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

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(Rueda et al., 1999; Maranga et al., 2000b). This type of chimera is

currently under development using HBV VLPs, yeast Ty particles, and

blue tongue virus VLPs, as well as other polioviruses.

A good example of the recent and future evolution of viral vaccines and

their concomitant issues of technology, complexity and competition, is the

rotavirus vaccine. This is of great relevance for the prevention of diarrhea,

which is often deadly in developing countries (half a million deaths per

year) and has high hospitalization costs in rich countries. After successive

failures of monovalent vaccines, multivalent vaccines based on the reshuf-

ﬂing of rotavirus strains comprising the attenuation properties of animal

strains with the external capsid of human serotypes were developed.

The ﬁrst vaccine (Rotashield

, developed by Wyeth Lederle) entered

the market in August 1998, after extensive clinical trials providing efﬁcient

protection (. 90%) and safety. After less than 1 year 15 cases of intussus-

ception were reported, leading to the withdrawal of this vaccine from the

market (Murphy et al., 2003, Fischer et al., 2004). Because this risk was

calculated as being lower than 1 in 5000 cases, the two major candidate

vaccines (pentavalent human-bovine reshufﬂing, Rotateq

Merck & Co.

and monovalent human attenuated Rotarix

, Glaxo Smith Kline) are

currently under the most extensive Phase III clinical trial record – over

60 000 patients each! This fact can give an idea of the increasing standard

of what is considered to be an acceptable risk in vaccination programs

(with the obvious exception of anticancer vaccines in initial stages and

therefore without clearly deﬁned acceptance standards). This shows an

evident fact in vaccine praxis: the ‘‘easy’’ vaccines have already been

Table 18.5 Viral vaccines: historical perspective

Year Disease Vaccine type

1796 Rubeola Live/attenuated*

1885 Rabies Killed/inactivated*

1928 Yellow fever Live/attenuated

1936 Flu Killed/inactivated*

1937 Encephalitis killed/inactivated

1945 Japanese encephalitis B Killed/inactivated

1955 Poliomyelitis Killed/inactivated

1958 Poliomyelitis Oral vaccine (live/attenuated)

1963 Measles Live/attenuated

1967 Mumps Live/attenuated

Rubeola Live/attenuated

1981 Hepatitis B Protein

1983 Varicella-zoster Live/attenuated

HB Protein/recombinant DNA subunit

Flu Killed/inactivated

Hepatitis A Subunit, with adjuvant

1997 Flu Subunit, with adjuvant

2000 HA-Typhoid fever Polysaccharide

2003 Flu Live/attenuated

Meningococcal disease Serotypes ACW – polysaccharide

2004 Rotavirus Live/attenuated

*Substituted by a new vaccine.

454 Animal Cell Technology

introduced in the market and the ‘‘difﬁcult’’ vaccines, namely viral

vaccines, are the only ones left.

For this reason, there are several groups in Europe (involving the

authors of this chapter) and in the USA looking for VLPs produced in

insect cells using multicystronic baculovirus presenting different comple-

mentary DNA encoding the critical viral proteins VP2, VP6, VP7, and,

eventually, VP4/VP8 in the same vector under the control of different

promotors (Vieira et al., 2005). Using this technology, after validation of

the humoral and cellular response, it is possible to consider systems

biology strategies to understand the replication, stability, mRNA kinetics,

protein production stoichiometry, and VLP packaging. Such knowledge

will allow the improvement of the molecular biology of the baculovirus

vector and the number of proteins generating appropriate VLPs.

The use of DNA vaccines could also bring additional beneﬁts since they

are based on technological platforms (such as the adenoviral platform

AdVac from Crucell), thus providing great ﬂexibility. It is possible in

these cases to add new antigens (derived from a virus, parasite of bacteria)

to obtain a greater efﬁcacy through the modulation of the immune

response to the mechanism of action of each pathogen. This type of

technology is particularly useful in cases where the production of antigen

is not only difﬁcult but also dangerous (HIV-1, Ebola virus, and West

Nile virus). Nevertheless, there are still important challenges, such as the

deﬁnition of formulations that signiﬁcantly increase the stability of the

vaccine.

