Oral Delivery of ACE2 Bioencapsulated in Plant Cells as Potential

Adjuvant Therapy to Reduce the COVID-19 Disease Severity

Eka B. Layadi

, Robert Sinto

, Friska W. Wijaya

and Oemar Ichsan

Division of Tropical Disease and Infection, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia,

Dr. Cipto Mangunkusumo National General Hospital, Jakarta, Indonesia

Keywords: SARS-CoV-2, COVID-19, ACE2, Bioencapsulated plant cell, Novel therapy

Abstract: COVID-19 has become a widespread pandemic and a devastating public health emergency. Numerous trials

have been conducted to search beneficial therapeutical interventions. ACE2 plays an essential role in the

pathogenesis of SARS-CoV-2 infection as this receptor become the entry point of virus to the cell. The

blockage of the ACE2 receptor and the delivery of ACE2 in the soluble form are some mechanisms that have

been proposed for potential therapy of COVID-19. Parenteral administration of ACE2 in soluble form has

been conducted in trials using the hrACE2 (human recombinant ACE2) and showed a favorable result.

However, the possibility of administering ACE2 through an oral route has not been extensively explored.

Bioencapsulated plant cell technique has shown resistant to digestive enzymes and gastric acid and able to

carry ACE2 to be absorbed safely into the circulation. Previous study showed promising utilization of

ACE2/Ang1-7 Bioencapsulated in Plant Cells to treat ocular inflammatory disorders in mice. Although no

clinical studies have been done yet, similar concept can be theoretically applied to hinder the development of

SARS-CoV-2 severe manifestation. The increasing soluble ACE2 may reduce the circulatory levels of

detrimental Angiotensin II effects as well as acting as a decoy to bind free virions from attaching to the target

cells.

1 INTRODUCTION

COVID-19 has become a global public health

emergency with the increasing emergence of new

cases daily in countries worldwide. There is an urgent

need for therapeutics now, more than ever, to control

SARS-CoV-2 infection (Vellingiri et al., 2020). The

biomedical community has made a massive effort to

find potential drugs by conducting many trials in

search of an effective cure for COVID-19. (The

Lancet Infectious Diseases, 2020).

ACE2 activator,

especially in the soluble form, is deemed one of the

plausible therapeutic strategies to control SARS-

CoV-2 infection, for this receptor acts as a gateway

of SARS-CoV-2 infection and the foundation of the

pathogenesis of COVID-19. This soluble form of

ACE2 can act as a competitive interceptor of SARS-

CoV-2 by binding the virus particles and limiting the

virus's attachment to the host's cell membranes

(Battle et al., 2020; Rodríguez-Puertas, 2020).

Parenteral administration of human recombinant

soluble ACE2 (hrsACE2), has been tested in patients

and has passed phase 2 clinical studies with a great

safety profile. A recent study by Zoufaly et al. (2020)

has shown that the administration of parenteral

hrsACE2 to a patient with severe COVID-19 resulted

in marked reductions in SARS-CoV-2 viral load,

serum levels of inflammatory cytokine, and serum

levels of Angiotensin II of the recipient (Abd El-Aziz

et al. 2020).

The route of administration of soluble

ACE2 through oral route has not been explored

extensively, despite successful trials of oral ACE2

bioencapsulated plant cell in animal studies with

favorable results (Shil et al., 2014). In this study, we

explore the possibility and feasibility of oral

bioencapsulated ACE2 in plant cells as a potential

adjuvant therapy to ameliorate COVID-19

manifestations in humans and how this drug would

potentially revolutionize the pharmaceutical industry,

____________________________________________________

https://orcid.org/0000-0001-5120-5848

https://orcid.org/0000-0003-3857-300X

https://orcid.org/0000-0001-6070-9079

https://orcid.org/0000-0002-8178-9335

330

Layadi, E., Sinto, R., Wijaya, F. and Ichsan, O.

Oral Delivery of ACE2 Bioencapsulated in Plant Cells as Potential Adjuvant Therapy to Reduce the COVID-19 Disease Severity.

DOI: 10.5220/0010492103300334

In Proceedings of the 1st Jenderal Soedirman International Medical Conference in conjunction with the 5th Annual Scientiﬁc Meeting (Temilnas) Consortium of Biomedical Science Indonesia

(JIMC 2020), pages 330-334

ISBN: 978-989-758-499-2

especially in developing countries all over the globe,

through its advantageous cost-efficiency.

