (P.1), ( E ) Delta (B.1.617.2), and ( F ) Omicron (B.1.1.529). Cells were stimulated for 48 hours with 10mM of 6 immunodominant CD4 + T cell peptides derived from SARS-CoV-2 structural (Spike, Envelope, Membrane) and nonstructural (orf1ab, ORF6, ORF7b, ORF8a, ORF10) proteins. The bar diagrams show the average/mean numbers (± SD) of IFN-γ-spot forming cells (SFCs) after CD8 + T cell peptide stimulation in lung tissues of vaccinated and mock-vaccinated mice. The dotted lines represent an arbitrary threshold set to evaluate the relative magnitude of the response. A strong response is defined for mean SFCs > 25 per 1 × 10 6 stimulated PBMCs. Results were considered statistically significant at P ≤ 0.05.

Article Snippet: The blocking solution was removed and 40μl/well of anti-SARS Nucleocapsid antibody (Novus Biologicals NB100–56576) at 1:1000 in 5% non-fat dry milk/PBS was added.

Techniques: Enzyme-linked Immunospot, Derivative Assay, Membrane

Journal: bioRxiv

Article Title: Diet Induced Obesity and Diabetes Enhance Mortality and Reduces Vaccine Efficacy for SARS-CoV-2

doi: 10.1101/2022.10.15.512291

Figure Lengend Snippet: A . Schematic overview illustrating diet schedule and infection for control and obese/diabetic mice. Briefly, at weaning (3 weeks), mice were placed on a normal diet (ND) or high fat diet (HFD). At 18 weeks of age, these mice were infected with either 1×10 3 or 1×10 5 pfu of SARS-CoV-2 Mouse Adaptive 10, monitored daily for weight changes, and a subset was euthanized at day 2, 4, and 7 to analyze lung viral titer and host immune response. The B . fasted blood glucose concentration and C . fasted serum insulin were determined prior to infection n=13 mice/group. These values were used to assess D . insulin resistance by HOMA-IR score {HOMA-IR = [26 * serum insulin (ng/ml) * blood glucose (mg/dL)]/405}. The E . starting weight of ND and HFD mice prior to infection n=45 mice/group. * p <0.5 and **** p < 0.0001 as determined by an unpaired two-sided t-test. The data are presented as mean ± SD.

Article Snippet: Endogenous peroxidase activity was blocked with DAKO Peroxidase-Blocking Reagent for 5 min before incubation with rabbit polyclonal SARS Nucleocapsid Protein Antibody [NB100-56576] (Novus Biologicals, Centennial, CO), 1:400 for 20 minutes at room temperature, followed by DAKO Anti-rabbit HRP Detection Reagent for 20 minutes.

Techniques: Infection, Control, Concentration Assay

Journal: bioRxiv

Article Title: Diet Induced Obesity and Diabetes Enhance Mortality and Reduces Vaccine Efficacy for SARS-CoV-2

doi: 10.1101/2022.10.15.512291

Figure Lengend Snippet: The A . survival, percent weight loss for B . low dose, C . high dose, grams lost for D . low dose, and E . high dose. D . Viral lung titers were assessed for control and obese/ diabetic mice throughout a SARS-CoV-2 MA10 infection. For A . * p < 0.05 as determined by a Log-rank (Mantel-Cox) test for survival analysis between ND and HFD for low and high infection dose with the data presented as mean ± SD with n=10 mice/group. For B-D the data are presented as mean ± SD with n =3-5 mice/group.

Article Snippet: Endogenous peroxidase activity was blocked with DAKO Peroxidase-Blocking Reagent for 5 min before incubation with rabbit polyclonal SARS Nucleocapsid Protein Antibody [NB100-56576] (Novus Biologicals, Centennial, CO), 1:400 for 20 minutes at room temperature, followed by DAKO Anti-rabbit HRP Detection Reagent for 20 minutes.

Techniques: Control, Infection

Journal: bioRxiv

Article Title: Diet Induced Obesity and Diabetes Enhance Mortality and Reduces Vaccine Efficacy for SARS-CoV-2

doi: 10.1101/2022.10.15.512291

Figure Lengend Snippet: Control and obese/diabetic mice were infected intranasally with either 1×10 3 or 1×10 5 pfu/ mouse. Lungs were collected on 2, 4, and 7 dpi and fixed in 10% neutral buffered formalin for greater than 24 hours. Tissue was embedded in paraffin. A . 5 µm sections were cut and stained with hematoxylin and eosin, and B . immunostained with anti-SARS Nucleocapsid Protein antibody. Images are shown at original magnification x10 and are representative of n = 3-5 mice/group.

Article Snippet: Endogenous peroxidase activity was blocked with DAKO Peroxidase-Blocking Reagent for 5 min before incubation with rabbit polyclonal SARS Nucleocapsid Protein Antibody [NB100-56576] (Novus Biologicals, Centennial, CO), 1:400 for 20 minutes at room temperature, followed by DAKO Anti-rabbit HRP Detection Reagent for 20 minutes.

