Aerosol Combination Therapy of All-trans Retinoic Acid and Isotretinoin as A Novel Treatment for Inducing Neutralizing Antibodies in COVID -19 Infected Patients Better Than Vaccine : An Innovative Treatment
Aerosol Combination Therapy of All-trans Retinoic Acid and Isotretinoin as A Novel Treatment for Inducing Neutralizing Antibodies in COVID -19 Infected Patients Better Than Vaccine : An Innovative Treatment
Aerosol Combination therapy of All-trans retinoic acid and Isotretinoin as A novel Treatment
 for Inducing Neutralizing Antibodies in COVID -19 Infected Patients better than vaccine : An
 innovative Treatment

 Mahmoud ELkazzaz(1),Tamer Haydara(2), Mohamed Abdelaal(3), Ahmed M. Kabel(4), Abedelaziz
 Elsayed(5) ,Yousry Abo-amer(6) ,Hesham Attia(7)

 1. Department of chemistry and biochemistry, Faculty of Science, Damietta University,Egypt.

 2. Department of Internal Medicine,Faculty of Medicine, Kafrelsheikh University, Egypt

 3. Department of Cardiothoracic Surgery,Faculty of Medicine, Kafrelsheikh University, Egypt

 4. Department of Clinical Pharmacy, Faculty of Medicine , Tanta University,Egypt.

 5. Department of Pharmaceutical Biotechnology, Faculty of Pharmacy,Tanta University,Egypt.

 6. Hepatology,Gastroenterology and Infectious Diseases Department, Mahala Hepatology
 Teaching Hospital,Egypt

 7. Department of Immunology and Parasitology, Faculty of Science, Cairo University, Egypt.

 -  Study Chair ((( Dr.Tamer Hydara))), Department of Internal Medicine,Faculty of
 Medicine, Kafrelsheikh University, Egypt Contact: Dr.Tamer Hydara-Tel:
 00201142233340 Mail: tamerhydara@yahoo.com

 -  Principal Investigator ((( Mahmoud Elkazzaz))), Faculty of Science, Damietta
 University,GOEIC,Egypt Contact:Tel: 00201090302015 Mail:
 mahmoudramadan2051@yahoo.com

 -  Study coordinator ((Prof/Dr Mohamed Abdelaal)), Department of Cardiothoracic
 Surgery,Faculty of Medicine, Kafrelsheikh University, Egypt Contact:Tel:
 00201001422577 Mail: Malaal2@hotmail.com

 Abstract

 The pandemic of COVID-19 which is caused by severe acute respiratory syndrome coronavirus 2
 (SARS-COV-2) has infected over 20,000,000 people causing over 700,000 deaths .It has no
 currently approved treatments. In this clinical study we confirm that combination of
 isotretinoin and All trans retinoic acid can be used in the treatment of SARS-COV-2 better
 than vaccine according to the findings of previous studies and researches. Retenoic acid can
 induce neutralizing antibodies in case of corona virus (COVID-19) by restoring inhebited and
 exhausted T cells via inhebiting both CD13 and Angiotensin-converting enzyme-2 (ACE2). CD13
 amyloid receptor which abundantly overexpressed on cell surface of lymphocyte, Dentritic
 cell, Macrophage, granulocytes and monocytes and is ubiquitous in respiratory tract
 epithelial cells, smooth muscle cells, fibroblasts, epithelial cells in the kidneys and small
 intestine, activated endothelial cells, and platelets In addition inhibing of
 Angiotensin-converting enzyme-2 (ACE2) , Angiotensin T1 protein and Angiotensin II-mediated
 intracellular calcium release pathway which is responsible for COVID-19 cell fusion and
 entry.ACE2-expressing cells are prone to SARS-CoV-2 infection as ACE2 receptor facilitates
 cellular viral entry and replication. A study demonestrated that patients with hypertension
 and diabetes mellitus may be at higher risk of SARS-CoV-2 infection, as these patients are
 often treated with ACE inhibitors (ACEIs) or angiotensin II type-I receptor blockers (ARBs),
 which have been previously suggested to increase ACE2 expression.Butisotretinoin was found to
 be the strongest down-regulator of ACE 2 receptors.and this will give hope for diabetic
 patients or patients with hypertension infected with COVID-19.Therefore we suggest that
 Retinoic Acid will help in inhabiting factors which may enhance antibody dependent
 enhancement (ADE), A phenomenon caused by covid-19 which expected to lead to failure of
 vaccination specially in case of corona virus (covid-19) as a hyper mutatated COVID-19
 antigens can lead to (ADE) phenomenon in which IgG antibodies facilitate viral entry and
 fusion with infected cell through uptake of the virus-IgG complex via the Fc receptors and
 later viral fusion with antigen presenting cells like Dentric cells, macrophages and B cells
 via FcR , through the neonatal FcR instead of antibodies induced viral agglutination and this
 is known as antibody dependent enhancement (ADE)(2) ADE can hamper vaccine development, as a
 vaccine may cause the production of antibodies which, via ADE, worsen the disease the vaccine
 is designed to protect against. ADE in COVID-19 infection can be caused by high mutation rate
 of the gene that encodes spike (S) protein. In this clinical study we suggest that Hyper
 mutated spike protein ,lymphopenia, and impaired dentreic cells all these factors can help in
 and lead to delayed antibodies response and appearing after a period of covid -19 symptoms
 onset and this may be responsible for antibody dependent enhancement (ADE)