2 BACKGROUND

2.1 SARS-CoV-2 Infection and ACE2

Molecular Pathogenesis

SARS-CoV-2 are single-stranded RNA viruses and

contain two groups of proteins, namely structural

protein such as spike (S) proteins that bind to the

receptors on the host cell, nucleocapsid (N) that

protects the genetic information of virus, matrix (M)

and envelope (E), and non-structural proteins such as

proteases (nsp3 and nsp5) and RdRp (nsp12)

(Chatterjee et al., 2020). SARS-CoV depends upon

ACE2 receptors expressed in human epithelial cells,

endothelial cells, and most abundantly in the lung

parenchyma. This receptor recognition is one of the

significant steps in viral infection of host cells and the

prelude of its pathogenesis (Chatterjee et al., 2020).

The S protein in the SARS-CoV-2 membrane

promotes virus entry into the host cell through ACE2

receptors widely spread among many cells in the

various organ system. Despite ACE2's existence in

various cells, 83% of total ACE2 in humans is

expressed in alveolar epithelium type 2, making this

virus's prominent tropism in the respiratory system.

Typically found in membrane-bound form, ACE2

comes in a smaller fraction in the soluble form

(Verdecchia et al., 2020). SARS-CoV-2 entry to the

cell will cause a depletion of ACE2 expression.

ACE2, which functions in cleaving AngII

(Angiotensin II) into Ang1-7 (Angiotensin 1-7), helps

regulate the balance of the two. An increase of AngII

due to deficiency of ACE2 will promote pro-

inflammatory, pro-oxidative, pro-fibrotic processes,

and vasoconstriction, which cumulatively contribute

to the deterioration of COVID-19 manifestations

(Zhang et al., 2020).

AngII plays a vital role in signaling cellular and

molecular events critical in the pathogenesis of

pulmonary fibrosis. The first mechanism is to

promote pro-inflammatory cytokines such as IL-6

and IL-8 by macrophages; the second is by producing

reactive oxygen species (ROS) among infected

epithelial cells and followed by its apoptosis and

lastly by proliferation, migration, and differentiation

of fibroblast to myofibroblast. Thus, the higher serum

AngII among patients with COVID-19 pneumonia,

the higher the risk of developing respiratory failure

and other adverse events (Delpino & Quarleri, 2020).

2.2 Bioencapsulated Plant Cell to

Deliver Protein-based Drug

Incorporating protein-based drugs into plant cells

has become a cutting-edge method called

bioencapsulated plant cells through complex

biotechnological engineering. There are two methods

to make the desired protein expressed in the plant cell,

through nuclear manipulation or chloroplast

manipulation. The chloroplast manipulation is

considered more superior to nuclear manipulation to

increase the levels of transgene expression. Each

plant contains about 10.000 copies of the chloroplast

genomes, and collectively they expressed up to 70%

of total leaf protein. A plethora selection of proteins,

ranging from minuscule antimicrobial peptides or

hormones to large-sized proteins encoded by

bacterial, viral, fungal, and human genes, have been

successfully expressed in chloroplasts (Kwon &

Daniell, 2015).

The genes that optimized the desired protein

expression are usually fused with CTB (cholera toxin

type B) to facilitate transepithelial transport in the gut.

After the fusion, the combined material will be cloned

into the chloroplast transformation vectors. Shoots

emerging with the modified chloroplast are further

investigated by using PCR to confirm the site-specific

integration of the chloroplast genome. After the

confirmation, the plants will be transferred to the

greenhouse for propagation and mass production. The

leaves of the plant that contain the desired protein will

go through a process called lyophilization.

Lyophilized plant cells are stable at a certain range of

temperature for many years, can further withstand the

digestive enzymes and denaturation from gastric acid,

maintaining the protein drug's structure and functions

(Park et al., 2020).

Plant cells' walls are composed of sturdy lignin

and cellulose, which cannot be broken down by

digestive enzymes. Combined with the effects of

lyophilization, layers of protection screen protect the

desired protein-based drug. After the bioencapsulated

drug arrives in the intestinal lumen, the intestinal

bacteria, especially from the Bacteroides spp. and

Firmicutes species, will break down lignin and

cellulose from the plant's cell wall cell, releasing the

drug into the intestinal lumen. In this step, the CTB

helps in absorption of the drug by translocating the

drug through the gut epithelium. After the absorption,

the drug will be released into the circulation (Kwon

& Daniell, 2016).

