NGUYỄN THỊ THÚY THE CURRENT STATUS OF mRNA AND OTHER VACCINES MANUFACTURING TECHNOLOGIES IN VIETNAM KHÓA LUẬN tốt NGHIỆP dược sĩ

mRNA TECHNOLOGY AND CURRENT STATUS OF mRNA TECHNOLOGY IN VACCINE MANUFACTURING IN VIETNAM.... IPV Inactivated polio vaccineIVAC Institute of Vaccines and Medical Biologicals IVT In vitro

BỘ Y TẾ

TRƯỜNG ĐẠI HỌC DƯỢC HÀ NỘI

NGUYỄN THỊ THÚY

THE CURRENT STATUS OF

mRNA AND OTHER VACCINES

MANUFACTURING TECHNOLOGIES

IN VIETNAM KHÓA LUẬN TỐT NGHIỆP DƯỢC SĨ

HÀ NỘI – 2022

BỘ Y TẾ

TRƯỜNG ĐẠI HỌC DƯỢC HÀ NỘI

NGUYỄN THỊ THÚY

MÃ SINH VIÊN: 1501484

THE CURRENT STATUS OF

mRNA AND OTHER VACCINES

MANUFACTURING TECHNOLOGIES

IN VIETNAMKHÓA LUẬN TỐT NGHIỆP DƯỢC SĨ

Foremost, I would like to express my sincere gratitude and give my warmestthanks to my supervisors and Associate Professor Dr Dam Thanh Xuan and Dr.Nguyen Khac Tiep who made this work possible Their guidance, support and advicecarried me through all the stages of writing my project I could not have undertakenthis journey without their help and patience

I would also like to thank school committee and professors for teaching,helping and especially inspiring me to see the values that pharmacists can create andcontribute to the society Thanks to all professors and lecturers with their dedicationand support, my 5-year program at Hanoi University of Pharmacy cannot be moreamazing and precious

Finally, I would like to give special thanks to my family and friends as a wholefor their continuous support and understanding

Due to the limited time and shortage of my knowledge, the thesis may haveshortages I sincerely hope to have your feedback on my thesis to make it better

Best regards,

Hanoi, June 2022Student

Nguyen Thi Thuy

TABLE OF CONTENTS

ACKNOWLEDGEMENT

TABLE OF CONTENTS

LIST OF ABBREVIATIONS

LIST OF TABLES

LIST OF FIGURES

ABSTRACT 1

CHAPTER 1 VACCINE DEFINITION AND CLASSIFICATION 2

1.1 Definition 2

1.2 Classification 2

CHAPTER 2 VACCINE MANUFACTURING TECHNOLOGY OVERVIEW IN VIETNAM 3

2.1 Live attenuated vaccine (LAV) technology 5

2.1.1 Overview 5

2.1.2 POLYVAC’s technology 5

2.1.2.1 Rotavirus vaccine (vaccine trade name: Rotavin-M1) 6

2.1.2.2 Measles vaccine vaccine trade name: MVVAC) 7

2.1.2.3 Measles-rubella vaccine (vaccine trade name: MRVAC) 8

2.1.2.4 Polio vaccine (vaccine trade name: bOPV) 8

2.1.3 IVAC’s technology 8

2.2 Inactivated vaccine technology 8

2.2.1 Overview 8

2.2.2 VABIOTECH’s technology 9

2.2.2.1 Cholera vaccine (vaccine trade name: mORCVAX) 9

2.2.2.2 Japanese encephalitis vaccine (vaccine trade name: Jevax®) 9

2.2.2.3 Hepatitis A vaccine (vaccine trade name: Havax®) 10

2.2.3 IVAC’s technology 10

2.3 Toxoid vaccine technology 10

2.3.1 Overview 10

2.3.2 IVAC’s technology 11

2.4 Subunit vaccine technology 11

2.4.1 Overview 11

2.4.2 VABIOTECH’s technology 12

2.4.3 DAVAC’s technology 12

2.4.4 Nanogen’s technology 12

CHAPTER 3 mRNA TECHNOLOGY AND CURRENT STATUS OF mRNA TECHNOLOGY IN VACCINE MANUFACTURING IN VIETNAM 14

3.1 Overview 14

3.1.1 History of mRNA vaccine 14

3.1.2 Applications of mRNA vaccine 17

3.1.3 Advantages and disadvantages of mRNA vaccine 18

3.1.4 mRNA vaccine classification 18

3.1.5 Mechanism of immune response induced by mRNA vaccines 20

3.1.6 mRNA vaccine technology 21

3.1.6.1 Engineered mRNA 21

3.6.1.2.The manufacturing process 23

3.1.6.3 Purification 25

3.1.6.4 Conventional and self-amplifying mRNA design 25

3.1.6.5 Lipid nanoparticles (LNP) technology 26

3.1.7 COVID-19 and mRNA COVID-19 vaccine 29

3.2 mRNA technology in COVID-19 vaccine manufacturing in Vietnam 31

3.2.1 Technology 31

3.2.1.1 STARRTM mRNA technology 31

3.2.1.2 LUNAR® delivery system 32

3.2.2 Advantages and Disadvantages of ARCT-154 34

3.2.3 Clinical Trials 34

3.2.3.1 Phase I (2 trials in total and one of them was executed in Vietnam) 35

3.2.3.2 Phase II (2 trials in total and one of them was executed in Vietnam) 35

3.2.3.3 Phase III (1 trial in total and it is being executed in Vietnam) 35

3.2.3.4 Outcome measures 36

CHAPTER 4 mRNA VACCINE IN THE FUTURE 38

CONCLUSION AND RECOMMENDATION 40 REFERENCES

CHO Chinese hamster ovary

CIGB Center for Genetic Engineering and BiotechnologyCOVID-19 Coronavirus disease of 2019

