Development of an Encapsulin Nanoparticle-Based Platform for a Next-Generation COVID-19 Vaccine BMED7022

A new COVID-19 vaccine platform depends on encapsulin protein nanoparticle

by Siriporn Maneekobkulong

Thesis submitted in fulfilment of the requirements for
the degree of

Master of Science, Biomedical engineering

under the supervision of Dr. Andrew Care

University of Technology Sydney

Faculty of Life Science

December 2022

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I declare that a thesis has never been submitted for any degree and it is not being delivered as a component of a different degrees application. Secondly, I verify that I wrote it and that I acknowledge any sources involved as well as any assistance I have already gotten through its preparation.

I gave my supervisors considerable time to review many revisions of my thesis and received much criticism.

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Student name: _Siriporn Maneekobkulwong___

Student ID: ___14356745____

Thesis Title: A new SARS-COV-2 (COVID-19) vaccine platform depends on encapsulin protein nanoparticles

The masters thesis mentioned satisfies all submission conditions, the writer thus declares.

Principal supervisor name: _Dr Andrew Care________

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Acknowledgements

First of all, I could not have undertaken this journey without my mentor, Dr Andrew Care, whose knowledge has been crucial in enabling this study. His encouragement and cooperation have given me research tools and motivation throughout my master's project. I would also want to thank my co-mentors, Caitlin Sives, Henrico Adrian and India Boyton from the Care laboratory research team at UTS for assistance and support during working in the laboratory.

I also would like to thank you the design future students from design faculty at UTS to collaborate ideas for producing the vaccine style based on fundament of this experiment.

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) has pandemic in worldwide and it has mutated too variant of concerns (VOCs). The currently vaccine in the market cannot prevent the new VOCs enough. We have to create a new SARS-COV-2 vaccine platform that is effective to against the new VOCs. In this experiment, SpyTag/SpyCatcher (ST/SC) technology is used to assemble on a protein nanoparticle platform in E. coli.

Content

Acknowledgements. .i

Abstract..ii

Abbreviations.iii

Introduction...1-2

Materials and Methods..2-4

Results.4-12

Discussion.13

Conclusion13

References..14

Appendix..15

Abbreviations

COVID-19 Coronavirus disease 2019

His-Tags Histidine tags

MX-Enc Myxococcus xanthus encapsulin

Qt Quasibacillus thermotolerans

QT-Enc Quasibacillus thermotolerans encapsulin

QT-Enc-HisTags Quasibacillus thermotolerans encapsulin with C-terminal His tag

RBD Receptor-binding domain

SARS-COV-2 Severe acute respiratory syndrome coronavirus 2

SC Spy-Catcher

ST Spy-Tag

SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis

TM-Enc Thermotoga maritima encapsulin

1. Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) or known as the coronavirus disease pandemic of 2019 (COVID-19) is one of the respiratory pandemic diseases that causes 6,566,676 people's death in worldwide(8,15). The total number of COVID-19 cases is 621,147,972(8,15). In December 2019, Wuhan, China, received its initial reports? and it became a pandemic in 2020.

In the family Coronaviridae, the genus Betacoronavirus, SARS-CoV-2 is an encapsulated virus with a single-stranded positive-sense RNA genome (30kb)(11). The virus RNA codes for 16 non-structural and nine helper proteins, as well as the four main components spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins(5). Comparable to the SARS-CoV, the SARS-CoV-2 begins host cells by interacting with the human angiotensin-converting enzyme 2 (ACE2) via the receptor-binding domain (RBD) within the S1 subunit and then causing endocytosis through the S2 subunit(5).

SARS-COV-2 has raised mutation in its characteristics to survive in the environment. It depends on changed genome sequences to increase spreading as an example (11). This leads to making many variants of concern (VOCs) (11). So, GISAID, Nextstrain, and Pango have been established by the WHO as nomenclature systems for labelling and recording SARS-CoV-2 genetic lineages (11). The current COVID-19 VOCs are alpha, beta, gamma, delta and omicron (8,15).