The drug administration technique through

bioencapsulated drug in plant cells is considered cost-

efficient because it does not need complex cold-chain

Oral Delivery of ACE2 Bioencapsulated in Plant Cells as Potential Adjuvant Therapy to Reduce the COVID-19 Disease Severity

331

storage and the drug contained within the plant cells

through lyophilization can still be viable for years.

Clinical advancement of this concept would

revolutionize protein drug production and delivery

for many metabolic and genetic disorders (Hu et al.,

2020).

2.3 The Potential Capacity of

Bioencapsulated ACE-2 As

COVID-19 Adjuvant Therapy

This Bioencapsulated technique has been used to

treat various diseases from Gaucher's disease,

diabetes mellitus, hypertension to Alzheimer's

disease. In Gaucher's disease, there is a trial using

carrot cells expressing human glucocerebrosidase

administered orally to rats, although it was found that

the concentration of glucocerebrosidase post-

intervention in the serum was 10-fold lower than the

control group with the IV formulation. In diabetes

mellitus, design for ideal oral insulin through

bioencapsulated plant cell is still in the path of

development (Kwon & Daniell, 2016). There is also

a study by Park et al. (2020) using IGF-1

bioencapsulated in lettuce cells to promote fracture

healing that showed promising results. There is still

an endless potential of this novel drug delivery

system that needs to be explored, and COVID-19 can

be one of them.

SARS-CoV-2 enters the cell through the ACE2

receptor facilitated by the S-protein spike of the virus

(Li et al. 2020). ACE2 is mostly bound to cell

membranes and only scarcely present in the

circulation in a soluble form (Verdecchia et al., 2020).

The entry of the virus is followed by downregulation

of ACE2 (Gheblawi et al., 2020). The

downregulation of ACE2 causes the levels of Ang II

to rise. The increased levels of AngII create a

detrimental chain of events that support

vasoconstriction, pro-inflammatory, pro-oxidative,

and pro-fibrotic conditions, leading to acute lung

injury in COVID-19 patients. (Lugito et al., 2020;

Bourgonje et al., 2019; Kuba et al., 2005). It explains

current potential therapeutic strategies to manage

SARS-CoV-2 infection, which are portrayed in figure

1, could be achieved by making spike protein-based

vaccine, inhibition of transmembrane protease serine

2 (TMPRSS2) activity, blocking ACE2 receptor, and

delivering an excessive soluble form of ACE2 (Zhang

et al., 2020).

There have been numerous trials concerning the

management of COVID-19 through the involvement

of the ACE2 receptor. One of them is a trial using

rhACE2 (recombinant human ACE2), which was

administered parenterally. This drug has completed

clinical trials and efficiently lowered plasma

angiotensin II and increased angiotensin 1-7 levels,

respectively (Gheblawi et al., 2020; Zoufaly et al.

2020). There has not been any study or trials

mentioning a possible ACE2 administration through

the oral route, but a proposed model uses

bioengineered probiotic, Lactobacillus paracasei,

that secretes soluble ACE2 to help ameliorate

COVID-19 manifestations (Senapati et al., 2020).

The administration through oral route with

bioencapsulated plant cells has never been

highlighted as a proposed COVID-19 therapy model.

A study conducted by Shil et al. (2014) used

ACE2/Ang-(1–7) Bioencapsulated in Plant Cells,

administered orally, as a cost-effective therapeutic

strategy for ocular inflammatory diseases in mice.

They succeeded in creating ACE-2 fused with CTB

in lyophilized bioencapsulated plant cell. The ACE2

activity assay through ELISA (enzyme-linked

immunosorbent assay), using protein extracts isolated

from plant leaves showed that the plant cells

successfully expressed human ACE2 which is

Figure 1: ACE2 as SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic targets.

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )

332

enzymatically active, and they found that the ACE2

protein can be detected in both serum and retina of the

mice subjects 5 hours after oral gavage (Shil et al.,

2014). There was also a similar study conducted by

Shenoy et al. (2014) using oral ACE2/Ang-(1–7)

Bioencapsulated in Plant Cells that successfully

prevented the progression of monocrotaline-induced

pulmonary hypertension in rats.