It is therefore expected that subunit vaccines will proliferate in the

market because they are predominantly safe, as long as their evolution is

supported by the development of new technologies that ensure efﬁcacy.

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458 Animal Cell Technology

Bioinsecticides

rcia Regina da Silva Pedrini and

Ronaldo Zucatelli Mendonc¸a

19.1 Introduction

This chapter describes the application of animal cells, particularly insect

cells, for baculovirus production and its use as a bioinsecticide. The use of

baculovirus in the control of insect pests and its production in vitro are

discussed.

Brazil is one of the largest agricultural producers in the world. Accord-

ing to data from the Institute of Agriculture (IEA), in 2003 the economic

balance of the Brazilian agro business was US$ 17.61 billion (Vicente et

al., 2003). In 2005, the Brazilian production was about 113 million tons of

grains, with soy accounting for about 48% of this production (IBGE,

2005).

Pesticides have an important role in the development of agricultural

production, in controlling pests and insuring a sustainable agriculture.

However, the constant use of chemical pesticides, in an indiscriminate

way, causes a reduction of the population of beneﬁcial organisms and

induces resistance of the targeted insect pests, which creates even more

dependence on chemical products. Therefore, the reduction of the usage of

these pesticides in agriculture is of great importance for commercial and

environmental reasons.

The biological control of insect pests in agriculture is an old practice.

More than 120 years ago, an infestation of citrus fruit production by

Icerya purchasi in Florida State (USA) was treated using a ladybug from

Australia, a procedure that was later used in other countries, including

Brazil (Carvalho et al., 1999). Currently, several bioinsecticides are used

for biological control, the most common being insects (wasps, acarids,

etc.), bacteria (Bacillus thuringiensis), fungi, or virus (baculovirus).

Bioinsecticides based on baculovirus have had a strong impact on the

production of grain. Brazilian research on the use of baculovirus was

initiated by EMBRAPA (Brazilian Agricultural Research Corporation)

during the 1970s (Moscardi, 1993). Because of this research, currently, soy

farming is maintained entirely without the use of chemical pesticides. The

biologically treated area exceeds two million hectares, distributed among

several different states of Brazil. This is the best worldwide example of

large-scale viral biopesticide usage (Ageˆncia Brasil, 2004).

In such a system, a species-speciﬁc virus is used. In the case of soy

cultivation, this virus is Baculovirus anticarsia, which speciﬁcally kills a

caterpillar found on the soy – Anticarsia gemmatalis – a defoliating pest

that substantially reduces farming productivity. With this bioinsecticide,

the use of millions of liters of chemical insecticide per harvest is avoided,

thus generating an enormous environmental and economical beneﬁt to the

country (EMBRAPA, 2002).

In addition to the economic and environmental gains, other advantages

of this product are the facts that the manipulation of chemicals can be

avoided and that the virus is species-speciﬁc. It only affects invertebrates

and causes no harm to other insects that could be natural bioinsecticides to

other pests. Moreover, other factors that contribute to its success are: the

high virulence of the virus and its infection efﬁciency, the low cost of the

product compared with chemical insecticides, and the fact that this is a

unique pest in most producing areas. The sum of these advantages makes

the baculovirus very attractive as a bioinsecticide (Moscardi, 1999;

Moscardi and Souza, 2002; Szewczyk et al., 2006).

Nevertheless, the bioinsecticide has some limitations. Although the high

speciﬁcity is very important in ecological terms, this restricts its market.

The bioinsecticide has low activity. However, this can be improved

through modiﬁcation of the baculovirus genome, suppressing or incorpor-

ating genes from other organisms, such as those that express hormones or

toxins. According to Szewczyk et al. (2006), genetically modiﬁed baculo-

virus will be gradually introduced into countries that have a general

acceptance of genetically modiﬁed organisms (GMOs).