 Keywords: COVID 2019 , Retinoic acid, Lymphopenia ,T Cells, Dentric cells , ADE, Vaccine
This is a Phase 2, , randomized (1:1:1), placebo-controlled, 2-weeks, proof-of-concept study
 to evaluate the safety and tolerability as well as the mechanistic effect of oral
 administration of potent inhibitor of neutrophil elastase(Inhaled All trans retinoic acid and
 inhaled isotretinoin in subjects infected with COVID -19

 Unfortunately, all of the vaccinated monkeys treated with the Oxford vaccine known as ChAdOx1
 nCoV-19, is undergoing human trials in Britain.became infected when challenged as judged by
 recovery of virus genomic RNA from nasal secretions," said Dr William Haseltine, a former
 Harvard Medical School professor who had a pivotal role in the development of early HIV/Aids
 treatments. and in general future COVID-19 vaccination which depends on inactivated viral
 vaccine will be restricted to healthy people with strong immunity and It will not be given to
 patients with History of contact with a SARS-CoV-2 infection (positive in nucleic acid test).
 In addition to the COVID-19 viral antigens lead to stimulate antibodies formation of IgM in
 acute phase and IgG type in chronic phase which is facilitate viral entry and fusion with
 infected cell through uptake of the virus-IgG complex via the Fc family of receptors and
 later viral fusion with antigen presenting cells like macrophages, B cells, monocytes via FcR
 family, and vascular endothelium through the neonatal Fc receptor (nFcR) instead of
 antibodies induced viral agglutination and this is known as antibody dependent enhancement
 (ADE)(2).

 There are various hypotheses on how ADE happens and there is a likelihood that more than one
 mechanism exists. In one such pathway, some cells of the immune system lack the usual
 receptors on their surfaces that the virus uses to gain entry, but they have Fc receptors
 that bind to one end of antibodies. The virus binds to the antigen-binding site at the other
 end, and in this way gains entry to and infects the immune cell. Dengue virus can use this
 mechanism to infect human macrophages, if there was a preceding infection with a different
 strain of the virus, causing a normally mild viral infection to become life-threatening.(3)
 An ongoing question in the COVID-19 pandemic is whether-and if so, to what extent-COVID-19
 receives ADE from prior infection with other coronaviruses.

 ADE can hamper vaccine development: ADE can hamper vaccine development, as a vaccine may
 cause the production of antibodies which, via ADE, worsen the disease the vaccine is designed
 to protect against. Vaccine candidates for Dengue virus and feline infectious peritonitis
 virus (a cat coronavirus) had to be stopped because they elicited ADE.(4)

 ADE in coronavirus infection can be caused by high mutation rate of the gene that encodes
 spike (S) protein. A thorough analysis of amino acid variability in SARS-CoV-2 virus
 proteins, that included the S-protein, revealed that least conservative amino acids are in
 most exposed fragments of S-protein including receptor binding domain (RBD).(5) Delayed
 antibodies response and secretion after covid -19 symptoms onset is responsible for antibody
 dependent enhancement (ADE)

 A study shows the first seroconversion day of IgA was 2 days after onset of initial symptoms,
 and the first seroconversion day of IgM and IgG was 5 days after onset. The positive rate of
 antibodies in the 183 samples was 98.9%, 93.4% and 95.1%, for IgA, IgM and IgG, respectively.
 The seroconversion rate for IgA, IgM or IgG was 100% 32 days after symptom onset. According
 to the cumulative seroconversion curve, the median conversion time for IgA, IgM and IgG was
 13, 14 and 14 days, respectively. (6)

 Because this immune response takes a while to show up, antibody tests will be negative for
 those newly infected with COVID-19, which is why they're not used for diagnosis.