CQA Critical quality attribute

CTL Cytoxic T lymphocyte

DAVAC Vaccine Company Limited of Dalat Pasteur

DMEM Dulbecco’s Modified Eagle’s

DNA Deoxyribonucleic Acid

DOPE Dioleoylphosphatidylethanolamine

DOTAP 1,2-dioleoyloxy-3-trimethylammoniumpropaneDPT Diptheria, tetanus toxoids and pertussis

dsRNA Double-stranded RNA

E.U.LPS Endotoxin Units lipopolysaccharide

FDA Food and Drug Administration

FFU Fluorecent Focus-Forming Unit

FPLC Fast protein liquid chromatography

HbsAg Hepatitis B surface antigen

HIV Human immunodeficiency virus

HPLC High-performance liquid chromatography

IPV Inactivated polio vaccine

IVAC Institute of Vaccines and Medical Biologicals

IVT In vitro transcribed

LAV Live attenuated vaccine

LNP Lipid nanoparticle

MDA5 Melanoma differentiation-associated gene 5

MERS-CoV Middle East Respiratory Syndrome

MMR Measles, mumps and rubella

MSV Master seed virus

NOD2 Nucleotide Binding Oligomerization Domain Containing 2NRM Non-replicating Mrna

ORF Open-reading frame

PEG Polyethylene glycol

PFU Plaque-forming unit

pKa The acid dissociation constant

PKR Protein kinase R

Pmkc Primary monkey kidney cells

POLYVAC Center for Research and Production of Vaccines and BiologicalsRIG-I Retinoic acid-inducible gene I

RNA Ribonucleic acid

RT-PCR Real time - polymerase chain reaction

SAM Self-amplifying Mrna

SARS-CoV-2 Severe Acute Respiratory Syndrome 2

SARS-CoV Severe Acute Respiratory Syndrome

siRNA Small interfering RNA

SPF Specific Pathogen Free

ssRNA Single-stranded RNA

TAA Tumor-associated antigen

TFH T follicular helper

TLR Toll-like receptor

TNF Tumor Necrosis Facto

USA United States of America

UTR Untranslated region

VABIOTECH Vaccine and Biological Production Company No 1WSV Working seed virus

LIST OF TABLES

Table 2.1 Six Vietnam Vaccine Manufacturers……….3Table 2.2 Six Vietnam Vaccine Manufacturers and their Products……… 4Table 3.1 Primary outcome measures……… 36

LIST OF FIGURES

Figure 3.1 Development timeline of mRNA vaccine [38], [44], [45]……….16Figure 3.2 Two categories of mRNA constructs from designing stage to antigen

expressing stage [54]……….……19Figure 3.3 How mRNA vaccines elicit immunity [59]……… 21Figure 3.4 Five critical quality attributes (CQAs) dictating the performance of mRNAconstruct to express the gene of interest efficiently [54]……… 22Figure 3.5 In vitro transcription reaction component [80]……… 23Figure 3.6 mRNA caps [81]……….24Figure 3.7 Conventional (A) and self-amplifying mRNA (B) vaccine designs [115].26Figure 3.8 Major LNP and polymer delivery methods for mRNA vaccines……… 27Figure 3.9 COVID-19 mRNA vaccine manufacturing process [133]……….30Figure 3.10 Arcturus’s COVID-19 vaccine technology……… 31

The impact of vaccines in reducing the health burden of infectious diseases hasbeen one of the greatest achievements in public health [1] Smallpox, a disease whichcaused more than 300 million deaths since 19thcentury [2], has been eradicated in theworld [1] Although smallpox is the only human disease which has been eradicated,with the advances in technology, there are more diseases which are believed andtargeted for global eradication in the near future

Advances in vaccine manufacturing technology plays an important role inallowing more countries to be able to produce vaccines as well as helping scientists toinvent more vaccines, thereby leading to millions of lives could be saved frominfectious diseases Up to now, mRNA vaccine is the latest type of vaccine which isinvented and approved There are only three approved COVID-19 mRNA vaccines;however, there are 50 mRNA vaccines which are in clinical trials [3] That a lot ofmRNA vaccines are being researched, not only for COVID-19 but also for otherdiseases shows the huge potential of this new vaccine type

As a Global Alliance for Vaccines and Immunization (GAVI) – eligiblecountry, Vietnam has been able to produce the majority of vaccines used in theNational Immunization Program [4] Currently, Vietnam is also executing clinicaltrials in COVID-19 mRNA vaccines [5] Therefore, Vietnam is a country that isproactively producing a lot of vaccine types requiring different manufacturingtechnologies However, there is no any official document about the current status ofvaccine manufacturing technologies being used and mRNA vaccine technology beingresearched in Vietnam Therefore, this thesis is written with these three main purposes:

1 Summarize the current status of vaccine manufacturing technologies used in allVietnamese vaccine manufacturers

2 Provide the overview of the mRNA vaccine technology

3 Provide the basic information about the mRNA vaccine being researched inVietnam

CHAPTER 1 VACCINE DEFINITION AND CLASSIFICATION

1.1 Definition

According to Vietnam Pharmacopeia 5th edition, human vaccine is defined as apreparation containing antigens which can stimulate adaptive immune response toprevent diseases Vaccines are produced from bacteria, Rickettsia or viruses Humanvaccine includes: live attenuated, inactivated microbes and antigenic components ofthe pathogens However, with the fast development of the vaccine manufacturing field,especially with the birth of mRNA vaccines and other modern vaccines, this definitionappears to be not appropriate and updated

