Recent Progress of Influenza Vaccine Production

Haochen Yu

Faculty of Biology and Food and Engineering, Changshu institute of technology, Changshu, 215500, China

Keywords: Influenza Vaccine, Vaccine Production, MDCK Cells.

Abstract: In today's world of relatively complete public health, people still struggle with flu epidemics. Influenza

vaccine is a good means of protection against influenza. However, there are still some shortcomings in today's

influenza vaccine production technology. This paper focuses on influenza vaccine production. It first

introduces the characteristics of influenza virus and the importance of vaccination. Then it summarizes two

common production methods: the traditional chicken embryo preparation and cell substrate culture taking

Madin-Darby Canine Kidney Cells (MDCK) cells as an example). The chicken embryo method has a long

history, but is limited by low production efficiency and potential antigenicity changes. MDCK cell-based

production has advantages like cell receptors similar to human cells, yet faces challenges such as poor cell

growth and safety concerns. By comparing these methods, the study aims to identify existing problems. The

outlook suggests further research on mammalian cell cultivation and gene--editing to improve vaccine

production, with the ultimate goal of combining big data and artificial intelligence (AI) to predict virus

mutations and enhance vaccine efficiency.

1 INTRODUCTION

Influenza virus belongs to the family

Orthomyxoviridae and is a single-stranded, negative-

stranded RNA virus whose structure includes a core

and an envelope. The core consists of nucleoprotein

and RNA polymerase. The nucleoprotein is type-

specific, and influenza viruses can be categorized into

four types: A, B, C, and D. The envelope contains two

important glycoproteins, hemagglutinin (HA) and

neuraminidase (NA). The envelope contains two

important glycoprotein spines, hemagglutinin (HA)

and neuraminidase (NA). HA binds to host cell

surface receptors and mediates viral entry into the

cell, while NA contributes to the release and diffusion

of newly formed viruses from infected cells, and both

glycoproteins are susceptible to mutation. Influenza

viruses are highly contagious, mainly through droplet

transmission, but also indirectly through contact with

contaminated hands and daily utensils. The average

person is susceptible and usually develops the disease

1-4 days after infection. Symptoms include high

fever, headache, malaise, muscle aches, cough, etc. In

severe cases, the disease can lead to pneumonia,

respiratory failure, and other serious illnesses. In

severe cases, it can lead to complications such as

pneumonia and respiratory failure, and even be life-

threatening. Influenza viruses mutate easily. The HA

and NA of influenza A viruses often mutate, leading

to the emergence of new subtypes and pandemics.

Influenza B viruses also undergo antigenic mutation,

but to a lesser extent, usually causing localized

epidemics. Influenza C viruses are generally

disseminated and relatively mild. Influenza D viruses

infect mainly pigs and cattle and are less pathogenic

to humans. Despite significant improvements in

healthcare and public health, viral infections remain

one of the leading causes of human and animal

disease worldwide. There is no doubt that influenza

poses a considerable risk and potential threat to

human health. Influenza is transmitted by airborne

droplets, is labile and highly contagious, and most

people are susceptible to infection (Jin et al., 2024).

For example, the influenza A virus poses a global and

ongoing threat to human health, causing between

290,000 and 600,000 deaths and as many as 5 million

cases of severe illness each year (Sekiya et al. 2021).

Although people nowadays pay more attention to

public health and prevent influenza by wearing masks

and other behaviors, this only prevents mass

transmission and infection but does not reduce the

likelihood of individuals being infected. It has been

reported that the use of masks etc. does not reduce the

risk of influenza, confirmed viral respiratory

infections, influenza-like illness, or any clinical

respiratory infection. However, in the case of

influenza vaccination, the body's immune system is

192

Yu, H.

Recent Progress of Inﬂuenza Vaccine Production.

DOI: 10.5220/0014446200004933

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of the 1st International Conference on Biomedical Engineering and Food Science (BEFS 2025), pages 192-197

ISBN: 978-989-758-789-4

effectively activated, which can protect an individual

and also control the mass spread of influenza.

Therefore, influenza vaccination is a relatively

economical and convenient way of control (Aimi et

al, 1990). Influenza vaccines are made from

artificially cultured influenza viruses that have been

inactivated and then processed, with the main

components being proteins such as the HA proteins

described above. The traditional method of preparing

influenza vaccines on a large scale is the production

of chicken embryos. However, in the face of a global

pandemic of highly pathogenic influenza, especially

avian influenza, this method does not meet market

demand. Therefore, research organizations and

vaccine companies are collaborating to develop new

production technologies such as cell culture.

Compared with the production of influenza vaccine

from chicken embryos, cell production of influenza

vaccine has the advantages of high production

efficiency and good results. In this paper, we

summarize two common large-scale production

methods, compare them, and analyze their

advantages and disadvantages.