Another aspect to be considered is the need for increased production.

At the moment, the production of the bioinsecticide is accomplished by

harvesting infected caterpillars in the areas infected or by growing the

caterpillars in a laboratory. In the ﬁrst case, there is great variability in

productivity from year to year, since the production depends on the insect

abundance during each harvest, which varies with multiple factors. How-

ever, there has been considerable progress in the production of the virus

under laboratory-controlled conditions (Moscardi and Santos, 2005). In

March of 2005 EMBRAPA ﬁnished the construction of a pilot plant with

the capacity to inoculate about 30 000 caterpillars per day, and another

institution, the COODETEC (Cascavel, PR), is enlarging its production

capacity to 600 000 caterpillars per day. However, the caterpillars have to

be fed with artiﬁcial diets and the cost of production of a dose of the

biopesticide, based on Baculovirus anticarsia, using raw material produced

locally, is approximately 90% higher than that obtained by direct harvest-

ing from the ﬁeld. The use of alternative formulations, such as substituting

agar for carrageen as a gelling agent, has reduced production cost, making

it more economically feasible to produce the bioinsecticide in the labora-

tory (Santos, 2003). However, this type of process has high labor demands.

Large-scale virus production in cell culture in bioreactors is a desirable

development, since it allows virus multiplication in a smaller area and it

reduces labor requirement, in comparison with in vivo production in the

laboratory.

19.2 Baculovirus as a bioinsecticide: mechanism of action

Baculovirus belongs to the Baculoviridae family, which has two different

genera: Nucleopolyhedrovirus (NPV) and Granulovirus (GV). Baculo-

460 Animal Cell Technology

virus exists as two phenotypes designated extracellular virus or budded

virus (BV) and the occlusion bodies, also known as OBs, COPs, GDP, or

polyhedra (Figure 19.1). The occlusion bodies are structures that are

formed inside the nucleus of the infected insect cells by baculovirus

(Friesen and Miller, 2001). These are highly resistant and able to stay

viable in the environment for several years under different climatic condi-

tions. The protected viral particles (virions) are known as ODVs (occlu-

sion-derived virus) (Harrap, 1972).

The protein contained in the GV OBs is called granulin and that in the

NPV is called polyhedrin (Rohrmann, 1999; Winstanley and O’Reilly,

1999). GVs are smaller than NPVs and possess rounded OBs with just one

virion occluded (Winstanley and O’Reilly, 1999). NPVs present a poly-

hedral form and they can be multiple type (MNPV) (Figure 19.1A)or

simple type (SNPV) (Figure 19.1B ), depending on the number of capsids

per virion (Rohrmann, 1999). Since most of the baculovirus used as

biopesticidas are NPVs, in this chapter the OBs will be referred as

polyhedra.

The names of different baculovirus species come from the host name in

which the virus was initially identiﬁed in nature. In this way, each

baculovirus species receives the name of this host and the name of the

family to which the virus belongs.

The infection cycle is shown in Figure 19.2. When the polyhedra are

dispersed in the environment, the viral particles inside them (ODVs) are

deposited on the plant leaves. When the caterpillars feed on the virus-

contaminated foliage, they ingest the polyhedra. The alkaline environment

in the caterpillar medium intestine causes breakdown of the polyhedra and

the viral particles are released from the polyhedra. The infection of the

cells occurs via a receptor-mediated fusion process. Once in the cytoplasm,

the nucleocapsids without membrane are transported to the nucleus of the

cell, where gene expression and genome replication begins.

MNPV

SNPV

Figure 19.1

Electron micrographs illustrating polyhedra produced by nucleopolyhedrovirus in

insect cell culture with multiple nucleocapsids per envelope (A), or with single

nucleocapsids per envelope (B).