Techniques: Control, Infection, Staining

Journal: bioRxiv

Article Title: Diet Induced Obesity and Diabetes Enhance Mortality and Reduces Vaccine Efficacy for SARS-CoV-2

doi: 10.1101/2022.10.15.512291

Figure Lengend Snippet: After SARS-2 MA10 challenge, lungs were harvested, cells isolated, and stained for surface markers for A . Natural Killer cells, B . Total T cells, C . T helper cells, D . Cytotoxic T cells, and E . B cells at day 2 and 4 post-challenge. * p < 0.05 as data were log-transformed and analyzed by mixed-effect analysis followed by Tukey for multiple comparisons n =3-5 mice/group

Article Snippet: Endogenous peroxidase activity was blocked with DAKO Peroxidase-Blocking Reagent for 5 min before incubation with rabbit polyclonal SARS Nucleocapsid Protein Antibody [NB100-56576] (Novus Biologicals, Centennial, CO), 1:400 for 20 minutes at room temperature, followed by DAKO Anti-rabbit HRP Detection Reagent for 20 minutes.

Techniques: Isolation, Staining, Transformation Assay

Journal: bioRxiv

Article Title: Diet Induced Obesity and Diabetes Enhance Mortality and Reduces Vaccine Efficacy for SARS-CoV-2

doi: 10.1101/2022.10.15.512291

Figure Lengend Snippet: A . Schematic overview illustrating vaccination and challenge for ND and HFD mice. Briefly, at 18 weeks of age, mice were vaccinated as well as 14 days later. On day 28 post-vaccination, mice were bled to determine B . serum neutralizing antibodies prior to infection, n = 12-15 mice/group. On day 32, these mice were challenged with 1×10 5 pfu of SARS-CoV-2 MA 10, monitored daily for weight changes, and a subset was euthanized on day 2 and 4 to analyze lung viral titer and host immune response. The C . mortality, weight loss of D . ND, E . HFD, and F . viral titer of sham and vaccinated mice after SARS-2 MA10 challenge. n= 5-10 mice/group. For D , * p <0.5 and *** p < 0.001 as determined by a mixed-effect analysis followed by a Sidak test for multiple comparisons. For F . p *** < 0.001 and p **** < 0.0001, data were log-transformed and analyzed by mixed-effect analysis followed by Sidik for multiple comparisons. The data are pooled from 2 independent experiments and presented as mean ± SD.

Article Snippet: Endogenous peroxidase activity was blocked with DAKO Peroxidase-Blocking Reagent for 5 min before incubation with rabbit polyclonal SARS Nucleocapsid Protein Antibody [NB100-56576] (Novus Biologicals, Centennial, CO), 1:400 for 20 minutes at room temperature, followed by DAKO Anti-rabbit HRP Detection Reagent for 20 minutes.

Techniques: Infection, Transformation Assay

Journal: bioRxiv

Article Title: Diet Induced Obesity and Diabetes Enhance Mortality and Reduces Vaccine Efficacy for SARS-CoV-2

doi: 10.1101/2022.10.15.512291

Figure Lengend Snippet: After SARS-2 MA10 challenge, lungs were harvested, cells isolated, and stained for surface markers for A . neutrophils, B . CD11b + DCs, C . monocyte-derived DCs, D .CD103 + cDCs, E . plasmacytoid DCs at day 2 and 4 post-challenge. * p < 0.05 and ** p < 0.01 as data were log-transformed and analyzed by mixed-effect analysis followed by Tukey for multiple comparisons n =3-5 mice/group.

Article Snippet: Endogenous peroxidase activity was blocked with DAKO Peroxidase-Blocking Reagent for 5 min before incubation with rabbit polyclonal SARS Nucleocapsid Protein Antibody [NB100-56576] (Novus Biologicals, Centennial, CO), 1:400 for 20 minutes at room temperature, followed by DAKO Anti-rabbit HRP Detection Reagent for 20 minutes.

Techniques: Isolation, Staining, Derivative Assay, Transformation Assay

Journal: Vaccines

Article Title: Geographical Differences in SARS-CoV-2 Antibody Response Dynamics and Neutralisation Profiles to Mild COVID-19: Lessons from a UK-Uganda Comparison.

doi: 10.3390/vaccines13040336

Figure Lengend Snippet: Figure 4. Correlations between neutralisation titres and SARS-CoV-2 D614G spike, RBD, and nu- cleocapsid IgG binding antibody OD responses stratified by nationality. Figure 4 illustrates the correlations between the SARS-CoV-2 D614G neutralisation titres and IgG optical density (OD) responses targeting spike (S), receptor-binding domain (RBD), and nucleocapsid (N) antigens, strati- fied by the UK and Uganda populations. Scatter plots and correlation coefficients demonstrate the strength and direction of associations for each antigen-specific response across the two populations. The analysis reveals distinct patterns in the relationship between neutralisation titres and D614G antigen-specific IgG responses by nationality.

Article Snippet: ELISA plates (Greiner Bio-One, #655001, Kremsmünster, Austria) were coated with 3 μg/mL of spike or nucleocapsid antigen (R&D Systems, #10474-CV-01M, #10474-CV-01M, Minneapolis, MN, USA), optimised to achieve maximum sensitivity and specificity.

Techniques: Binding Assay