According to the USA CDC, vaccine is a preparation that is used to stimulatethe body’s immune response against diseases Vaccines are usually administeredthrough needle injections, but some can be administered by mouth or sprayed into thenose [6]

1.2 Classification

Vaccines can be classified based on different criteria as follows

Based on effects, there are single vaccines and combination vaccines A singlevaccine is used for only one disease (eg: BCG vaccine, influenza vaccine,…), whereas,

a combination vaccine is two or more different vaccines that have been combined into

a single shot and the combined effect is at least the same or better than the singlevaccine (eg: DPT vaccine, MMR vaccine,…) [7]

Based on pathogens, there are virus vaccines (eg: polio vaccine, COVID-19vaccine,…) and bacterial vaccines (BCG vaccine, cholera vaccine,…)

Based on administration routes, there are oral vaccines (eg: polio vaccine),injectable vaccines (MMR vaccine) and others such as inhalable,…

Based on generation, there are first generation vaccine (live attenuated vaccine

or LAV, inactivated vaccine and toxoid vaccine), second generation vaccine (proteinsubunit vaccine and recombinant vector viral vaccine) and third generation vaccine(combination of recombinant vaccine and new excipients in order to stimulate desiredimmune response)

Based on history, there are traditional/conventional vaccines (LAV, inactivatedvaccine, toxoid vaccine, subunit vaccine) and modern vaccines (LAV produced bygene technique, recombinant antigen, DNA vaccine,…)

CHAPTER 2 VACCINE MANUFACTURING TECHNOLOGY OVERVIEW

IN VIETNAM

To date, in Vietnam, there are four vaccine manufacturers which areVABIOTECH, POLYVAC, IVAC and DAVAC These four manufacturers havemanufactured the majority of vaccines used in Expanded Program on Immunization(EPI) in Vietnam Besides, there are two new companies which are doing research onCOVID-19 vaccines as their very first vaccine which sets a milestone of their entryinto vaccine manufacturing field Below are these manufacturers with their products

Table 2.1 Six Vietnam Vaccine Manufacturers

Name

abbreviations

Full names in English Full names in Vietnamese

VABIOTECH Vaccine and Biological

Production Company No 1

Công ty TNHH MTV Vacxin vàSinh phẩm số 1 – Bộ Y Tế (Hà Nội)

POLYVAC Center for Research and

Production of Vaccines and

VinBioCare VinBioCare Biotechnology Joint

Stock Company

Công ty cổ phần Công nghệ sinh học

VinBioCare (Hà Nội)

Nanogen Nanogen Pharmaceutical

Biotechnology Joint Stock

Company

Công ty cổ phần Công nghệ sinh học

dược Nanogen

Table 2.2 Six Vietnam Vaccine Manufacturers and their Products

VABIOTECH Oral cholera, japanese

encephalitis, hepatitis B, hepatitis

VinBioCare Covid-19 vaccine Clinical trial (not approved

2.1 Live attenuated vaccine (LAV) technology

2.1.1 Overview

Live, attenuated vaccines contain a version of the living pathogenic microbethat has been attenuated or weakened in the lab so that it has lost its significantpathogenicity [8]

Manufacturing principle: bacteria or viruses are cultured in special conditions

in order to weaken or remove their pathogenicity, then live attenuated cells arecollected There are two kinds of live attenuated vaccines The first kind of LAV isproduced by isolating pathogenic microbes from animals whose diseases are similar tohumans’ diseases Therefore, this type of vaccine can stimulate protective immuneresponses without causing diseases in humans The second kind of LAV is produced

by isolating pathogenic microbe from patient, then culturing them in vitro in cells andfinally, weakening them by different methods such as heat method for heat-sensitivemicrobes (oral Sabin polio vaccines,…) and biological method of serial passagesthrough a foreign host to mutate genes (measles vaccines,…) [8]

According to vaccine classification based on history, this is the traditionalvaccine Therefore, one disadvantage is the fact that this vaccine requires a cultureprocess Moreover, microbes in LAV are live which means that there is potential forthe vaccine virus to revert to a form capable of causing disease to humans, especiallyimmunocompromised people This vaccine also requires to be kept cool In terms ofadvantages, this vaccine is so similar to the natural infection that only a small amount

of LAV can help prevent and creates a stronger and longer-lasting immune responsecompared to inactivated and subunit vaccines Therefore, for the majority of LAV, 1 or

2 doses can give people a lifetime of protection against a microbe and the disease itcauses [9]

Some LAV are BCG, oral polio, measles, mumps, rotavirus, and yellow fevervaccines

In Vietnam, there are two manufacturers producing LAV, which are POLYVACand IVAC

2.1.2 POLYVAC’s technology

There are the total of 4 vaccines which are produced by POLYVAC and all ofthem are LAV, namely rotavirus, measles, polio and measles-rubella vaccines

2.1.2.1 Rotavirus vaccine (vaccine trade name: Rotavin-M1)

Rotavin-M1 is an oral LAV which is produced on Vero cells Each 2ml vaccinedose contains at least 2x106 PFU rotavirus strain G1P[1], the most common Rotavirusstrain causing acute gastroenteritis and diarrhea in children in Vietnam [10]

POLYVAC has conducted research on rotavirus since 1998 through these 5stages: (1) monitoring diarrhea caused by the rotavirus at the Pediatric hospitals inVietnam during 1998-2011, thereby identifying pathogenic circulating strains by theRT-PCR method; (2) Establishing an attenuated virus seed strains system by cloningthe virus, multiple culture passages of virus lines, Vero cell adaptive virus line, andvirus purification; (3) Establishing a laboratory rota vaccine production process andanimal experiment evaluation; (4) Conducting three phase clinical trials; (5) Licensingand vaccine production [11]