Despite the successful creation of ACE2 drug

bioencapsulated in plant cell for animal studies and

how the bioencapsulated protein drug technique has

been successfully developed to treat some human

diseases, ACE2 drug bioencapsulated in plant cell has

never been tested to human subjects. This new drug

administration, theoretically speaking, could be one

of the therapeutical strategies to deliver a soluble

form of ACE2. This ACE2 bioencapsulated plant cell

will be released into the circulation right after the

cellulose on the plant carrier's outer cell wall is

digested by intestinal bacteria. The fusion of ACE-2

with CTB helps to translocate ACE2 into the gut

epithelium cells. The ACE2 drug that circulates in

plasma can act as a decoy for SARS-CoV-2 binding

so that some active viruses might not attach to the

ACE2 receptor in various cells in the body. If SARS-

CoV-2 already infects the patient, this drug's

administration can ameliorate the severity of the

infection. Soluble ACE2 has protective effects and

can cleave AngII (which brings pro-inflammatory,

pro-oxidant, and pro-fibrosis) into Ang1-7 (which has

beneficial effects). Thus, through these chains of

events, ACE2 that is administered bioencapsulated in

plant cells would be able to decrease the severity of

COVID-19 infection and lower the incidence of

cytokine storm by regulating the RAS system and

keeping the AngII and Ang1-7 in the right balance.

3 CONCLUSIONS

Bioencapsulated ACE2 in plant cells could be a

therapeutical strategy to deliver the soluble form of

ACE2 in COVID-19 patients. By acting as a

competitive interceptor that limits the attachment of

SARS-CoV-2 to membrane cells, this soluble ACE2

could prevent SARS-CoV-2 entry and replication in

the target cells. Aside from this effect, ACE2 also acts

by cleaving AngII, which exerts detrimental

properties that aggravate the severity of COVID-19

manifestations. In addition to those benefits,

bioencapsulated drug in plant cells is considered cost-

efficient because it does not need complicated cold-

chain storage, and the ACE2 contained within the

plant cells through lyophilization could still be viable

for years. Advancement of this bioencapsulated

protein drugs in plant cell technique could be just

what Indonesia, or other developing countries, need

as a potential cost-efficient strategy to ameliorate

SARS-CoV-2 infection.

REFERENCES

Vellingiri, B., Jayaramayya, K., Iyer, M., Narayanasamy,

A., Govindasamy, V., Giridharan, B., et al. (2020).

COVID-19: A promising cure for the global panic. Sci

Figure 2: The process of making bioencapsulated ACE2 and its desired effects on SARS-CoV2.

Oral Delivery of ACE2 Bioencapsulated in Plant Cells as Potential Adjuvant Therapy to Reduce the COVID-19 Disease Severity

333

Total Environ, 725;138277. doi:

10.1016/j.scitotenv.2020.138277.

The Lancet Infectious Diseases (2020). Curing COVID-19.

Lancet Infect Dis, 20(10):1101.

https://doi.org/10.1016/S1473-3099(20)30706-4

Batlle, D., Wysocki, J., Satchell, K. (2020). Soluble

angiotensin-converting enzyme 2: A potential approach

for coronavirus infection therapy? Clin Sci,134(5):543–

545.

Rodríguez-Puertas, R (2020). ACE2 activators for the

treatment of COVID 19 patients. J Med Virol,

92(10):1701-1702. https://doi.org/10.1002/jmv.25992

Zoufaly, A., Poglitsch, M., Aberle, J.H., Hoepler, W., Seitz

T., Traugott M., et al (2020). Human recombinant

soluble ACE2 in severe COVID-19. Lancet Respir

Med, 8(11):1554-1558. doi: 10.1016/S2213-

2600(20)30418-5

Abd El-Aziz, T.M., Al-Sabi, A., Stockand, J.D. (2020).

Human recombinant soluble ACE2 (hrsACE2) shows

promise for treating severe COVID-19. Signal

Transduct Target Ther,

5(1).https://doi.org/10/1038/s41392-020-00374-6

Shil P.K., Kwon K.C., Zhu P., Verma A., Daniell H., Li Q.

(2014). Oral delivery of ACE2/Ang-(1-7)

bioencapsulated in plant cells protects against

experimental uveitis and autoimmune uveoretinitis.

Mol Ther, 22(12):2069–2082. doi:

10.1038/mt.2014.179.

Chatterjee S.K., Saha S., Munoz M.N.M. (2020). Molecular

pathogenesis, immunopathogenesis and novel

therapeutic strategy against COVID-19. Front Mol

Biosci, 7:196. doi: 10.3389/fmolb.2020.00196

Verdecchia P., Cavallini C., Spanevello A., Angeli F.