2 MECHANISM OF ACTION

OFINFLUENZA VACCINE

Influenza vaccines can induce the body to produce

immune and cell-mediated immune responses. The

humoral response is mainly based on the systemic

response IgG and the local antibody response sIgA,

and the cellular immune response is primarily based

on the T-cell response. Both responses are capable of

inducing a cross-protective effect. Among them, IgA

mainly prevents the influenza virus from being

absorbed by the body, IgG mainly prevents the lower

respiratory tract from being infected with the virus,

and T cells mainly prevent further expansion of the

viral hazard by specifically recognizing and

removing the virus from infected tissues.

3 INFLUENZA VACCINE

PRODUCTION METHODS

The chicken embryo method of vaccine preparation

began in 1937 as the first successful method of

growing influenza viruses. In 1941, the U.S.

government approved the use of a vaccine prepared

by this method, and in 1945 it was used on a large

scale by the U.S. military, where it proved to be

effective.

This crude vaccine was usually prepared by

injecting the virus into the urocystic blastocysts of

chicken embryos, culturing them for some time, and

then passing them through erythrocytes and a series

of relatively crude isolations, followed by

formaldehyde inactivation of the virus to obtain the

inactivated influenza virus or the crude vaccine.

In the 1960s, thanks to the rapid development and

application of isolation techniques, people were able

to better purify the virus, and the whole virus vaccine

was born. Today, the commercially available chicken

embryo influenza vaccine is highly effective and

protects 90% of those vaccinated. (Belshe et al, 2001,

Belshe et al, 2004, Belshe et al, 2004, Nichol, 2003)

3.1 Inactivated Influenza Whole Virus

Vaccine

The influenza virus was injected into the allantoic

fluid of chicken embryos and incubated until stable.

After stabilization, the chicken embryo allantoic fluid

was removed and inactivated with formalin. After

passing the sterility test and inactivation test, the

inactivated allantoic fluid is separated by

ultracentrifugation or chromatography, and packaged

and reprocessed to obtain the vaccine. The whole

virus-inactivated vaccine obtained by this method has

high side effects, does not apply to children under 6

years of age, and has a relatively narrow application

range.

3.2 Influenza Virus Lysate Vaccine

Based on the whole-virus inactivated vaccine, the

inactivated virus is lysed by selecting a suitable

lysing agent, and only the biomolecules that can be

recognized by the immune system are retained, such

as HA (hemagglutinin protein), NA (neuraminidase

protein), and part of the M proteins (divided into two

kinds, M1 and M2, M1 is responsible for the

assembly of the virus and budding, and M2 is

responsible for the opening of the cellular ion channel

to make the virus survive in the cell, M1 is

responsible for the viral M1 is responsible for virus

assembly and budding, and M2 is responsible for

opening cellular ion channels to make the

intracellular environment acidic, which leads to the

fusion of the viral envelope with the endosomal

membrane. The fusion of the envelope with the

endosomal membrane facilitates the release of RNP

(ribonucleoprotein: wrapped around the genetic RNA

of the virus) and NP (nucleoprotein: tightly bound to

the viral RNA to form RNP) proteins from the virus.

This adaptation is relatively widespread (Luo

Recent Progress of Inﬂuenza Vaccine Production

193

2012.)3.3 Subunit inactivated influenza vaccines

Based on the lysed influenza virus vaccine, HA,

NA, and other proteins are broken down and purified

by appropriate lysis conditions to form a vaccine,

which can be adapted to a larger proportion of the

population than previous methods. This method can

be adapted to a larger proportion of the population,

especially children and the elderly with weakened

immunity, than previous methods.

4 CELL MATRIX CULTURE

Currently, MDCK, PER.C6, AGE.CR and

EB14/EB66 cells have been established and used for

influenza vaccine production (Wen et al, 2015, Chu

et al, 2009, van et al, 2011). Among these cell lines,

the MDCK cell line is currently more mature.

Therefore, in this paper, MDCK cells are used as an

example of cell-matrix culture.

4.1 MDCK Cell Production

Due to the evolution of the virus, vaccines produced

from chicken embryos do not completely prevent

influenza (this is due to some differences between the

receptors on chicken embryo cells and those n the 2

mammalian cells), so the World Health Organization

proposed the use of mammalian cells for vaccine

production in 1995. Among them, MDCK cells were

experimentally tested to be the most suitable for

vaccine production (Huang et al, 2015, Suderman et

al, 2021). MDCK cells, known as Madin-Darby

Canine Kidney Cells, were first established by SH

Madin and NB Darby of the Naval Research

Laboratory at the University of California, Berkeley,

USA, and were derived from the kidneys of healthy

Cocker Spaniel dogs. This cell surface receptor is

more similar to human cell surface receptors, so

influenza viruses do not need to adapt to changes in

cell receptors in culture, reducing the likelihood of

mutation.