The results of each stage are described as follow [11] Stage 1: Rotavirus strainscirculating in the country in 1998-2011 period were mainly P [1] and G1 Stage 2:Rotavirus strains G1P [1] have been chosen through selective cloning and by multipletissue culture passages; first in MA104 cells, then in primary monkey kidney cells(pMKC) and in Vero cells, then the purified virus on Vero cells was used to establishthe master seed virus (MSV) coded KH0118 and working seed virus (WSV) Stage 3:The vaccine production process: (a) culture Vero cell in 5% fetal calf serumsupplemented DMEM/37oC/3d, then wash with DMEM and incubate the harvestedcells in new DMEM/37oC/3h; (b) activate the WSV strains G1P [1] KH0118 using 15ug/ml trypsin/37oC/30m; (c) virus absorption: empty the cell culture bottles prepared

in step ‘a’, add 3 ml of virus suspension activated in step ‘b’ into the culture bottle toobtain concentrations of infected cells reached to 0.006 FFU/ml/ Absorb/37oC/1h; (d)add fresh medium to the infected cell cultures, then the culture vessels are met with 70

ml of 20 mg trypsin/ml supplemented DMEM pH 7.0 -7.15; then incubate thevessels/37 oC; (e) harvest the vaccine virus suspension at day 3: shake the culturebottles, put them at -20oC, thaw them, transfer the virus solution into plastic bottle, thepool cell solution is preserved at -60oC, and then thaw out once again; (f) after beingthawed, virus suspension is pooled in a big tank, vaccine virus suspension is filtered(0.65 um diameter), semi finished vaccine solutions are preserved at <-60oC; (g) dilute

to have G1P [1] virus concentration in each dose (2 ml) greater or equal to 106.3

PFU/ml with 35% sucrose; (h) fill the Rotavin-M1 in glass vials of single dose, 2ml/dose, storage at ≤-20 oC Preclinical trial: show good results on young rhesusmonkeys, Macaca mulatta Stage 4: Clinical trials: vaccines showed to have a high

immune response, equivalent of that of Belgium Rotarix after using vaccine on 30adults, and 1,000 infants each of 6-12 weeks old Stage 5: The vaccine was licensed tothe manufacture in May 2012.

Principle of attenuating rotavirus pathogenicity: serial passage through foreignhosts, first in MA104 cells, then in primary monkey kidney cells (pMKC) and in Verocells To be more specific, the MA104 cells in 10 g trypsin/ml supplemented DMEMwere infected with 0.25 ml of obtained genetically homogeneous rotavirus (culturecondition: 5% CO2 , 4 cycles/min, 72h, 37oC) Rotavirus lines with the highest titersobtained from six repeated subcultures were selected Then 12 passages wereperformed on primary monkey kidney cells in 10 mg trypsin/ ml supplementedDMEM (same incubation condition as above), again selected the rotavirus cultureclones with the highest titers Next, 0.25 ml of the attenuated virus suspension wasadapted in primary monkey kidney cells and passed 17 times on the Vero cells in 20micrograms of trypsin/ml supplemented DMEM (same condition) After this, rotaviruswas attenuated successfully and went through isolation and purification step toproduce MSV and WSV [11]

2.1.2.2 Measles vaccine vaccine trade name: MVVAC)

MVVAC is a freeze-dried LAV which is produced on specific free-pathogen(SPF) chicken embryo fibroblasts Each 0.5ml vaccine dose contains at least 1000 PFUmeasles virus strain AIK-C Administration route: subcutaneous injection [12]

MVVAC’s manufacturing technology was transferred to POLYVAC fromKitasato Daiichi Sankyo Vaccine Co., Ltd., Japan in 2006 [13] AIK-C strain wasdeveloped independently from wild Edmonston strain by isolating from sheep kidneycells and chicken embryo fibroblasts at 32.5oC WS was cultured on SPF for severaldays and then the supernatant was collected and freeze dried [14]

2-step – producing - AIK-C strain process by Kitasato Institute in 1974: (1)isolating 4 strains from Edmonston strain after 8 passages on sheep kidney cells, thenselecting one strain named AIK which can adapt to 33oC; (2) from this AIK, they

isolated one virus line which contains t marker like-AIK virus and performs higher

yield when cultured in chicken embryo This strain was named AIK-C and used as MS[14]

2.1.2.3 Measles-rubella vaccine (vaccine trade name: MRVAC)

MRVAC is a combination vaccine which consists of live attenuated measlesvirus (AIK-C strain) produced on primary SPF chicken embryo cells and liveattenuated rubella virus (Takahashi strain) produced on SPF primary rabbit kidneycells [15]

Principle of attenuating these two viruses pathogenicity: serial passage throughforeign hosts

2.1.2.4 Polio vaccine (vaccine trade name: bOPV)

bOPV is an oral polio vaccine including type 1 and 3 (Sabin strain) produced onMacaca mulatta monkey (Rhesus monkey) kidney cells and stabilized by magnesiumchloride Each 0.1ml bOPV contains Typ 1: ≥ 106CCID50; Typ 3: ≥ 105.5CCID50 [16]Principle of attenuating polio virus pathogenicity: serial passage on Macacamulatta monkey kidney cells

of Mycobacterium bovis, a related subspecies of M tuberculosis (> 90% homology) by

adding beef bile To be more specific, RD1 region which is crucial to the virulencewas deleted Finally, after 231 passages, they made a live attenuated strain namedBCG [18]

