Project title

Biomaterials enhance the immune response against the covid-19

antigens, display and processed by denteric cell (DC) for
.immune cells activation/proliferation in vitro/ex vivo

Background

Professional antigen-presenting cells (APC) of the immune

system such as DC, macrophages, B lymphocytes are responsible for
foreign antigen capture and processing to achieve immune defence
against these agents. However, denteric cells (DC) as part f innate
immunity and the first line of the immune response, play an important
role in processing and encounter pathogens ( e.g viruses, bacteria,
parasites ) in specializing different ways to activate T lymphocytes. The
recognition of pathogens depends on Toll-like receptors (TLRs) pathogen
recognition receptors (PRRs) on DCs and the pathogen-associated
molecular patterns on microbes(PAMPs).

A coronavirus (CoV) commonly known as SARS-CoV-2 (severe acute

respiratory syndrome coronavirus 2) and causing COVID-19 (coronavirus
disease of 2019) has become a pandemic following an outbreak in
Wuhan. Furthermore, the binding of the virus to the mannose receptor via
lipopolysaccharides (LPSs), toll-like receptors via LPS/proteins/RNA,
and sialic acid (Sia) via hemagglutinin, or sugar-acid segments of
glycans. HA-to-Sia binding is considered based on the innate Sia N-
acetylneuraminic acid and the acquired Sia N-glycolylneuraminic acid in
the epithelial cells and the sialidase/neuraminidase- or esterase-
hydrolyzed release and transmission of CoVs.

The RNA of the covid-19 virus are recognized by various pathogen-

associated molecular patterns (PAMPs) in the cell membrane, endosomes,
and cytoplasm. This recognition continues throughout the viral lifecycle
in a host cell. so the enveloped nature of SARS-CoV-2 leads to its
preferential endocytic membrane fusion through the recruitment of
clathrin/caveolin.

The Effective vaccination can be developed with a new strategic

design for obtaining high titer antibodies with a strong memory cell
response. Moreover, for preventing the outbreak spreading of pandemic
infection. on the other hand, with absolute healthy subjects, we need a
great immunity response against the infectious covid-19 virus.

The term biomaterial substance can be artificial or natural

substrates, such as synthetic polymer, lipids, peptides or other
macromolecules. recently, the nanoparticle can be added to antigen as
target molecules for antigen-presenting cells or DCs for immune
recognition. Although Inorganic biomaterials have been proposed such as
gold nanoparticles (AuNPs) because easily internalized via both denteric
cells and macrophages.

The glycoprotein of the covid-19 virus is a proteinaceous peplomer spike

structure. the membrane-binding proteins are β-spirals, similar to those in
SGP of SARS-CoV-2, or the coiled-coil α-helix, such as those in HA.
Nanoparticles can be displaying the viral antigen to host immune cells
such as the small nanovesicles expressing MERS virus S protein on its
surface, mimicking MERS-CoV pathogen. Recombinant viral proteins,
namely, Spike (S), Envelope (E), and membrane (M) proteins, were
transfected into Bm5 cells, and S-protein displaying nanovesicles were
obtained by both surfactant treatment as well as mechanical extrusion
method.

The aim of the study

We can be targeting the PRR of DCs in vitro/ex vivo by adding
biomaterial for covid-19 glycoproteins such as HA/LPS to optimize the
magnitude of the immune response for both humoral and cellular
immunity. A strong T-cell mediated or mucosal is important for effective
vaccination. This approach enables a more comprehensive phenotypic
profile of circulating DCs in the blood ( in vitro )and ex vivo detection of
antigen-specific of DCs subsets population.

The PAMPs of covid-19 are classified to surface protein and genetic

material leading to activation of signals pathways such as TLRs( 4,7,8,9),
CLR (c-type lectin receptors), NLR( nucleotide-binding receptor), RLR
(retinoic acid-inducible gene like receptors).

We can combine the antigen of interest and the biomaterial such as

nucleic acid nanoparticles complex as vehicles to direct the APC for T -
lymphocytes activation and proliferation. And understanding of the host
immunological factors.

Haemoaglutinin-esterase (HE), is a lectin with ligand-binding domains 1

and 2, is common to CoVs. The binding of a pathogen to a Sia, such as
the HA-based binding of CoVs to N-acetylneuraminic acid (Neu5Ac), is
the first step of infection, and viral replication. While the synthetic
nanomaterial can interact with cell membrane protein effectively.

The most common elements of biomaterials are liposomes, solid

polymers, and natural polymers. or gold and silica nanoparticles which
can be loaded with the virus antigen and engulfed by DCs and
macrophages to enhance anti-inflammatory phenotypes with therapeutic
options.

We can use engineered biomaterials for antigen delivery to enhance the

immunomodulation and also tissue repair after covid-19 infection and
immune response dysregulation.

The severe infection states for COVID-19 initiate by type I interferon in

response to plasmacytoid denteric cells (pDCs) and T & B plasma cells
lymphocytes, with activation of alveolar macrophages which activated
cytokines storms and inflammatory leukocytes.

There are generally two classes of pulmonary DCs responsible for antigen
presentation CD 103+, CD11b+ which are processed the SARS-COVID
antigen after migrating to lymph nodes

Until now, the mechanism of SARS-CoV access into DCs is still unclear
however, decrease function and diminished numbers of DCs have been
reported during COVID infection.

Ethical approval

All work with human cells and procedures may need appropriate
institutional approval before initiation

Material and methods

 1. Isolation for T and B lymphocytes from peripheral
blood iv vitro
Healthy control volunteer, 5-10 ml freshly whole blood is
collected in EDTA tubes and isolation of PBMCsby ficoll-
plaque for evaluation and stimulation in culture by DCs
activation inoculated with COVID antigen-biomaterial.
The subsets population analysed by flow cytometry were
defined as CD3+, CD19+, CD11b+.
Note: the absolute number of Dcs in peripheral blood is low
approximately 0.2%
2.Isolation of DCs and preparation ex vivo
A relatively homogenous population of functionally mature dendritic
cells can be generated from CD14+ blood monocytes by incubating them
with the proper cytokines (Zhou and Tedder, 1996). by characteristic
biomarker for CD marker by using flow cytometry monoclonal
antibodies mAb for CD33 /monocyte, CD83,86,80 for DCs and
APCs respectively and CD40/ B lymphocyte
RNA or protein antigen of covid virus used as nucleic acid
nanoparticles targeted ex vivo with DCs

1.2 Isolate peripheral blood monocytes from a leukapheresis pack or

buffy coat preparation using Ficoll-Paque density gradient
centrifugation and plastic adherence.

 0.2 mM EDTA in PBS, Ca2+ and Mg2+ free (APPENDIX 2)

 14.5% metrizamide solution (see recipe), room temperature
 Recombinant human granulocyte/macrophage colony-stimulating
factor (GM-CSF; see recipe)
 Recombinant human IL-4 (see recipe)
 Recombinant human TNF-α (see recipe)
 Inverted phase-contrast microscope
  Sorvall H1000B rotor (or equivalent)
 15-ml conical polypropylene centrifuge tube
 Tissue culture flask

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