Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy

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Viral vaccines: concepts,

principles, and

biopr ocesses

Isabel Maria Vicente Guedes de Carvalho Mello,

Mateus Meneghesso da Conceic¸a

o, Soraia Attie Calil Jorge,

Pedro Estilita Cruz, Paula Maria Marques Alves,

Manuel Jose

Teixeira Carrondo, and Carlos Augusto Pereira

18.1 Introduction

Animal cell culture technology has always played a major role in the

development of virology. Signiﬁcant progress in the propagation of viruses

and the consequent development of viral vaccines were only made possible

after the 1950s, upon the establishment of cell culture technology. Animal

cell culture gradually substituted live animals in the preparation of viral

antigens used in vaccination, such as the vaccines against smallpox and

rabies. At the same time, the production of viral antigens in cell cultures

led to considerable progress in bioprocesses technology. Cell culture

bioprocesses are now well established in bioreactors of up to 12 000 L.

Recent developments in virology and cell culture technology have

allowed research and development laboratories to engage in molecular

manipulation of viruses and cells for bioprocess production of viral gene

products. Examples are the establishment of recombinant viral vectors for

expression in animal cells that have signiﬁcant potential in producing

recombining vaccines and treatment by gene therapy. Thus, a recombinant

vaccine against hepatitis B virus (HBV) has been developed and several

others are in the ﬁnal phases of clinical trials.

This chapter concerns the development of cell culture technology for

viral vaccine production, which is related to: (a) a need for prophylaxis

and/or treatment of the most important viral diseases, such as AIDS,

hepatitis C, inﬂuenza, and papillomavirus; (b) the establishment of current

molecular technologies, and (c) a reduction in risk factors for the manip-

ulation of live viral particles.

Cell culture technology for the production of viral products is focused

on establishing protocols for recombinant products of low risk to prevent

viral diseases. In many countries, viruses are increasingly important as

biological agents for the control of agricultural pests (see Chapter 19).

18.2 Viral replication

The pathological effects of viral diseases are the consequence of various

factors, such as the toxicity of the products of metabolism of infected cells

and host reaction to infected cells expressing viral protein, as well as

changes in the genetic expression of host cells through structural or

functional interactions with genetic material or viral proteins. In some

cases, the symptoms or acute signs of diseases caused by a virus can be

directly related to the elimination of the infected cells.

For a better understanding of the pathological effects caused by viral

infections and of their control by vaccination or antiviral therapy, it is

important to understand how viruses infect cells, express their genes,

multiply, and change the cellular metabolism after the infection. The

genetic characteristics of the host as well as its sensitivity, are factors that

must be considered in evaluating the magnitude of viral replication.

Viruses are exclusively intracellular organisms and therefore depend on

the cells to multiply. A complete viral particle, or virion, consists of one

nucleic acid molecule (RNA or DNA) covered by a protein layer (nucleo-

capsid). Some virions have a lipid cover with a glycoprotein envelope. The

main function of the virion is to transport the viral genome to the interior

of the host cell to be replicated and ampliﬁed.

The infectivity of host cells varies considerably between viruses. A

speciﬁc virus may have a great diversity of host cells, while another may

be capable of infecting only one type of cell. The sensitivity deﬁnes the

capacity of the cell or animal to be infected.

Viral multiplication involves different ways of replication. However,

there are some common characteristics in the replicative cycles of viruses.

Initially, viruses insert their genetic material (RNA or DNA) into the cell,

and the size, composition, and genetic organization of this material vary

signiﬁcantly between viruses, as well as the proteins that are needed for

replication. After the infection, there is a period called the eclipse phase,

when only few viruses are found in the infected cells. During this phase,

the genome and all the viral machinery is exposed to the host, but the viral

progeny is still small. Afterwards, there is a pause when virions accumulate

inside or outside of the cell at an exponential rate. This pause is called the

maturation phase. After some hours, lytic viruses cause cellular lysis with

the cessation of all metabolic activity and the cells lose their structural

integrity. Cells infected by non-lytic viruses can continue virion synthesis

over a long period of time.

The reproductive cycle of viruses may take hours or days. The infection

of cells does not guarantee the production of viral progeny, which may be

productive, restricted, aborted, or latent. A productive infection occurs in

permissible cells and results in infectious viral particles. An abortive

infection may occur in two circumstances: ﬁrstly, although the cell is

sensitive to infection, it is not necessarily permissive, allowing the expres-

sion of only a few viral genes. The second circumstance is when a sensitive

cell, permissive or not, is infected by defective viruses that do not have all

the necessary viral genes for their replication. Furthermore, the cells can

be temporarily permissible. In this case, the viral particles may remain in

the cells until they become permissive or else some viral particles may be

436 Animal Cell Technology

produced for a limited period of time by a fraction of the cell population.