4.2 Advantages and Challenges of

Using MDCK Cells for Vaccine

Production

The receptors on the surface of MDCK cells are

similar to those of human somatic cells, which can

prevent viruses from mutating during the culture

process. However, it faces many problems in the

culture process, such as slow growth, high

differentiation, low susceptibility to infection, and

poor stability. In addition, it needs to be attached to

the surface of the carrier in the culture medium for

growth, which is easily limited by the surface area of

the substrate and difficult to realize large-scale

production. Therefore, it needs to be domesticated

during the production process to facilitate its mass

production.

4.3 Preparation of Vaccines from

MDCK Cells

4.3.1 Microcarrier Culture of MDCK Cells

In 1967, Van Wezel (van,1967) introduced the

microcarrier culture method so that the traditional

method was no longer a limitation. Microcarriers are

usually magnetic beads with a diameter of 90-350 μm

and a density slightly greater than that of water

(Alfred et al. 2011). It can support cell attachment

growth with a larger surface area to volume ratio

compared to single cells. Since microcarrier-based

cell culture can be performed in suspension mode,

metabolites, dissolved oxygen, nutrients, and pH of

the medium can be controlled in a timely and efficient

manner; moreover, cells cultured on microcarriers

can better maintain their cellular phenotype because

of the mechanical support provided to them by the

microcarriers during the culture process (Healthcare,

G. E et al ,2005). Some researchers compared the

growth of an influenza virus strain (A/PR8/34) in

MDCK cells (Tree et al, 2001) and compared the viral

yields of different porous and solid microcarrier

cultures, as well as those of conventional cell culture

methods and chicken embryo cultures. The higher

number of cells in solid vector culture may be

because solid vectors have a more suitable surface for

MDCK cell attachment, while porous vectors may

have other limitations such as oxygen and nutrient

transport limitations, and accumulation of wastes

within the structure, in addition to differences in the

concentration of solid and porous vectors per

milliliter of liquid and the speed of agitation that may

also affect the growth of MDCK cells. This effect of

different microcarriers in cell culture has been

demonstrated in other cell lines such as BHK cells

(Alves et al, 1996).

4.3.2 MDCK Cell Suspension Culture

Domestication Technology

This method mainly solves two problems in MDCK

cell culture, one is to solve the walling problem in

MDCK cell culture, and the other is to solve the

serum-dependent problem in MDCK cell culture. At

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present, there are two main ways to domesticate

MDCK suspension cells: (1) Before converting

MDCK cells into suspension cells, they are first

adapted to be cultured in a serum-free medium of

some known composition. This process can be

accomplished by both direct and indirect methods.

The direct method is a complete media change.

Specifically, the direct method of domesticating

MDCK cells is the gradual conversion of traditional

serum-rich medium to serum-free medium. This

process can be carried out in steps, gradually reducing

the proportion of serum while increasing the

proportion of serum-free medium, so that cultured

MDCK cells can gradually adapt to the serum-free

environment and grow stably. An indirect way to

domesticate MDCK cells is to introduce specific

small molecule compounds or biofactors during the

domestication process to facilitate the adaptation of

the cells to serum-free culture conditions. These

biokines and compounds mimic growth factors and

cell signaling pathways found in serum and help

MDCK cells adapt to the serum-free environment.

The goal of these domestication methods is to reduce

or eliminate the serum dependence of MDCK cells

and adapt them to serum-free culture conditions. This

will fulfill biosafety requirements, reduce the

potential risk of exogenous pathogenic factors, and

lower production costs. In conclusion, the

domestication methods of MDCK suspension cells

mainly include direct and indirect methods, which are

cultured under serum-free culture conditions to

gradually reduce the dependence on serum and

provide more applicable cell lines for further research

and production. (2) Gene Modification. It has been

documented that the expression of the full-length

human Siat7e gene correlates with the wall-

dependence of the cells. The Siat7e gene encodes the

human sialyltransferase ST6GalNAc V, which is

responsible for the synthesis of GD1α (glycosidic

aminoglycoside) from GM1b

(monosialylguanoside), and shows higher levels of

Siat7e gene expression in non-adherent-dependent

HeLa cells as compared to adherent-dependent HeLa

cells (Jaluria et al ,2007), and when inhibition of

Siat7e gene expression was used with SiRNA

technology, significantly enhanced adhesion

properties were observed in these cells. Siat7e gene

expression and a significant enhancement of the

adhesion properties of these cells were observed

when Siat7e gene expression was inhibited using

siRNA technology. This implies that the Siat7e gene

may have an inhibitory role in the cell wall

attachment process. This finding made it possible to

make mammalian cells grow without wall adherence

by genetic modification. Chu et al (Chu et al ,2009)