 "If it's the beginning of the infection, you don't pick it up, it's something that only
 develops later," Dr. Melanie Ott, a virologist and immunologist at the Gladstone Institutes
 and a professor at the University of California, San Francisco.(7)

 So, the principal investigator expects that delayed antibody response and delayed
 immunoglobulin class switching are the main reason for antibodies infectivity and
 non-specificity in the highly mutated covid-19 infection

 High-affinity IgG antibodies may efficiently resolve Covid -19 infection

 The more than 100 COVID-19 vaccines in development mainly focus on another immune response:
 antibodies. These proteins are made by B cells and ideally latch onto SARS-CoV-2 and prevent
 it from entering cells. T cells, in contrast, thwart infections in two different ways. Helper
 T cells spur B cells and other immune defenders into action, whereas killer T cells target
 and destroy infected cells. The severity of disease can depend on the strength of these T
 cell responses(1).

 Studies on humoral responses to infections have been mainly geared toward the production of
 high-affinity IgG antibodies that efficiently resolve an infection. It has been well
 recognised, however, that humoral immune response to infection can be a double-edged sword
 that either serves as a protective mechanism to resolve the infection or aggravates the
 tissue injury, e.g. IgG response causes fatal acute lung injury by skewing
 inflammation-resolving response in SARS .(8)

 In the case of respiratory infection, while IgM and IgG isotypes have been the primary
 emphasis in characterising immunity, mucosal and systemic IgA responses that may play a
 critical role in the disease pathogenesis, have received much less attention.(9),(11)

 High neutrophils in covid-19 infection may be the reason of delayed antibody response and
 severe complications

 Humoral immune response, especially production of neutralizing antibody, plays a protective
 role by limiting infection at later phase and prevents reinfection in the future. In
 SARS-CoV, both T and B cell epitopes were extensively mapped for the structural proteins, S,
 N, M and E protein.(12) A limited serology details of SARS-CoV-2 was reported. In a
 preliminary study, one patient showed peak specific IgM at day 9 after disease onset and the
 switching to IgG by week 2.25 Interestingly, sera from 5 patients of confirmed COVID-19 show
 some cross-reactivity with SARS-CoV, but not other coronavirus. Furthermore, all sera from
 patients were able to neutralize SARS-CoV-2 in an in vitro plaque assay, suggesting a
 possible successful mounting of the humoral responses.(13) Whether the kinetic/titer of
 specific antibody correlates with disease severity remains to be investigated.(14)

 In severe cases of coronavirus disease 2019 (COVID-19), viral pneumonia progresses to
 respiratory failure. Neutrophil extracellular traps (NETs) are extracellular webs of
 chromatin, microbicidal proteins, and oxidant enzymes that are released by neutrophils to
 contain infections. However, when not properly regulated, NETs have potential to propagate
 inflammation and microvascular thrombosis - including in the lungs of patients with acute
 respiratory distress syndrome. While elevated levels of blood neutrophils predict worse
 outcomes in COVID-19, the role of NETs has not been investigated. We now report that sera
 from patients with COVID-19 (n = 50 patients, n = 84 samples) have elevated levels of
 cell-free DNA, myeloperoxidase(MPO)-DNA, and citrullinated histone H3 (Cit-H3); the latter
 two are highly specific markers of NETs.(15)

 Neutrophils Are Sophisticated Cells Able to Adapt to Changing Inflammation

 Neutrophils are not simple bags of enzymes sent to kill pathogens before the adaptive immune
 cells move in. In fact, they are able to respond to altered inflammatory status; neutrophils
 can produce cytokines , alter their gene expression during inflammation and throughout
 "aging" and survive for significantly longer than traditionally thought, with one study
 placing lifespan from bone marrow at 5.4 days . As a consequence, neutrophils are able to
 adapt to changing conditions and direct other cells' behaviour-a task which they can perform
 with some sophistication.

 There are a number of murine models in which the T cell responses can be exacerbated by
 depleting neutrophils, implying they have a regulatory role . This suppression of T cell
 responses by neutrophils requires close contact and development of an immunological synapse
 -perhaps as the mediators thought responsible, reactive oxygen species and H2O2 do not
 diffuse far. Uptake of apoptotic or necrotic neutrophils also inhibits DC antigen
 presentation and co-stimulation, resulting in reduced T cell responses-a situation which can
 be exploited by pathogens. For example, neutrophils capture L. major and are subsequently
 engulfed by DCs, suppressing antigen presentation and T cell priming

 NE was also shown to inhibit maturation and function of dendritic cells, including expression
 of the costimulatory molecules CD40, CD80 and CD86 Inflammatory Lung Secretions Inhibit
 Dendritic Cell Maturation and Function via Neutrophil Elastase.(16)

 Inhibitor of elastase in covid-19 infection :

 1. Alvelestat was developed as treatment for lung diseases like Chronic Obstructive
 Pulmonary Disease. Alevelestat works by blocking certain proteins in the body that are
 responsible for inflammation and damage to the lungs that can lead to COPD symptoms.