This type of infection is called restricted. In a latent infection, the viral

genome persists in temporarily permissive cells without the destruction of

the infected cells.

The following sections outline the stages of the replication used by

viruses.

18.2.1 Adsorption

This stage consists of the virus binding to the cell. This involves the

speciﬁc binding of a glycoprotein that appears on the external structure of

a virus to a host cell receptor.

The sensitivity of a cell to a speciﬁc virus is frequently related to the

presence of these receptors in the cell membrane. The concept of sensitiv-

ity is not related to permissiveness. A cell may not be sensitive to a certain

virus due to lack of receptor, but may produce viral progeny if its viral

genome is introduced into its cytoplasm.

18.2.2 Internalizing and unwrapping the viral particle

After the binding, the virus may use various means of penetration: (a)

direct penetration, via the translocation of the entire virus through the

cytoplasmic membrane; (b) endocytosis, which is mediated by receptors,

resulting in the formation of intercytoplasmic vesicles containing many

viral particles; (c) direct fusion of the viral envelope with the cytoplasmic

membrane.

Non-enveloped viruses generally use the ﬁrst two penetration mechan-

isms, while the enveloped viruses enter a cell by endocytosis followed by

binding with the membrane of an endosome. In addition to this mechan-

ism, the enveloped viruses fuse directly with the cell membrane. The

fusion of the viral envelope with the cell membrane requires the inter-

action of the glycoproteins of the virus with a cell receptor. After the

internalization of the viral particle, the genome is freed for later expres-

sion. This process is known as unwrapping and it involves both cellular

and viral enzymes.

18.2.3 Structure and organization of viral genomes

Viral genomes consist of RNA or DNA, which can be single- or double-

stranded, and may consist of one or more fragments. During viral replica-

tion, both DNA and RNA viruses synthesize protein by translation of

messenger RNA. The mRNA is then translated by the cell into the viral

proteins that will constitute the viral particles.

Virus with single-strand RNA

There are three groups of virus with a single-stranded RNA genome

(ssRNA). The ﬁrst group comprises viruses with RNA that functions both

as genomic and messenger (for example, picornovirus and ﬂavovirus). The

ssRNA in these viruses is conventionally called positive-strand virus

Viral vaccines: concepts, principles, and bioprocesses 437

(+sRNA). After it enters the cell, the +sRNA functions as mRNA, binding

with cellular ribosomes to complete its translation into proteins. The

product of this translation is a polyprotein that will later be cleaved.

The second function of this RNA is genomic, to be used as a precursor for

the synthesis of a complementary negative RNA strand (RNA–) catalyzed

by a polymerase derived from the polyprotein cleavage. The negative

strand will be transcribed once again as genomic RNA (RNA+) through a

viral polymerase. During this process, several strands of genomic RNA

will be produced, as well as proteins that will be used to produce the viral

particles. One characteristic of the RNA+ virus replication is the capacity

of its genomic RNA to function as mRNA after the infection, thus leading

to the synthesis of the enzymes responsible for the viral genome replica-

tion, without need for the complete viral particle to carry the enzymes

(Figure 18.1). This allows the RNA extracted from the virus to become

infectious, although less than the complete viral particle.

Another group of ssRNA viruses consists of those that have negative

single-strand RNA (–sRNA). This group consists of orthomyxovirus,

paramyxovirus, and rhabdovirus, among others. The genomic RNA of

these viruses has two functions: to serve as a template for transcription and

for replication. The viral genome must translate its own mRNA in order

to synthesize viral proteins because the host cell does not have the

appropriate enzymes. Therefore the –sRNA virus will have a transcriptase

along with its genome. The isolated viral genome is not infectious, because

there must be a viral transcriptase to translate the genomic RNA into

several mRNA strands that will later be translated into viral proteins.

These are used to make a copy of all its genome, producing a large strand

Viral progeny

Viral RNA

strand )(⫹

Polyprotein coded by

viral genome

RNA

strand ( )⫺

Viral proteases

RNA

strand ( )⫹

Figure 18.1

Flow of events during the replication of a positive-strand RNA virus.

438 Animal Cell Technology

of +mRNA that will serve as a template for the synthesis of genomic –

RNAs (Figure 18.2).