Currently COVID-19 vaccine

Scientists investigate and produce a vaccine to prevent the disease. There have different three types of vaccines, which are messages RNA, vector and protein subunit (3): the mRNA vaccine employs genetically modified mRNA to train human cells to know how to produce the S protein located on the COVID-19 virus's surface, but the COVID-19 virus' genetic material is inserted into a modified form of another virus to create the vector vaccine (3). The protein subunit vaccine uses only the harmless S protein part of a virus to stimulate the immune system(3).

Encapsulin

Encapsulin is peptide structures that resemble the cellular membranes of bacteria or viruses and have an icosahedral form(). It is used as a nanocarrier particle for cargo protein vectorisation and customised epitope display?. Thermotoga maritima (TM) and Myxococcus xanthus (MX) are bacteria species that have homogeneous encapsulins, that self-assemble forming icosahedrally symmetry structural modules. TM and MX shell are 20-24 nm (form T=1) and 30-32 nm (form T=3) in diameters respectively?. TM-Enc is made up on sixty identical amino acids (AAs) while the MX shell is assembled to three time of TM-Enc size (180 AAs). Quasibacillus thermotolerans (QT) shell is larger size than TM and MX which is 42 nm in diameter and it is created by 240 AAs (T=4) .

Spy-Tag/ Spy-Catcher (ST/SC) protein ligation system

The SpyCatcher-SpyTag system is developed seven years ago as a method for protein ligation. It is based on a modified domain from Streptococcus progenies surface protein (SpyCatcher), which recognised a cognate 13-amino-acid peptide (SpyTag)(12). Upon recognition, the two form a covalent isopeptide bond between the side chains of lysine in SpyCatcher and aspartame in SpyTag. This technology has been used, among other applications to create covalently stabilised multi-protein complexes, for modular vaccine production, and to label proteins (e.g. for microscopy). The SpyTag system is versatile as the tag is a short, unfolded peptide that can be genetically fused to exposed positions in target proteins; similarly, SpyCatcher can be fused to reporter proteins such as GFP, and the epitope or purification tags.

HYPOTHESIS

AIMS:

1. Materials and Methods

Identification of suitable loop areas on encapsulin nanoparticles via computer simulation

For recognition of the encapsulins, the catalogue of specie names, shell types and sizes of the cargo are making into different code depends on Uniprot and RCSB Protein Data Bank (PDB) websites. These websites provide the protein sequences that make the loop region to build the cargo. So, the information from these websites are uploaded into differentiation of files types to analyse the flexibility of the loop section individual species and the similarity of the loop areas between them. These are done by MEDUSA and ESPRIPT website with UCSF ChimeraX program.

Analysed the similarity of encapsulin protein cages

Encapsulin of Thermotoga maritima (Tm) (PDB code: 3DKT), Myxococcus xanthus (Mx) (PDB code: 4PT2) and Quasibacillus thermotolerans (Qt) (PDB code: A0A0F5HPP7) were used the codes from Uniprot website to get the align sequence section in FASTA file. The codes were Q9WZP2, Q1D6H4 and A0A0F5HPP7 respectively. These FASTA files were converted into the text files to use in Espript websites to compare the similarity between these encapsulins via using the PDB codes.

Recognized the flexibility of encapsulin protein nanoparticle

The sequences of individual encapsulin cage from Uniprot were inserted into MEDUSA website. Then, they were analysed to provide the different classes of the resiliences.

Determine the loop region of these encapsulin

The PDB codes were used for downloading the images of biological assembly 1 in PDB-gz file. Next, these images were uploading into UCSF ChimeraX software to analyse the loop regions that were suitable for adding Spy-Tags (AHIVMVDAYKPTK) (8). The ChimeraX software also use for finding the internal and external of the loop region to add His-Tag (HHHHHH) in C-terminal, which is the outside of the cargo when the loop region perform a shell.