 Alvelestat Inhibitor of elastase stimulate murine B lymphocyte differentiation into IgG-
 and IgA-producing cells via immunoglobulin class switching and inducing also known as
 isotype switching, isotypic commutation or class-switch recombination (CSR), is a
 biological mechanism that changes a B cell's production of immunoglobulin from one type
 to another, such as from the isotype IgM to the isotype IgG

 A study demonstrated that depletion of neutrophils improves the production of mucosal
 IgA and IgG after sublingual immunization with Bacillus anthracis edema toxin as
 adjuvant. These past studies also demonstrated that an inverse correlation exists
 between the number of neutrophils and production of IgA by B cells. Using specific
 inhibitors of elastase, we addressed whether the elastase activity of neutrophil could
 be the factor that interferes with production of IgA and possibly other immunoglobulin
 isotypes. We found that murine splenocytes and mesenteric lymph node cells cultured for
 5 days in the presence of neutrophil elastase inhibitors secreted higher levels of IgG
 and IgA than cells cultured in the absence of inhibitors and also,This treatment
 however, increased the frequency of CD4+ T cells.(17) As a mucosal targeted virus,
 SARS-CoV-2 would be expected to generate secretory IgA (sIgA) and induce strong mucosal
 immunity. Indeed, the mucosal anti-viral immunity has been shown to result in part from
 the IgA-mediated interactions with the pathogenic microorganisms to prevent pathogens
 from adhering to the cell surface.(17) Upregulated IgA production may be the result of
 increased levels of TGF-β and IL-10 that promote antibody switching in SARS-CoV-2
 infection.(17) A study reported that depletion of neutrophils before systemic injection
 of vaccines increased the magnitude of antigen-specific CD4+ T cell responses, and the
 levels of antigen-specific serum IgG responses .(18)

 Alvelestat and Sivelestat were found to stimulate AID mRNA synthesis. The NE inhibitors
 also induced expression of BAFF, APRIL and IL-10 mRNA in a dose-dependent manner.
 Neutrophil elastase and the related serine proteases cathepsin G and proteinase 3, were
 shown to regulate cytokine responses through activation or degradation of cytokines,
 cytokine receptors, or toll-like receptor .(18) This is consistent with our finding that
 NE inhibitors stimulate IL-10 expression, but also BAFF and APRIL in cultures of
 splenocytes. IL-10 was reported to regulate expression of AID for induction of Ig CSR.
 (19) and it is wellestablished that IL-10 facilitates Ig class switching for production
 of IgA (20). Thus, these data suggest that NE inhibitors stimulate antibody production
 through stimulation of AID and inhibition of the activation/degradation of cytokines and
 cytokine receptors by elastase. NE was also shown to inhibit maturation and function of
 dendritic cells, including expression of the costimulatory molecules CD40, CD80 and CD86
 .(21). Thus, the presence of neutrophil inhibitors may have promoted the maturation of
 dendritic cells and their expression BAFF and APRIL and perhaps costimulatory molecules
 (i.e., CD40) for enhanced antibody production. Although Alvelestat and Sivelestat are
 specific NE inhibitors, one should consider that they might also inhibit related
 elastase-like enzymes. In this regard, activated human B cells were reported to express
 a trypsin-like serine protease on the cell surface .(22) Concluding remarks It was
 previously shown that neutrophil peptide defensins enhance IgG, but not IgA responses
 against nasally co-administered vaccine antigens .(23) Here, the study have shown that
 the serine protease activity of neutrophil elastase, and possibly other serine protease
 such as B cell elastase, also participate in the regulation of B cell biology and
 adaptive immunity. However, unlike neutrophil peptide defensins, NE negatively regulate
 differentiation of B lymphocytes into IgG and IgA secreting cells. The presence of AID
 generally improves antibody-based immunity.(24).

 High Neutrophils in COVID -19 infected patients are capable of causing severe damage to
 the lung tissue by releasing toxic proteases and reactive oxygen species if not
 counterbalanced by the antiproteases.