The retroviruses are the third group of single-strand RNA virus. These

viruses have a more complex strategy for the production of mRNA. The

retroviruses have a diploid genome associated with a DNA-dependent

RNA polymerase (reverse transcriptase) that transcribes RNA into a

hybrid DNA–RNA. The RNA strand is digested by the viral ribonuclease

H and a complementary DNA is synthesized, thus producing a double-

strand DNA (dsDNA) that will be inserted into the cellular genome with

the participation of the viral integrase enzyme, producing a provirus

(Figure 18.3). After the integration, the cellular-dependent polymerase

RNA–DNA will initiate the synthesis of the proteins that are essential to

replicate the viral genome followed by the synthesis of proteins to produce

the virus.

Virus with double-strand RNA

The genome of reoviruses is formed by double-strand RNA (dsRNA)

composed of several dsRNA fragments (10–12). The RNA fragments are

transcribed inside a capsid by a polymerase, giving rise to several mRNAs.

Here, the mRNA molecules have two functions: (a) they are translated as

monocistronic messengers, forming viral proteins; (b) they serve as a

template for the synthesis of a complementary RNA strand, giving rise to

double-strand RNA genomic fragments (Figure 18.4).

Viral RNA

strand ( )⫺

⫹mRNA

Proteins

RNA

strand ( )⫹

Progeny RNA

strand ( )⫺

Viral progeny

Viral enzymes

Figure 18.2

Flow chart of events during the replication of negative single-strand RNA virus.

Viral vaccines: concepts, principles, and bioprocesse s 439

Viral progeny

tRNA

viral enzymes

RNA progeny

mRNA

Linear DNA

Viral RNA

Integrate in

DNA

Proteins

Figure 18.3

Flow chart of event s during the replication of retroviruses.

Double-strand

RNA progeny

Viral progeny

Partial production

Double-strand

viral RNA

Proteins

Viral enzymes

sRNA

( ) strand⫹

Figure 18.4

Flow chart of events during the replication of reovirus.

440 Animal Cell Technology

Virus with DNA

The double-strand DNA viruses are transported into the nucleus where

they translate and replicate their genome using cellular enzymes to

produce mRNA. These mRNA strands are later translated into proteins

that direct the synthesis of the proteins and genomes of the virus. The

isolated DNA of these viruses is considered infectious (Figure 18.5). There

are some double-strand DNA viruses, such HBV, which have a different

replication strategy that includes an intermediary RNA and a reverse

transcription phase. In this case, after the viral DNA penetrates the

nucleus of the cell, it is converted into a covalently closed circular DNA

molecule (cccDNA). Before the viral genome replication occurs, a cellular

enzyme (DNA-dependent RNA II polymerase) translates several RNAs

from cccDNA and genomic and subgenomic RNAs (Figure 18.5). Most

DNA viruses replicate in the nucleus of the cell, except for poxviruses,

which replicate in the cytoplasm. These viruses are practically autonomous

in terms of translation factors. Parvoviruses have a single-strand DNA

genome and replicate inside the cellular nucleus using replicative-cycle

cellular polymerases.

Circular genomic

DNA

Covalently closed

DNA

mRNA

strand ( )⫹

Protein

Genomic DNA

Viral enzymes

Viral progeny

Genomic RNA

Figure 18.5

Flow chart of events during the replication of the hepatitis B virus.

Viral vaccines: concepts, principles, and bioprocesses 441

18.2.4 Production and maturation of viral particles

Viruses use three strategies to assemble, mature, and release from infected

cells. With the ﬁrst strategy, represented by picornavirus and adenovirus,

the assembly and maturation are completed inside the cell, either in the

cytoplasm or in the nucleus. As a rule, all non-enveloped viruses are

produced and become infectious inside the cell and depend on the cellular

lysis for release.

The second strategy is used by enveloped viruses, such as togavirus and

retrovirus, which are enveloped in the cytoplasmic membrane. They are

generally released by the budding of cellular membranes or exocytose

through a vacuole, usually without cellular lysis.

The third strategy is used by viruses whose nucleocapsids are produced

in the nucleus of the cell, with assembly and maturation involving the

nuclear membrane. The enveloped viruses accumulate in the endoplasmic

reticulum and are carried to the cell surface via vacuoles. Some viruses that

use this process, such as herpesvirus, are cytolytic (cause cell lysis).