2.2 Inactivated vaccine technology

According to vaccine classification based on history, this vaccine belongs to thetraditional vaccine Therefore, one disadvantage is that it requires culture process Due

to the fact that the microbes are killed, this vaccine produced short and weakimmunogenicity Thus, it requires multiple doses and a large volume per dose Interms of advantages, it is very easy to manufacture and safe to use on humans

Some products are influenza, polio (IPV), hepatitis A, COVID-19 vaccines,…

In Vietnam, there are two manufacturers producing inactivated vaccines, whichare VABIOTECH and IVAC

2.2.2 VABIOTECH’s technology

2.2.2.1 Cholera vaccine (vaccine trade name: mORCVAX)

mORCVAC is an oral inactivated cholera vaccine produced from O1 and O139strain Each cholera vaccine contains: V.Cholerae O1, El Tor, Phil.6973 (beinactivated by formaldehyde) 600 E.U.LPS; V.Cholerae O139, 4260B (be inactivated

by formaldehyde) 600 E.U.LPS; V.Cholerae O1, Cairo 50 (be inactivated byformaldehyde) 300 E.U.LPS; V.Cholerae O1, Cairo 50 (be inactivated by heat) 300E.U.LPS; V.Cholerae O1, Cairo 48 (be inactivated by heat) 300 E.U.LPS [19]

mORCVAX is the first cholera vaccine containing O1 and O139 strain in theworld This vaccine was researched and first licensed by Vietnam in 1997 and untilnow, Vietnam is the only country to use these two strains in cholera vaccine [20].Principle of inactivation: chemical or heat treatment

Manufacturing process: culturing bacteria in suitable medium, inactivating byformaldehyde or heat, centrifugation or filtering, cholera toxin elimination [21]

2.2.2.2 Japanese encephalitis vaccine (vaccine trade name: Jevax®)

Jevax® is an inactivated vaccine produced from Nakayama strain (belong togenotype III virus) on mouse brain Administration route: intradermal injection

Jevax® manufacturing technology is transferred from Research Institute ofOsaka University In 1935, Nakayama strain was isolated from an infected patient’scerebrospinal fluid and maintained by serial passage on mouse brain [22]

Principle of inactivation: chemical treatment

Manufacturing process: (1) insert virus into 3-week-old mice’ brains; (2) collectbrains when mice show symptoms; (3) centrifugate, ultrafilter, protamine sulfateprecipitation and zonal centrifugation on different sucrose concentrations ; (4) dilutewith phosphate buffer system (5) stabilize by adding gelatin, natri glutamate andthimerosal [23].

2.2.2.3 Hepatitis A vaccine (vaccine trade name: Havax®)

HAVAX® is a suspension containing inactivated hepatitis A virus (HM 175hepatitis A strain absorbed onto aluminum hydroxide This virus is cultured onprimary Maccaca mulatta monkey kidney and inactivated by formaldehyde Each mlvaccine contains purified Hepatitis A virus antigen less than 200ug Administrationroute: intramuscular injection [24]

2.2.3 IVAC’s technology

IVAC produce inactivated pertussis toxin which then is added to be a part inDPT vaccine (diphtheria- pertussis -tetanus)

No public information on technology is published by the manufacturer

2.3 Toxoid vaccine technology

2.3.1 Overview

Toxoids are bacterial toxins, usually the exotoxins, secreted by pathogens thatcause many of the disease symptoms after infection Toxoid vaccines (e.g vaccines fordiphtheria and tetanus) are made by purifying the bacterial exotoxin Toxicity ofpurified exotoxins is then suppressed or inactivated either by heat or withformaldehyde (while maintaining immunogenicity) to form toxoids [25]

Toxoid vaccines mechanism: this vaccines stimulate body to secrete anti-toxoidantibodies which can bind with the toxin and neutralize its effects Thus, this technique

is reserved for diseases in which the secreted toxins are the main cause of the illness[25]

According to vaccine classification based on history, this vaccine belongs totraditional vaccine Therefore, one disadvantage is that it requires culture process Italso generates weak immunogenicity, thereby requiring multiple doses Moreover, theprocedures for the production of toxoid vaccines ought to be strictly controlled toachieve detoxification/inactivation without excessive modification of the antigenic

epitope structure However, thanks to the fact that there are no live microbes, thisvaccine is safe to use on humans.

Some products are tetanus and diphtheria vaccines

In Vietnam, there is only one manufacturer that produces toxoid vaccines, which

is IVAC

2.3.2 IVAC’s technology

IVAC is producing one toxoid vaccine which is combination vaccine of tetanusand diphtheria with the trade name of Td

Tetanus toxoid’s manufacturing process: (1) culture C.tetani on Muller-Miller

medium; (2) inactivate tetanus toxoid by formaldehyde; (3) purify tetanus toxoid bysuperfiltering and fractional salt precipitation, eliminate salt by columnchromatography; (4) add aluminum hydroxy as adjuvant and adsorb on aluminiumphosphate [26]

Diphtheria toxoid’s manufacturing process: (1) culture Corynebacterium diphtheriae 1 to 3 days; (2) centrifugate, separate supernatant, superfilter, precipitate

toxoid by ammonium sulfate, purify toxoid by column chromatography, inactivatetoxoid by formalin or formaldehyde; (3) adsorb on aluminium phosphate [27]

2.4 Subunit vaccine technology

2.4.1 Overview

Subunit vaccine are composed of protein or glycoprotein components of apathogen that are capable of inducing a protective immune response after eliminatingall other pathogen parts which are not antigenic [28]

Manufacturing principle: identify and remove pathogen parts which are toxic butnot antigenic, isolate and purify pathogen parts which are antigenic Subunit vaccinescan be produced by conventional biochemical or recombinant DNA technologies [29]