 2. All-trans retinoic acid (RA) has beneficial effects when used in a variety of
 inflammatory skin conditions. In a study, the authors found that RA inhibited superoxide
 anion production and proteolytic enzyme release by human and rat neutrophils.
 Concomitantly, the authors found that RA-treated neutrophils were less able than
 untreated neutrophils to injure endothelial cells in culture even though the adhesion of
 the RA-treated neutrophils to endothelial cell monolayers was not diminished. Inhibition
 of cytotoxicity occurred over the same range of concentrations that inhibited oxygen
 radical formation and protease release. In additional studies, it was observed that
 pretreatment of endothelial cells with RA-induced resistance to subsequent injury by
 activated neutrophils. Finally, in vivo studies showed that pretreatment of rats for 3
 days with RA (1-10 mg/day, IP) reduced the degree of injury in the lungs and skin sites
 after treatment with bovine serum albumin and antibodies to bovine serum albumin in the
 reverse-passive Arthus reaction. Thus, RA can modulate neutrophil-mediated endothelial
 cell injury by an effect on both the neutrophils and their target cells. Together, these
 effects may underlie the reduction in immune complex-mediated injury seen in
 experimental animals. The beneficial effects that retinoids have in a variety of
 inflammatory skin diseases may likewise be a reflection of their effects on the
 physiology of both neutrophils and endothelial cells.

 Oral retinyl palmitate or retinoic acid corrects mucosal IgA responses toward an intranasal
 influenza virus vaccine in vitamin a deficient mice. Retinoids inhibit inflammatory TH17 T
 cell responses, promote regulatory T cell (Treg) responses, and regulate expression of
 toll-like receptors (TLRs) .(57)

 So, the principal investigator expects that retinoic acid will be effective in covid -19
 patients better than the other neutrophil elastase inhibitor like Alvelestat because Attia et
 al (2018) demonstrated that Sivelestat reduced secretion of IgM in a dose-dependent manner ,
 IgM type of antibodies which is very important in viral neutralization and on the other hand
 retinoic acid stops neutrophil-mediated endothelial cell injury by an effect on both the
 neutrophils and their target cells.

 Seconed drug is Isotretinoin(13cis RA) and ATRA may be able to inhibit COVID 2019 infection
 via inducing the antiviral immunity and this is discussed as follow :

 A study demonstrated that 13 cis retinoic is used in treating Emphysema (emphysema is a lung
 condition that causes shortness of breath), Since an effective immune response against viral
 infections depends on the activation of cytotoxic T cells that can clear infection by killing
 virus-infected cells , boosting the numbers and function of T cells in COVID-19 patients is
 critical for successful recovery. A recent study reported that the 82.1% of COVID-19 cases
 displayed low circulating lymphocyte counts. A CoV infects macrophages, and then macrophages
 present CoV antigens to T cells. This process leads to T cell activation and differentiation,
 including the production of cytokines associated with the different T cell subsets (Th17),
 followed by a massive release of cytokines for immune response amplification. The continued
 production of these mediators due to viral persistence has a negative effect on NK, and CD8 T
 cell activation.(25)

 Recent study of 522 COVID patients and 40 healthy controls from two hospitals in Wuhan, China
 demonstrated that T cell numbers are negatively correlated to serum IL-6, IL-10 and TNF-α
 concentration, with patients in decline period showing reduced IL-6, IL-10 and TNF-α
 concentrations and restored T cell counts. T cells from COVID-19 patients have significantly
 higher levels of the exhausted marker programmed cell death protein(PD-1) as compared to
 health controls. Moreover, increasing PD-1 and Tim-3 expression on T cells could be seen as
 patients progressed from prodromal to overtly symptomatic stages, further indicative of T
 cell exhaustion. T cell exhaustion is a progressive loss of effector function due to
 prolonged antigen stimulation, characteristic of chronic infections. Dendritic cell
 inhibition is connected to exhaustion of CD8+ T cell polyfunctionality during chronic
 hepatitis C virus infection. CD8 T cells produce very effective mediators to clear
 nCoV2019.(26)

 Dendritic cells (DCs) play a key role in innate immune and adaptive immune responses. As the
 strongest antigen presenting cells in the organism, they effectively stimulate the activation
 of T lymphocytes and B lymphocytes, thus combining innate and adaptive immunity. Immature DCs
 have strong migration ability, and mature DCs can effectively activate T cells in the central
 link of startup, regulation, and maintenance of immune responses. Thus, once the maturation
 process of DCs is blocked, it directly affects the initiation of subsequent adaptive immune
 response. MERS-CoV-2 is able to affect human dendritic cells and macrophages in-vitro
 .Productive replication of Middle East respiratory syndrome coronavirus in monocyte-derived
 dendritic cells modulates innate immune response.(27)