Design the parts of the encapsulins to insert the Spy-Tag and His-Tag

All information from three parts before were used to make four samples from QT encapsulins due to the size of the QT encapsulin was not too small to add the mixture of Spy-catcher, linker and His-Tag. Also, it had more flexibility of loop region. After designed four different parts of the loop regions to insert the Spy-tag/Spy-Catcher technology and Histidine tags (His-Tags), they were made and deliveried by the professional scientists from Twist Biosciences and Integrated DNA Technology (IDT) companies.

Loop segment interaction with nanoparticle is confirmed in silico

For confirmation of the inserted combination of Spy-catcher, linker and His-Tag into QT encapsulins, the four different DNA synthesiss parts of QT samples are processed into transformation step. This step is the process to produce many plasmids samples in competent cell E. coli (NEB 5-Alpha). Then, they are grown and selected only colonies, that contained the plasmids samples, in the selecting colonies process. After that, they are grown in BL21(DE3) competent E. coli cells to express protein. Next, they were run in the gel to show the molecular weight protein in the expression evaluation step. The last two steps were cell lysis and purification processes.

Cells Transformation

The four different DNA synthesiss parts of QT were Qt Enc-68-69-spycatcher-histag (716ng/ul of stock), Qt Enc-133-134-sypcatch-histag (691ng/ul of stock), Qt Enc-197-198-sypcatcher-histag (694ng/ul of stock) and Qt Enc-222-223-sypcatcher-histag (708ng/ul of stock). They added neuclease-free water to make the total concentration of each sample into 10 ng/ul. This enzyme was used for helping plasmid to not break down. Then, mixed each samples tube well by spinning them three times. Next, 50 ul of the competent E. coil cell (NEB 5-Alpha) were added into each new tube for four tube. Also, 2 ul of individual samples were add into the new tubes, that were labeled their name. Next, the mixed between sample and competent cells tubes were incubated on the ice for 30 minutes. After that, they were put into the water bath at 42C to heat shock before put them back on the ice for 5 mins to incubate. Also, BL21 (DE3) competent E. coli cell are made the same process with NEB 5-alpha competent cells.

Selecting plasmid

After the transformation, NEB 5 Alpha competent cells that contains the samples (50 uL) were added into the plates in the order of labling name. The plates contained antibiotic to select the colonies that contained the sample plasmids. Then, the sample were spreader down on the plates by strips. Next, the plates were incubatedat 37C for overnight.

Protein expression

A flask contains 100 ml of LB media with 100 uL of Kanamycin antibiotic (Kan) (50 mg/mL of stock) in each for four flasks. Then, BL21 (DE3) competent cells contains the four samples (1% v/v) are add into the flasks under the labelling singly. Next, the flasks are put in the shaking machine at 37C at 200rpm for 2 hours to grow the bacteria. The samples were then started growing aerobic conditions at 37C until they attained an uv spectrophotometer at 600nm (OD600) of 0.5-0.6. After that, 0.1mM of isopropyl-?-d-thiogalactopyranoside (IPTG) (1 molelar of stock) are added into all flasks to stimulate protein expression.

All sample flasks are divide into twelve flasks and they are put into the shaking machine at different temperature (20C, 30C and 37C respectively) at 120 rpm for overnight.

Cell expression

All samples (1 mL) with different temperature conditions, are the stock in the flasks, are taken into the tubes and the tubes are spin in the centrifuges for 10 minutes at 4C at 1,400 rmp. Then, Coomassie dry stain (10 uL) are added into twelve tube of samples and all tubes spin in small centrifuges for a couple seconds. Next, all samples are put in thermometer at 95C for 10 minutes to heat the samples. Then, the samples are spin again for a couple seconds. Then, the mixture of water (630mL) with 10X SDS loading buffer (70 mL) is poured into the gel tank. Next, all samples (10 uL) are taken from the stock and added them into the gel in order of temperatures distinctions. After that, the gel is run at 200 speed for 35 minutes and then 5 minutes later. The gel is taken off the SDS buffer and it is add into the box for fixed step. The fixed step is starting by pouring the water to cover the gel in the box and heating the gel in the microwave for 1-2 minutes to see the air bubbles on the waters surface. After that, the gel is taken and put it on the shaking machine for 5 mins. These steps are making for three times before staining the gel for overnight.