In term of disadvantages, subunit vaccines may be more expensive and mayrequire specific adjuvants to enhance the immune response Moreover, these vaccineshave less strong immune response compared to LAV However, subunit vaccinesperform excellent stability profile They have no live components which means there

is no risk of inducing diseases [28]

Some products are Hepatitis B, typhoid, COVID-19 vaccines,…

In Vietnam, there are two manufacturers which produce subunit vaccines, whichare VABIOTECH and DAVAC Nanogen is also conducting clinical trials on theirCOVID-19 subunit vaccine This part will mention these three companies althoughproduct of Nanogen is not approved yet

Manufacturing process: (1) Identify the sequence of gene which codes forHbsAg antigen; (2) insert plasmid containing this gene into S.cerevisiae; (3) culturethis recombinant yeast in the medium containing yeast extract, peptone, dextrose,amino acids and salt; (4) Lysis the yeast by hydrophilic interaction and size exclusionchromatography to extract HBsAg, then HbsAg is wrapped by 22nm lipoprotein andpurified by a series of physical and chemical methods; (5) Purified protein is treated inphosphate buffer system with formaldehyde, sterile filtration and precipitation withaluminium to make vaccine having aluminium sulfate as the adjuvant [31]

S-CoV-2 was then absorbed into aluminum hydroxide gel adjuvant (Alhydrogel®; Croda,Denmark) [33], [34].

Manufacturing process (not industrial scale): (1) Identify gene coding forantigenic protein S, M, E, N in SARS-COV-2; (2) insert plasmid containing this geneinto CHO cells; (3) culture CHO cells in suitable condition; (4) adsorb onto aluminiumand add adjuvant [33]

CHAPTER 3 mRNA TECHNOLOGY AND CURRENT STATUS OF mRNA TECHNOLOGY IN VACCINE MANUFACTURING IN VIETNAM

3.1 Overview

3.1.1 History of mRNA vaccine

Although mRNA vaccines are newly available to the public and the COVID-19vaccines made by Pfizer/BioNTech are known to be the first mRNA vaccine tocomplete all clinical trial stages and be licensed for use in December 2020 [35], thetechnology has been studied since 1970s [36]

mRNA was first discovered in the early 1960s [37] Through a longdevelopment history with countless researches, there were some milestones In late

1987, a scientist named Robert Malone mixed strands of messenger RNA withdroplets of fat, to create a kind of molecular stew which he used to bath the human cell[38] Then he discovered that the human cells absorbed the mRNA, and beganproducing proteins from it, which inferred that RNA can be treated potentially as adrug [39] It was the first time anyone had used fatty droplets to ease mRNA’s passageinto a living organism [38] In 1984, Krieg and other scientists at Harvard Universityused an RNA-synthesis enzyme (taken from a virus) to produce biologically activemRNA in the lab, which then was injected into frog eggs and showed to produceprotein like the real thing [40], [41] Years later, Malone added a new kind of liposomethat carried a positive charge and was developed by Philip Felgner In 1989, he andInder Verma showed that the lipid–mRNA complexes could spur protein production inmice [42], which was the very first time mRNA could be potentially treated as avaccine In 1993, a team led by Pierre Meulien was the first to show that an mRNA in

a liposome could elicit a specific antiviral immune response in mice [43] In 1997, EliGilboa proposed taking immune cells from the blood, and coaxing them to take upsynthetic mRNA that encoded tumour proteins; next the cells would then be injectedback into the body where they could marshal the immune system to attack lurkingtumours [38] Inspired by Gilboa, Hoerr founded CureVac and succeeded elicit animmune response in mice and then executed the human testing In 2007, not only wasvaccine firm BioNTech founded by two spouses scientists; biochemist Katalin Karikóand immunologist Drew Weissman at the University of Pennsylvania (UPenn) inPhiladelphia also founded RNARx after making a key finding: that altering part of themRNA code helps synthetic mRNA to slip past the cell’s innate immune defences[38] .The first mRNA vaccine which successfully entered preclinical trial was fluvaccine in the 1990s [36] Not until two decades later could human-beings observe the

first mRNA vaccine, rabies vaccine tested in humans in 2013 [36] Finally, vaccinetechnology has a huge breakthrough when the first mRNA vaccine was approved forhuman use against SARS-CoV-2 in 2020 The detailed history timeline is shown in thefigure below:

Figure 3.1 Development timeline of mRNA vaccines [38], [44], [45]

3.1.2 Applications of mRNA vaccine

During the last two decades, mRNA vaccines have been investigatedextensively for not only infectious diseases but also cancer prophylaxis and therapy.The basic principle of mRNA therapeutics involves delivery of in vitro transcribedmRNA into a target cell, where cellular machinery translates the mRNA into afunctional protein In vaccine applications, an mRNA encoding a viral protein willelicit a protective immune response, whereas in therapeutic applications, an mRNAencoding an absent (or dysfunctional) protein in a patient provides functional proteinexpression Thus, mRNAs can be widely used in vaccine development, proteinreplacement therapy, and the treatment of genetic diseases