 Another study proposed that C -C chemokine receptor type 4 (CCR4) contributes to T cell lung
 homing imprinting. It was found that lung DCs induce the expression of CCR4 on T cells. Lung
 DCs-activated T cells traffic more efficiently into the lung and protect against influenza
 more effectively compared with T cells activated by DCs from other tissues. Lim and
 colleagues suggested that CXCR4 plays a role in CD8+ T cell migration to airway tissues.(28)

 Dendritic cell inhibition is connected to exhaustion of CD8+ T cell polyfunctionality during
 chronic hepatitis C virus infection. CD8 T cells produce very effective mediators to clear
 nCoV2019.(29)

 Presence of RA in different tissues is very imprtant for immune induction and fighting viral
 infection, for example, RA is present at high concentrations in the small intestine due to
 metabolizing dietary vitamin A by gut epithelial cells. In this local environment, RA
 activate and primes dendritic cells (DCs) to become CD103+ DCs that produce RA.3, 4CD103+ DCs
 are migratory cells that activate naive T cells in mesenteric lymph nodes to become effector
 T cells that contribute to both intestinal homeostasis and immunity.and also RA is an
 important signal that induces IgA-producing B cells. The gut homing T cells and B cells play
 essential roles in protecting the digestive tract from pathogens.(30) Retinoic acid (atRA)
 can inhibit the spontaneous apoptosis of activated human T lymphocytes in vitro. 13-cis RA
 activates Th2 cytokine production Enhanced circulating dendritic cell numbers.(40)

 So, according to this previous studies the principal investigator suggests that T cells
 lymphopenia and exhaustion may be resulted by Dendritic cells (DCs) infection and inhibition
 by MERS-CoV-2.

 Retinoic acid has profound effects on cellular proliferation and differentiation. Moreover,
 it has been reported that ATRA exhibits both anti-inflammatory and immunoregulatory effects.
 Recent studies have shown that FOXP3 expression and the immune function of Regulatory T cells
 (Tregs ) can be enhanced by ATRA in the immune system of both patients and mic.13Retinoic
 acid (RA) is produced by a number of cell types, including macrophages and dendritic cells,
 which express retinal dehydrogenases that convert vitamin A to its main biologically active
 metabolite, all-trans RA. All-trans RA binds to its nuclear retinoic acid receptors that are
 expressed in lymphoid cells and act as transcription factors to regulate cell homing and
 differentiation. RA production by CD103+ dendritic cells and alveolar macrophages functions
 with TGF-b to promote conversion of naive T cells into Foxp3+ regulatory T cells and,
 thereby, maintain mucosal tolerance.(50) So, principal investigator expects high inducing of
 Dendritic cells (DCs) by retinoic acid treatment which will lead to T cells activation and
 migration with less exhaustion phenomenon.

 Researchers from Wenzhou, China looked at clinical laboratory features including lipid levels
 of patients with COVID 19. They found dramatic reductions in the cholesterol levels of
 patients infected with COVID 19, compared with healthy controls .The study provides data to
 suggest that cholesterol levels decline quite rapidly during the early stages of infection
 and increase as the patient starts to recover. Therefore, indicating that cholesterol may
 have an important role to play in defending the body against such infections According to our
 protocol depending on previous studies the principal investigator demonstrated that there is
 a strong relation between immune system and cholesterol levels . :-

 Cellular cholesterol is a component of the plasma membrane and is also essential in cell
 proliferation. Regulation of intracellular cholesterol levels has been proposed as a
 mechanism to regulate T cell and macrophages proliferation. Intracellular cholesterol level
 is regulated by two competing pathways, cholesterol uptake and efflux, and ABCA1 plays a
 major role in the cholesterol efflux pathway. The ATRA induces ABCA1 expression and
 ABCA1-dependent cholesterol efflux in activated primary human CD4+ T cells implying that RA
 could affect T cell functions by regulating the cellular cholesterol levels.(51) ATRA
 upregulates ABCA1 expression only in activated CD4+ T cells, indicating that induction of
 ABCA1 by ATRA and 13 cis Retinoic Acid may play an important role in immune response.