(2020). The pivotal link between ACE2 deficiency and

SARS-CoV-2 infection. Eur J Intern Med, 76:14-20.

doi: 10.1016/j.ejim.2020.04.037

Zhang H., Penninger J.M., Li Y., Zhong N., Slutsky A.S.

(2020). Angiotensin-converting enzyme 2 (ACE2) as a

SARS-CoV-2 receptor: molecular mechanisms and

potential therapeutic target. Intensive Care Med,

46(4):586–590. https://doi.org/10.1007/s00134-020-

05985-9

Delpino M.V., Quarleri J. (2020). SARS-CoV-2

Pathogenesis: Imbalance in the Renin-Angiotensin

System Favors Lung Fibrosis. Front Cell Infect

Microbiol, 10:340. doi: 10.3389/fcimb.2020.00340

Kwon K. C., Daniell H. (2015). Low-cost oral delivery of

protein drugs bioencapsulated in plant cells. Plant

Biotechnol J, 13(8):1017–

1022.http://doi.wiley.com/10.1111/pbi.12462

Park J., Yan G., Kwon K.C., Liu M., Gonnella P.A., Yang

S., et al. (2020). Oral delivery of novel human IGF-1

bioencapsulated in lettuce cells promotes

musculoskeletal cell proliferation, differentiation and

diabetic fracture healing. Biomaterials, 1;233:119591.

https://doi.org/10.1016/j.biomaterials.2019.119591

Kwon K.C., Daniell H. (2016). Oral delivery of protein

drugs bioencapsulated in plant cells. Mol Ther.

24(8):1342–1350. doi: 10.1038/mt.2016.115

Hu X., Yang G., Chen S., Luo S., Zhang J. (2020).

Biomimetic and bioinspired strategies for oral drug

delivery. Biomaterials Science, 8(4):1020–1044.

Available from:

https://pubs.rsc.org/en/content/articlehtml/2020/bm/c9

bm01378d

Li X., Geng M., Peng Y., Meng L., Lu S. (2020). Molecular

immune pathogenesis and diagnosis of COVID-19.

Journal of Pharmaceutical Analysis, 10(2):102-108.

https://doi.org/10.1016/j.jpha.2020.03.001

Gheblawi M., Wang K., Viveiros A., Nguyen Q., Zhong

J.C., Turner A.J., et al. (2020). Angiotensin-Converting

Enzyme 2: SARS-CoV-2 Receptor and Regulator of the

Renin-Angiotensin System: Celebrating the 20th

Anniversary of the Discovery of ACE2. Circ Res,

126(10):1456-1474. doi:

10.1161/CIRCRESAHA.120.317015

Lugito N.H., Kurniawan A., Damay V., Chyntya H.,

Sugianto N. (2020). The role of gut microbiota in

SARS-CoV-2 infection: Focus on angiotensin-

converting enzyme 2. Curr Med Issues, 18(3):261.

Available from:

http://www.cmijournal.org/text.asp?2020/18/3/261/28

9426

Bourgonje A.R., Abdulle A.E., Timens W., Hillebrands J.,

Navis G.J., Gordijn S.J., et al. (2020). Angiotensin‐

converting enzyme 2 (ACE2), SARS‐CoV‐2 and the

pathophysiology of coronavirus disease 2019 (COVID‐

19). J Pathol, 251(3):228–48.

https://doi.org/10.1002/path.5471

Kuba K., Imai Y., Rao S., Gao H., Guo F., Guan B., et al.

(2005). A crucial role of angiotensin converting

enzyme 2 (ACE2) in SARS coronavirus–induced lung

injury. Nat Med, 11(8):875–879. doi: 10.1038/nm1267

Senapati S., Dash J., Sethi M., Chakraborty S. (2020).

Bioengineered probiotics to control SARS-CoV-2

infection. Res Ideas Outcomes,

6:54802.https://doi.org/10.3897/rio.6.e54802

Shenoy V., Kwon K.C., Rathinasabapathy A., Lin S., Jin

G., Song C., et al (2014). Oral delivery of angiotensin-

converting enzyme 2 and angiotensin-(1-7)

bioencapsulated in plant cells attenuates pulmonary

hypertension. Hypertension, 64(6):1248-1259. doi:

10.1161/HYPERTENSIONAHA.114.03871.

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )

334