Cancer mRNA vaccines were designed to express tumor-associated antigensthat stimulate cell-mediated immune responses to clear or inhibit cancer cells [46].mRNA cancer vaccines combine the potential of mRNA to encode for almost anyprotein with an excellent safety profile and a flexible production process Duringvaccination, naked or vehicle loaded mRNA vaccines efficiently express tumorantigens in antigen-presenting cells (APCs), facilitate APC activation andinnate/adaptive immune stimulation The vaccines that target tumor-associated ortumor-specific antigens (TAAs or TSAs) can specifically attack and destroy malignantcells that overexpress the antigens and achieve chronic therapeutic response because ofimmunologic memory Therefore, cancer vaccines offer specific, safe, and tolerabletreatment compared to other immunotherapies [47] This is exemplified by theRNActive® technology, which induces balanced humoral and cellular immuneresponses in animal models and is currently evaluated in several clinical trials foroncologic indications [48] There are four types of cancer vaccines, including tumor orimmune cell-based vaccines, peptide-based vaccines, viral vector-based vaccines, andnucleic acid based vaccines [49] Up to now, over twenty mRNA-basedimmunotherapies over all above four cancer vaccine types have entered clinical trialswith some promising outcomes in solid tumor treatments and U.S FDA has recentlyapproved two prophylactic vaccines, one for human papillomavirus (HPV) thataccounts for 70% of cervical cancers, and another for hepatitis B virus that can causeliver cancer More encouragingly, PROVENGE (Sipuleucel-T), an immune cell-basedvaccine has been approved by the U.S FDA in 2010 as the first therapeutic cancervaccine for treating hormone-refractory prostate cancer patients [47] Besides, a recentpublication by Pardi and colleagues showed that mRNA encoding the light and heavychains of a broadly neutralizing anti-HIV antibody encapsulated in lipid nanoparticles

suggest that the utilization of nucleoside-modified mRNA can be expanded for passiveimmunotherapy against HIV, cytomegalovirus (CMV), human papillomavirus, etc [51].

3.1.3 Advantages and disadvantages of mRNA vaccine

Vaccination is considered as the most effective way to prevent infectiousdiseases However, regarding traditional vaccines, one of the most noticeabledisadvantages is that these conventional vaccines require a lot of time for the growth

of highly pathogenic organisms at a large scale Therefore, they are not goodcandidates for emerging diseases That is when a quick-manufactured mRNA vaccinecan become the powerful tool which can fill the gap between a disease epidemic and adesperately needed vaccine One advantage of mRNA vaccines are egg and cell free,rapid and scalable production as making mRNA vaccines do not require egg or cellculture Therefore, within weeks, clinical batches can be generated after theavailability of a sequence encoding the immunogen Moreover, due to the lack of viralstructural proteins, the replicon does not produce infectious viral particles, whichmeans mRNA vaccines are non-infectious Additionally, mRNAs cannot potentiallyintegrate into the host genome and will be degraded naturally during the process ofantigen expression [51] Finally, new targets requiring multi-antigen approaches willbenefit from the speed in which mRNA can render multiple constructs

However, the biggest challenges are about its instability and lowimmunogenicity In terms of instability, mRNA could be taken up by the body andquickly degraded before it could be translated into proteins in the cells The solution tothis problem is the nanotechnology which uses fatty droplets (lipid nanoparticles) towrap the mRNA, thereby delivering mRNA safely to the cells and the optimal design

of mRNA which will be discussed in details in the next chapter Once inside the cell,the mRNA could be translated into proteins which then are presented on the cellsurface and recognized by the immune system Due to low immunogenicity, mRNAvaccines usually require a boost dose after the first dose However, past studies haveshown that the immunostimulatory ability of mRNA can be influenced by theintroduction of modified nucleosides aside from complexing mRNA with variouscarrier molecules [52]

3.1.4 mRNA vaccine classification

Currently, two forms of mRNA vaccines have been developed: conventionalmRNA vaccines and self-amplifying mRNA vaccines, which are derived from positivestrand RNA viruses Compared with the rapid expression of conventional mRNAs,

published results have shown that vaccination with self-amplifying mRNA vaccinesresults in higher antigen expression levels, although delayed in time, which persist for

several days in vivo Equivalent protection is conferred but at a much lower RNA dose

[53] The biggest difference between these two kinds of vaccines is the mRNAconstruct either conventional mRNA (non-replicating mRNA or NRM) or self-amplifying mRNA (SAM), thereby leading to different translation process in cells

Figure 3.2 Two categories of mRNA constructs from designing stage to antigen

expressing stage [54]

Non-replicating mRNA (NRM) constructs encode the coding sequence (CDS),and are flanked by 5′ and 3′ untranslated regions (UTRs), a 5′-cap structure and a 3′-poly-(A) tail [54] The self-amplifying mRNA (SAM) construct encodes additionalreplicase components able to direct intracellular mRNA amplification [54] Following

is the detailed flow of mRNA from designing stage to antigen expressing stage: (1)NRM and SAM are formulated in this illustration in lipid nanoparticles (LNPs) thatencapsulate the mRNA constructs to protect them from degradation and promotecellular uptake; (2) Cellular uptake of the mRNA with its delivery system typicallyexploits membrane-derived endocytic pathways; (3) Endosomal escape allows release

of the mRNA into the cytosol; (4) Cytosol-located NRM constructs are immediatelytranslated by ribosomes to produce the protein of interest, which undergoes subsequentpost-translational modification; (5) SAM constructs can also be immediately translated

by ribosomes to produce the replicase machinery necessary for self-amplification ofthe mRNA; (6) Self-amplified mRNA constructs are translated by ribosomes toproduce the protein of interest, which undergoes subsequent post-translational

modification; (7) The expressed proteins of interest are generated as secreted,

trans-membrane, or intracellular protein; (8) The innate and adaptive immune responsesdetect the protein of interest [54]