 Retinoic acid and liver X receptor agonist synergistically inhibit HIV infection in CD4+ T
 cells by up-regulating ABCA1-mediated cholesterol efflux.(52)(53)

 RA also acts directly on macrophages at both mucosal sites and other immunological sites.
 AtRA modulates peritoneal macrophage activation by endotoxin and IFN-γ by suppressing TNF
 production and nitric oxide (NO) synthesis .In addition, at RA inhibits the expression of
 PGE2 and COX-2 and the release of TNF, which are induced by bacterial lipopolysaccharide
 (LPS) in murine peritoneal macrophages .(54)

 A study reported recently that substance (ATRA) have preventive effects on pulmonary fibrosis
 by inhibiting IL-6-dependent proliferation and TGF-β1-dependent trans differentiation of lung
 fibroblasts. Also, another studies demonstrated that 13-cis-retinoic acid and other retinoid
 analogs inhibit IL-1-induced IL-6 production and that this effect is analog-specific and, at
 least partially, transcriptionally mediated. This effect was dose-dependent with an IC50 of
 10(-7) M RA and significant inhibition being noted with doses of RA as low as 10(-8) M. IL-10
 production was inhibited by ATRA administration.(55)

 A study demonstrated that TLR3(-/-), TLR4(-/-), and TRAM(-/-) mice are more susceptible to
 SARS-CoV than wild-type mice but experience only transient weight loss with no mortality in
 response to infection. In contrast, mice deficient in the TLR3/TLR4 adaptor TRIF are highly
 susceptible to SARS-CoV infection, showing increased weight loss, mortality, reduced lung
 function, increased lung pathology, and higher viral titers . New studies showed that the
 high level of IFN-α/β produced from the TLR3-IRF3/IRF7 pathway and IFN-β is the reason for
 inhibiting DENV replication. 13Cis retinoic Acid induced significant upregulation of
 toll-like receptor 3 (TLR3) resulting in an immune response to dsRNA intermediate which can
 be partially generated during CoV-2 replicationTLR3 sensitized by dsRNA and cascades of
 signaling pathways (IRFs and NFκB activation, respectively) are activated to produce type I
 IFNs.(56)

 .

 Oral retinyl palmitate or retinoic acid corrects mucosal IgA responses toward an intranasal
 influenza virus vaccine in vitamin a deficient mice. Retinoids inhibit inflammatory TH17 T
 cell responses, promote regulatory T cell (Treg) responses, and regulate expression of
 toll-like receptors (TLRs) .(57) In vitro treatment of normal human monocytes with all-trans
 retinoic acid (ATRA) down-regulates TLR-2 expression (58), but this effect has not been
 examined in patients treated with systemic retinoids. Isotretinoin (13-cis retinoic acid;
 13-cis RA) is the only drug capable of inducing a long-term or permanent remission of acne;
 however, the mechanisms leading to this durable response are unknown. Given the role of TLR-2
 in initiating immune responses to P. acnes, we hypothesized that down-regulation of TLR-2 on
 monocytes and/or the induction of Treg responses in vivo may represent mechanisms by which
 isotretinoin exerts its long-term effects.

 Isotretinoin(13cis RA) may be able to inhibit COVID 2019 entry via down regulation of ACE2 ,
 AT1 protein and Ang II-mediated intracellular calcium release rather than inhibition of
 interleukin-6 (IL-6) and this is discussed as follow :

 Many authors misunderstands the difference between ARBs, which block ATR1 receptors and the
 hypothesised drug that could block the COVID-19 binding site on ACE2 receptor, which is
 totally different from ATR1 and 2.

 The COVID-19 pandemic caused by SARS-COV-2 has infected over 2,000,000 people causing over
 150,000 deaths.(59) A key host cellular protein required for the virus entry is
 angiotensin-converting enzyme 2 (ACE2) whose expression has been demonstrated in many tissues
 including alveolar epithelial type II cells in lungs, oral mucosa and intestine, heart,
 kidney, endothelium and skin. ACE2-expressing cells can act as home cells and are prone to
 SARS-CoV-2 infection as ACE2 receptor facilitates cellular viral entry and replication.(60) A
 study demonestrated that patients with hypertension and diabetes mellitus may be at higher
 risk of SARS-CoV-2 infection, as these patients are often treated with ACE inhibitors (ACEIs)
 or angiotensin II type-I receptor blockers (ARBs), which have been previously suggested to
 increase ACE2 expression.(61) In another study by Sinha et al who analyzed a publicly
 available Connectivity Map (CMAP) dataset of pre/post transcriptomic profiles for drug
 treatment in cell lines for over 20,000 small molecules, isotretinoin was the strongest
 down-regulator of ACE 2 receptors. On the other hand, they found 6 drugs in CMAP that are
 currently being investigated in clinical trials for treating COVID-19 (chloroquine,
 thalidomide, methylprednisolone, losartan, lopinavir and ritonavir, from clinicaltrials.gov),
 none of which was found to significantly alter ACE2 expression (P>0.1) Moreover, another
 study demonstrated that isotretinoin is a Potential papain like protease (PLpro) inhibitors
 which is a protein encoded by SARS-CoV-2 genes and considered one of the proteins that should
 be targeted in COVID-19 treatment by performing target-based virtual ligand screening.As
 Principal Investigator discussed before that (13cRA) is the strongest down-regulator of ACE2.