constructed MDCK cell lines stably expressing

Siat7e by altering the adherence-dependence of

MDCK cells through a genetic engineering approach

so that they could be cultured in suspension; they

utilized human Siat7e genes to express Siat7e genes

in suspended MDCK cells. They transformed the

eukaryotic expression plasmid of the full-length

human Siat7e gene into E. coli DH5α, purified and

extracted the plasmid ST6GalNac V for transfection

by the QIAprep Spin Miniprep kit (Qiagen), and then

transfected the MDCK cells with the transfection

reagent Lipofectamine2000 Regent to express the

Siat7e in MDCK cell lines. MDCK cell lines

expressing Siat7e were altered to be non-adhesion

dependent. Results of subsequent production

experiments performed after infection of the

modified cells with influenza B/Victoria/504/2000

virus showed that the cell-derived virus was

antigenically similar to the chicken embryo-derived

virus and its nucleotide sequence was identical. Cells

expressing Siat7e produced hemagglutinin

(expressed as hemagglutination units per 106 cells)

with approximately 20-fold higher specific yields of

hemagglutinin than those produced by parental

MDCK cells.

4.3.3 MDCK Cell Culture for Influenza

Virus Vaccines

Based on the suspension culture technique described

above, it is possible to obtain different MDCK cells

suitable for different production conditions by

obtaining the virus from cells at different stages. If

the virus is cultured in chicken embryos, it needs to

be cultured in MDCK cells first to adapt to the cells.

The cultured virus is added to the cells for co-culture,

allowing the virus to enter the cells and pass on until

the virus is stabilized. Finally, the cell suspension is

collected, clarified filtered, purified by formaldehyde

inactivation, and lysed to obtain the influenza virus

vaccine.

5 TECHNICAL SUMMARY

COMPARISON OF

PRODUCTION METHODS

(COMPARISON OF

DISCOVERED VACCINE

PRODUCTION METHODS)

In terms of production operation, the influenza

Recent Progress of Inﬂuenza Vaccine Production

195

vaccine cultured in the chicken embryo has relatively

less complexity of operation than the influenza

vaccine cultured in MDCK cell culture, and it is

found to be early, has a long period of use, and has a

guaranteed safety, but in terms of product quality the

cells cultured in MDCK cell culture are closer to the

receptors of human cells. In addition, during viral

proliferation, the instability of its RNA leads to

changes in the aspartic acid residue sites of the

expressed proteins, causing glycosylation shifts or

leading to other mutations. This makes the produced

vaccine weak or even ineffective. However, through

the study of Li et al ( Li et al 2021), it was found that

the glycosylation sites and potential sites of viral HA

proteins produced by MDCK cells were more than

those of vaccines produced by chicken embryos, so

MDCK cells were gradually selected for vaccine

production.

6 CHALLENGES AND

PROSPECTS

Limitations of utilizing chicken embryos for

influenza vaccine production include (1) Lower

production efficiency compared to cell culture

making it difficult to meet the demand in case of an

influenza pandemic. (2) Some findings suggest that

the chicken embryo-adapted influenza virus strains

used for vaccine production have three amino acid

mutations in the antigenic sites B [H156Q, G186V]

and D [S219Y] of the strains themselves compared to

the prototype strains recommended by the World

Health Organization and that these mutations may

result in significant changes in the antigenicity,

immunogenicity, and efficacy of the vaccine.

(Skowronski et al, 2014) These challenges may allow

further study of glycosylation of viral surface

proteins. A limitation of influenza vaccine production

with MDCK cells is that its safety needs to be

improved. MDCK cells have been reported to have a

potential risk of tumorigenesis in immunodeficient

mice (Jin et al., 2024). The tumor genesis and

influencing factors can be further investigated.

7 CONCLUSION

The purpose of this study is to summarize and

introduce the production methods of influenza

vaccine and to identify the problems of the existing

production methods. Through this study, it was found

that the process of human vaccine production is a race

against the mutation of pandemic viruses, so in the

future, more in-depth research can be conducted on

the cultivation of influenza vaccines by other

mammalian cells similar to MDCK cells, as well as

due to the extensive use of gene technology, or can be

genetically edited on existing MDCK cells, so that its

receptor can be more compatible with that of human

cells or can be combined with influenza viruses more

smoothly, which can prepare for the emergence of

more potent influenza viruses in the future. It is also

hoped that in the future, it will be possible to make

mammalian cells more resistant to influenza viruses

so that they can be prepared for the emergence of

more potent influenza viruses. It is also hoped that in

the future, the vaccines produced by mammalian cells

will be safer, more stable, and simpler, and can be

customized for the mass production of different types

of influenza. The ultimate goal is to be able to

combine big data and artificial intelligence

technology to predict the mutation of the virus so that

the efficiency of the vaccine produced can be

increased or even reach 100% effectiveness.

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