3.1.5 Mechanism of immune response induced by mRNA vaccines

The core principle behind mRNA as a technology for vaccination is to deliverthe transcript of interest, encoding one or more immunogen(s), into the host cellcytoplasm where expression generates translated protein(s) to be within the membrane,secreted or intracellularly located [51] In reality, the immune response mechanisminstigated by mRNA remains to be elucidated The process of mRNA vaccinerecognition by cellular sensors and the mechanism of sensor activation are still notclear Intracellularly, two kinds of RNA sensors, endosomal toll-like receptors (TLRs)and the RIG-I-like receptor family, have been identified [51] Upon entry into cells,single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) are recognized bythese receptors TLR bind to ssRNA in the endosome, while components of theinflammasome such as MDA5, RIG-I, NOD2 and PKR bind to ssRNA and dsRNA inthe cytosol, resulting in cellular activation, and production of type I interferon [55]–[57] and multiple inflammatory mediators [58] Dendritic cells (DCs) are activatedand present antigen and co-stimulatory molecules to antigen-specific naive T cells,which become activated and differentiated into effector cells to form cytotoxic Tlymphocytes or helper T cells T follicular helper (TFH) cells help S protein-specific Bcells to differentiate into antibody-secreting plasma cells and promote the production

of high affinity anti-antigen antibodies Following vaccination, antigen-specificmemory T cells and B cells develop and circulate along with high affinity antibodies,which together help prevent subsequent infection with the diseases

Figure 3.3 How mRNA vaccines elicit immunity [59]

3.1.6 mRNA vaccine technology

There are two most important factors in terms of mRNA technology, whichare the synthetic mRNA and the lipid nanotechnology That is the reason why theGrand Prize of Vietnam-Founded Vinfuture Prize 2022 valued at $US3 million wasawarded to three scientists: Katalin Kriko (USA) - Drew Weissman (USA) as thefounders of mRNA application in vaccine and medicine; and Pieter Cullis (Canada) asthe founder of lipid nanoparticle technology (LNP) in mRNA vaccine [60]

3.1.6.1 Engineered mRNA

Regarding the first factor, mRNA can now be synthetically produced through

a cell-free enzymatic transcription reaction Both conventional mRNA (non-replicatingmRNA or NRM) and self-amplifying mRNA (SAM) have in common a cap structure,5′ and 3′ untranslated regions (UTRs), an open-reading frame (ORF), and a 3′ poly(A)tail [58] SAM differs with the inclusion of genetic replication machinery derived frompositive-stranded mRNA viruses, most commonly from alphaviruses such as Sindbisand Semliki-Forest viruses [61], [62] Generally, the ORF encoding viral structuralproteins is replaced by the selected transcript of interest, and the viral RNA-dependentRNA polymerase is retained to direct cytoplasmic amplification of the repliconconstruct [54]

Figure 3.4 Five critical quality attributes (CQAs) dictating the performance of

mRNA construct to express the gene of interest efficiently [54]

Five principal CQAs include (1) 5′ capping efficiency and structure; (2) UTRstructure, length, and regulatory elements; (3) modification of coding sequence; (4)poly-A-tail properties; (5) mRNA purity [54] To be more specific, first, the purity ofthe mRNA is a crucial determinant of yields, and it is known that the DNA-dependentRNA polymerases yield smaller oligoribonucleotide impurities as a result of abortiveinitiation events [63], as well as double-stranded (ds) RNA generated by self-complementary 3′ extension, [64] which can result in type I interferon andinflammatory cytokine production through pattern recognition receptors Karikó et al.[65] demonstrated that removal of contaminants in mRNA preparations reduced innateimmune responses and resulted in significantly higher levels of reporter proteinexpression in vitro Second, the 5′ and 3′ UTR regions are important for maximizinggene expression The length of the 3′ UTR, [66] 5′ UTR structures, and regulatoryelements in both UTRs [67] all impact efficiency Third, the 5′ 7-methylguanosine(m7G) cap of the mRNA molecule, linked via a triphosphate bridge to the firsttranscribed nucleotide, is essential for efficient translation, and blocks 5′–3′exonuclease-mediated degradation [54] The specific cap structure plays a critical role

in both protein production and immunogenicity, with incomplete capping (5′triphosphate) and Cap 0 structures shown to stimulate RIG-1 [68]–[70] In addition, 2-O′-unmethylated capped RNA can be sequestered by cellular IFN-induced proteinswith tetratricopeptide repeats (IFIT1) that prevent the initiation of translation, [71] or

detected by the cytoplasmic RNA sensor MDA5 [72] Manufacturers of mRNAvaccines pay careful attention to the choice of enzyme and reaction conditions, inorder to catalyze the highest percentage of cap formation Fourth, the poly (A) tail andits properties such as length, are crucial for translation and protection of the mRNAmolecule [73], [74] Last, codon optimization and modification of nucleotides havecontributed to translation efficiency For example, optimization of guanine andcytosine (GC) content can have a significant impact [75] and has been well establishedwith DNA vaccines The innate immune activation to mRNA can also influence itsutility as a delivery system The use of modified nucleosides, such as pseudouridine orN-1-methylpseudouridine to remove intracellular signaling triggers for protein kinase

R (PKR) activation, resulted in enhanced antigen expression and adaptive immuneresponses [76]–[78]

3.6.1.2.The manufacturing process

The manufacturing process begins with the generation of a plasmid DNA(pDNA) containing an RNA polymerase promoter, such as T7 [79] and the

corresponding sequence for the mRNA construct The in vitro transcription reaction

includes a linearized plasmid DNA encoding the mRNA vaccine, as a template, anRNA polymerase, and nucleoside triphosphate as essential components [54]

Figure 3.5 In vitro transcription reaction component [80]

A cap structure - an N7-methylated guanosine linked to the first nucleotide ofthe RNA via a reverse 5′ to 5′ triphosphate linkage, is enzymatically added to thetranscriptional product at the end of the reaction or as a synthetic cap analog in asingle step procedure [54] In addition to its essential role of cap-dependent initiation