Knowledge of viral replication strategies has helped to optimize the

approaches for the genetic modiﬁcation of the viruses for better expression

of the heterologous genes. It has also led to the introduction of biopro-

cesses for the production of infectious viral particles or viral-like particles,

as will be discussed next.

18.3 Production of viral particles by cell culture

Viral particle production from cell cultures has several differences from

other bioprocesses. The production of molecules like enzymes, toxins, or

other proteins synthesized by bacteria, fungi or animals, depend upon

culture parameters, such as pH, temperature, dissolved oxygen, or nutri-

ents. Product formation may occur through secondary metabolic path-

ways, which are not related to the development or growth of the cell. In

these situations, research and technological development must be directed

to the speciﬁc cell and this involves the improvement of the cell as a better

molecular production unit. So, there is a direct relation between nutrient

conversion, cell growth, and the expected improvement of the ﬁnal

productivity.

Viral particle production processes by cell culture infection, cannot be

characterized in such a simple way, since the ﬁnal product – ‘‘virus’’ –

does not result from a secondary metabolic pathway. However, it can be

better described as a process redirecting the cell machinery towards viral

particle production, which only happens after viral infection. The virus

production process can be divided into two different steps. The ﬁrst

involves cell multiplication, which results from the conversion of culture

medium substrates into cell mass. At the instant of viral infection, the

cellular production unit no longer exists, since the viral genetic material

forms a new production unit, initiating the second step of the virus

production process. This production unit is the infected cell and is the

producer of new viral particles. This production phase requires nutritional

and metabolic conditions that are not observed during cell growth. These

conditions are normally studied separately. Nevertheless, virus production

442 Animal Cell Technology

essentially requires the development of the cell that is ‘‘consumed’’ during

the virus production phase, and the metabolic status of the cell at the

moment of infection is a key factor for the success of the viral particle

production process.

Industrial production of viral vaccines preferably requires the use of

continuous or immortalized cell lines as the basis for viral multiplication.

BHK-21, Vero, MDCK, MDBK, CHO, or HeLa cells are the main

platforms used in the production of a huge variety of viral vaccines, since

they are considered stable, do not suffer signiﬁcant genetic modiﬁcations

even after numerous generations of growth, and have a great capacity for

in vitro growth. In addition, these cell lines are susceptible to infection by

several different viruses, irrespective of their origin. As an example, the

Vero cell line has been obtained from an African green monkey kidney.

Although this cell immortalization process results in metabolic and func-

tional changes not observed in normal cells, Vero cells still preserve some

typical kidney cell characteristics. Despite this, certain viruses, like rabies

or poliovirus, which naturally infect nervous tissues, are easily adapted to

infect Vero cells (Frazzatti-Galina et al., 2001). The multifunctional

property of continuous cell lines is important for establishing the best

platforms for an industrial production process.

Even though there are many cell lines capable of cultivation in suspen-

sion, the majority of cells isolated from animal tissues retain their adherent

physiological characteristics and must be grown on a solid surface. These

are named adherent cells.

The basic need for a solid support guides all production choices invol-

ving industrial processes for adherent cells. A large variety of vessels has

been developed for adherent cell cultures. Petri dishes, Roux bottles,

T-ﬂasks, and roller bottles are examples of cell culture vessels with a glass

or polystyrene surface. The system of choice is dependent on the scal-

ability of multiple steps, as well as the cost of equipment and qualiﬁed

operators.

As mentioned in Chapter 9, since production scale-up is related to the

increase of cell culture surface for adherent cells, consideration must be

given to the relationship between the surface area available for cell growth

and the bioreactor volume (Kent and Mutharasani, 1992).

The ﬁrst adherent cell culture industrial process was conducted in roller

bottles, a system with limited scale-up possibilities. However, a great

advance in scale-up of high density cultures was achieved by Van Wezel in

1967. This allowed the culture of adherent cell lines on the surface of

microspheres, called microcarriers. Even under low agitation conditions,

microcarriers remain suspended in medium, which allows the culture to be

homogeneous and to be controlled easily to maintain optimal physiologi-

cal conditions, such as pH, temperature, and aeration. Besides, this is the

only system for adherent cells that allows constant monitoring of cell

growth. This enables cell morphology to be monitored and combines all

the advantages of suspension culture systems with the requirements of

adherent cells (Grifﬁths et al., 1987). So, the process can be well monitored

and controlled with high density cell cultures obtained in small bioreactor

volumes.

Vaccine production based on cells can use different methods of culture.

Viral vaccines: concepts, principles, and bioprocesses 443