Bioinsecticides 461

The nucleocapsids are envelopes that contain a nucleoprotein in the

center, which transports the viral DNA to the cell nucleus. After replica-

tion, new copies of the viral genome are encapsulated in the nucleus,

creating new nucleocapsids. The nucleocapsids then migrate to the cyto-

plasm and leave the cell by a budding process. In this process, the

nucleocapsids become enveloped by the acquisition of the host cell plasma

membrane. The resulting virions are extracellular (budded virus, BV) and

these constitute the main products of the ﬁrst stage of the infection process

in the caterpillar, known as primary infection.

The BVs are taken to other tissues of the larva by the tracheal system,

where they enter the cells by endocytosis, starting with the secondary

infection process in which the infection is spread to all cells of the host

organism. Two virion types are produced in this stage of the infection.

Some of the newly synthesized nucleocapsids migrate to the cytoplasm

and are released from the cytoplasmic membrane, forming new BVs.

Other nucleocapsids are enveloped again in the nucleus. The virions that

are occluded (ODVs), are surrounded by the protein crystal structure,

forming the polyhedra. In the ﬁnal phase of infection, the larva becomes

cream in color and the outer covering (tegument) becomes fragile. The

Insect feeding on

virus-contaminated

foliage

Polyhedra

Polyhedra are

released upon cell

lysis and death

of the host

Lysis

Insect cell

Replication

Nucleus

Budding

Secondary

infection

endocytosis

Budded virus

Primary

infection

Fusion

Cell

Dissolution of polyhedra

in the midgut releasing the

virions (occlusion-derived

virus)

Figure 19.2

Diagram of the life cycle of a nucleopolyhedrovirus.

462 Animal Cell Technology

rupture of the tegument causes the release of the polyhedra, which are

then available for the infection of other caterpillars (Federici, 1986;

Granados and Federici, 1986; Adams and Bonami, 1991; Rohrmann, 1999;

Winstanley and O’Reilly, 1999; Friesen and Miller, 2001).

19.3 Animal cell cultur es for baculovirus production

The insect baculovirus–cell system has been widely used, mainly for the

production of recombinant proteins (Maiorella et al., 1988; Jarvis, 1997).

This system has several advantages, including the ability to produce

functional recombinant proteins that are immunogenically active, the

ability to make post-translation modiﬁcations, and the fact that it contains

a powerful promoter (polyhedrin), as well as the fact that the virus is not

pathogenic to plants and vertebrates (Caron et al., 1990; Nguyen et al.,

1993; Godwin et al., 1996).

However, for bioinsecticide production there are speciﬁc factors to be

taken into account:

(i) The large-scale cell production to be used in virus production, since

large volumes of cells are necessary, at a competitive cost.

(ii) Economic production processes and cheap culture media are needed

to make production viable.

(iii) It is necessary to establish the most effective cell line for viral

production with high virus per cell productivity. The productivity

depends on both the cell type and virus.

(iv) Mutant generation must be monitored and avoided. The risk increases

with the number of passages of the virus in cells.

(v) It is necessary to maintain viral virulence when cultured in vitro.It

has been shown that there is a tendency for a loss of virulence with

viral passage in cell culture.

(vi) The activity of the polyhedra produced in vitro should be compared

with those obtained in caterpillars.

19.4 Effect of culture medium, cell line, and virus isolate on

biopesticide production

Lepidoptera-derived insect cell lines are used for biopesticide production.

Sf21 cells, originated from pupal ovarian tissue of the fall armyworm

Spodoptera frugiperda, and Sf9 cells, a subclone of the Sf21 cells, are the

most widely used cell lines for biopesticide production (King and Possee,

1992) (Figure 19.3). The availability of insect cell lines from Invitrogen (La

Jolla, EUA) was an important step for increasing the use of these cell lines

worldwide.

The establishment of insect cell line cultures allowed a detailed study of

the infection cycle of many baculoviruses. These cell lines are easily

cultured in vitro and their maintenance is relatively simple. The majority

of the cell lines can be cultivated using a temperature of 25–308C. How-

ever, the best temperature for the growth and infection of Sf9 cells is

around 27–288C (King and Possee, 1992).

Bioinsecticides 463