Cell lysis and protein purification

These processes are made by Henrico, who is assistant in this experiment from Cares laboratory member.

Analyse Spy-Tag(ST)/Spy-Catcher(SC) technique in encapsulin with RBD Antigen

This step is made by India, who is assistant in this experiment from Cares laboratory member.

2. Results

Identification of the loop sectors from three species

According to the Uniprot, the number of similar TM encapsulins sequences compared with MXs encapsulins amino acids was 58 (figure 1). It was the same amount of amino acids when comparison between TM and QT encapsulin (figure 1). When contrast MX with QT shell sequences, they had more number of similarity (137) (figure 1). The total similarity of these three sequences were 40 (figure 1).

The results that analysed the three encapsulin nanocompartment via Espript websites were; Both TM and MX had the same formal identification code, which was 58 (figure 2.A). MX and TM had 110 amino acids in common among groups, whereas only 52 amino acids did so within groups (figure 2.A). TM and QT carried the same restrictive identity number (58) (figure 2.B) with TM contrast with MX as well (figure 2.A). 107 amino acids were shared across the group between TM and QT, compared to 58 amino acids shared within the group. MX against QT has a sum of 137 AA for strict matching, 42 AA in group resemblance, and 179 AA of group similarity (figure 2.C). The results for the three enclosed numerals had a rigors match of 40 (figure 2.D) as the same with results from Uniport. The similarity within the group of these encapsulin are 40 while the similarity over group are 80 (figure 2.D).

Discussion

According to the results of the experiment, QT-Enc with ST dose not fused well in the high temperature (37C) which is the range of human body temperature (figure 10). It can cause an issue for our vaccine in the future. We do not know why it happen like that so, we need to study further to find the reason and solution of it. Interesting point of this experiment is that the presented bands above 250 MW from the gel (figure 10). We guess that these bands could be incomplete of hexameric or pentameric protein units to assemble. QT-Enc-Sc attached with ST-RBD presented the unclear band from the SDS-PAGE gel, but it suggested that it can have some combination between them. This also needs further discovery.

However, the previous study showed the success to use the virus-like-particles (VLPs) or encapsulins platform to produce a vaccine to against diseases. a human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis E virus (HEV) vaccines are the example of it?. Referred to the early study, it had similar test of using the ST/SC system to be success for a mammalian approach to display COVID-19 associated consequences linked with a C-terminal SC on ferritin with an N-terminal ST?1;?.

Our hypothesis is rejected because our results showed that QT-Enc dose not assemble into a shell. So, it needs to re-check the areas that inserted ST to ensure its flexible part in the QT-Enc. We might made a mistake to insert ST into the loop regions.

The advantages of this experiment are increased effective vaccine to the variants of COVID-19 spike, low cost to produce vaccine and saved money for delivery and storage of vaccine, if this experiment is successful in the clinical trail and using in the commercial.

The limitation of this experiment is short time to performance in the laboratory and it might be some miss take for analyse the areas, that we want to add combination of the Spy-Tag, linker and His-Tag into QT-Enc.

Future directions

This experiment is more successful to appear the combination of the QT cargo with the ST and His-Tags, it should go to the pre-clinical trail to test. Therefore, it can be use for COVID-19 vaccine in the future. This vaccine could be develop to make it like the insulin implant in a pharmacy shop when it passes through all requirements and process of safety from food and drug administration (FDA).