 The principal investigator expects that 13cRA can inhibit or dowenrgulat ACE2 by direc
 interaction and binding with the transmembrane ACE2, Suggesting its therapeutic potential in
 preventing the entry of COVID 2019 to the host cell.

 Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) and
 SARS-CoV FP FP have shown that calcium (Ca2+) plays an important role for fusogenic activity
 via a Ca2+ binding pocket with conserved glutamic acid (E) and aspartic acid (D)
 residuesdemonstrated that intracellular Ca2+ enhances MERS-CoV WT PPs infection by
 approximately two-fold and that E891 is a crucial residue for Ca2+interaction. Electron spin
 resonance revealed that this enhancement could be attributed to Ca2+ increasing MERS-CoV FP
 fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry titration showed that
 MERS-CoV FP binds one Ca2+, as opposed to SARS-CoV FP which binds to two Ca2+ ion.(62)

 Angiotensin II increases the intracellular calcium activity in podocytes of the intact
 glomerulus. The L-type Ca2+ channel blocker nicardipine did not influence the Ang II-mediated
 [Ca2+] increase and it has been postulated that SARS-CoV-2 binding to ACE2 may attenuate
 residual ACE2 activity, skewing the ACE/ACE2 balance to a state of heightened angiotensin II
 activity leading to pulmonary vasoconstriction and inflammatory and oxidative organ damage,
 which increases the risk for acute lung injury (ALI). AngII via AT1 receptors upregulates
 many proinflammatory genes, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular
 adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6).30 but 13cis RA specifically
 down-regulated the AT1 protein in a dose- and time-dependent manner. Down-regulation of the
 AT1 expression leads to reduced AngII-mediated intracellular calcium release Similarly with
 receptor down-regulation, Treatment with 13cRA resulted in a significant reduction in AT1
 mRNA .(63)

 Finally, the principal investigator expects that Isotretinoin will help in antibody induction
 against COVID-19 via inducing different types of antiviral immune factors and also,
 decreasing of COVID -19 hyper mutation by blocking its ACE2 receptors and preventing the
 virus from cell entry and replication and this will give the immune system time to recognize
 COVID -19.
Inclusion Criteria:

 Adult SARI patients with 2019-ncov infection confirmed by PCR; Absolute value of
 lymphocytes < 0. 6x 109/L; Severe respiratory failure within 48 hours and requires
 admission to ICU. (severe respiratory failure was defined as PaO2/FiO2 < 200 mmHg and was
 supported by positive pressure mechanical ventilation (including non-invasive and invasive
 mechanical ventilation, PEEP>=5cmH2O))

 Exclusion Criteria:

 Age < 18 Pregnant Allergic to experimental drugs and patients have the following
 conditions:

 1. Hypercholesterolemia

 2. Hypertriglyceridemia

 3. Liver disease

 4. Renal disease

 5. Sjögren syndrome

 6. Pregnancy

 7. Lactation

 8. Depressive disorder

 9. Body mass index less than 18 points or higher than 25 points

 10. Contraindications for hormonal contraception or intrauterine device.

 11. Autoimmune diseases A history of organ, bone marrow or hematopoietic stem cell
 transplantation

 12. Patients receiving anti-hcv treatment

 13. Permanent blindness in one eye

 14. History of iritis, endophthalmitis, scleral inflammation or retinitis 15-90 days of
 retinal detachment or eye surgery

 15. The competent physician considered it inappropriate to participate in the study

 16. HIV infection or other immunodeficiency or with an absolute neutrophil count ≤1.0 ×
 109/L

 17. Abnormal liver biochemistry (alanine aminotransferase, aspartate aminotransferase,
 gamma-glutamyl transferase) >1.5 × upper limit of normal or total bilirubin > upper
 limit of normal (unless Gilbert's disease with normal conjugated bilirubin)

 18. Any of the following laboratory abnormalities are present at baseline:

 -  Platelet count <150×109/L

 -  Serum albumin ≤ 3.5 g/dL

 -  INR ≥1.2

 -  CPK ≥ ULN.

 19. Significant liver fibrosis as evidenced by Fibrosis-4 (FIB-4) score >3.25

 20. History of hypersensitivity to retinoic acid or any of its excipients or the class of
 neutrophil elastase inhibitors Known hypersensitivity to medications used in the study
 procedures (e.g. midazolam, fentanyl, and lidocaine for bronchoscopy)