Download Recombinant Newcastle Disease Virus as a Vaccine

Recombinant Newcastle Disease Virus as a Vaccine Vector
Z. Huang, S. Elankumaran, A. Panda, and S. K. Samal1
Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland,
8075 Greenmead Drive, College Park, Maryland 20742-3711
(Key words: reverse genetics, RNA virus, paramyxovirus, Newcastle disease virus, vectored vaccine)
2003 Poultry Science 82:899–906
introducing multiple gene deletion mutations in the genome of a virus, it is possible to generate a new class of
attenuated vaccines that are safe and may not result in
reversion to virulence. Recombinant DNA technology has
also made it possible to generate vaccines utilizing viruses
as vectors for the expression of protective antigens of
other viruses. This new class of vaccine is called “vectored
vaccine.” Vectored vaccines are also advantageous in that
DIVA is made easier by using marker genes. Although
there are numerous examples of vectored vaccines reported in literature, few have been investigated as veterinary vaccines (Sheppard, 1999). To date, only three viral
vector vaccines, all of which are based on pox virus vectors, have been licensed for commercial use (Sheppard,
1999). The only poultry vaccine to be licensed for use is
a fowl pox virus vectored vaccine carrying Newcastle
disease virus genes (Boyle and Heine, 1993; McMillen
et al., 1994). Recently, recombinant replication-defective
Semliki forest virus vectored vaccine against infectious
bursal disease virus of chickens has been reported to
produce a strong antibody response, but the efficacy of
the vaccine after challenge is unknown (Phenix et al.,
INTRODUCTION
The use of vaccines against infectious diseases has been
one of the true success stories of modern medicine. Vaccines are the most effective and inexpensive prophylactic
tools in veterinary medicine. Traditionally, there are two
major strategies for the production of viral vaccines: one
employing modified live attenuated virus and the other
employing chemically inactivated virus. However, vaccines produced by conventional means are imperfect in
many respects with regard to safety, efficacy, and cost.
Differentiating vaccinated animals from their infected
(DIVA) counterparts is another major constraint in using
live attenuated vaccines and may cause problems in disease control measures. The unsatisfactory efficacy, safety,
antigenic variation, and the emergence of new diseases
dictate the need to develop newer and more effective
vaccines. This is particularly true for developing vaccines
against poultry diseases, in which cost constraint and the
need to vaccinate en masse are primary concerns.
Through the use of recombinant DNA technology, it is
now possible to generate live virus vaccines that promise
significant improvements in safety, efficacy, and cost. By
Abbreviation Key: CAT = chloramphenicol acetyl transferase; DIVA
= differentiating vaccinated from infected animals; HEp-2 = human
epidermoid carcinoma; HPIV 3 = human parainfluenza virus type 3;
IBV = infectious bronchitis virus; NDV = Newcastle disease virus; NSV
= nonsegmented negative-sense virus; rNDV = recombinant NDV; RNP
= ribonucleoprotein.
2003 Poultry Science Association, Inc.
Received for publication August 28, 2002.
Accepted for publication December 20, 2002.
1
To whom correspondence should be addressed: ss5@umail.
umd.edu.
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good potency, the intrinsic ability of attenuated strains
to revert in virulence makes control of this disease by
vaccination difficult. Armed with the knowledge of virulence factors of this virus, it is now possible to produce
genetically stable vaccines and to engineer mutations that
enhance immunogenicity. The modular nature of the genome of this virus facilitates engineering additional genes
from several different pathogens or tumor-specific antigens to design contemporary vaccines for animals and
humans. This review will summarize the developments
in using NDV as a vaccine vector and the potential of
this approach in designing next generation vaccines for
veterinary use.
ABSTRACT Veterinary vaccines remained conventional for more than fifty years. Recent advances in the
recombinant genetic engineering techniques brought forward a leap in designing vaccines for veterinary use. A
novel approach of delivering protective immunogens of
many different pathogens in a single virus vector was
made possible with the introduction of a “reverse genetics” system for nonsegmented negative-sense RNA viruses. Newcastle disease virus (NDV), a nonsegmented
negative-sense virus, is one of the major viruses of economic importance in the poultry industry throughout the
world. Despite the availability of live virus vaccines of
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HUANG ET AL.
2001). Several studies in recent years highlighted the potential of Newcastle disease virus (NDV) to be used as a
vaccine vector for avian diseases (Krishnamurthy et al.,
2000; Huang et al., 2001; Nakaya et al., 2001). This review
will focus on the methodologies employed and success
achieved for this approach and analyze the means of
improving NDV-vectored vaccines to prevent diseases
of poultry.
NDV
REVERSE GENETICS SYSTEMS
FOR NONSEGMENTED NEGATIVE-SENSE
RNA VIRUSES
The development of methods to recover NSV entirely
from cloned cDNA, established in recent years, opened
up the possibility of genetically manipulating this virus
group, including NDV (Conzelmann, 1998; Roberts and
Rose, 1998). This unique molecular genetic methodology,
termed “reverse genetics,” provides a means not only to
investigate the functions of various virus-encoded genes
(Palese et al., 1996; Nagai, 1999) but also to allow the use
of these viruses to express heterologous genes (Bukreyev
et al., 1996; Mebatsion et al., 1996; Schnell et al., 1996;
Hasan et al., 1997; He et al., 1997; Sakai et al., 1999). This
provides a new method of generating improved vaccines
and vaccine vectors.
Rabies virus was the first NSV to be recovered entirely
from cloned cDNA. The strategy used to recover rabies
virus was to transfect cells with four plasmids encoding
the full-length antigenomic viral RNA, the rabies virus
nucleoprotein (N), and RNA polymerase proteins (L and
P), all under the transcriptional control of a T7 RNA
polymerase promoter. The T7 RNA polymerase was provided by a recombinant vaccinia virus vector (VvT7). This
transfection resulted in the encapsidation of the antigenomic RNA by the N protein to form RNP and in the replication and transcription of these RNP by the viral RNA
polymerase, leading to the generation of recombinant rabies virus. Reverse genetics systems now exist for numerous nonsegmented and segmented negative-sense RNA
viruses, including NDV (Schnell et al., 1994; Garcin et al.,
1995; Collins et al., 1995; Lawson et al., 1995; Radecke et
al., 1995; Whelan et al., 1995; Bridgen and Elliot, 1996;
Kato et al., 1996; Palese et al., 1996; Baron and Barrett,
1997; He et al., 1997; Hoffman and Banerjee, 1997; Jin et
al., 1998; Leyrer et al., 1998; Buchholz et al., 1999; Fodor
et al., 1999; Neumann et al., 1999; Peeters et al., 1999;
Römer-Oberdörfer et al., 1999; Krishnamurthy et al., 2000;
Huang et al., 2001). However, it should be noted that
recovery of recombinant virus is governed by the individual concentrations of each expression plasmid, and in
some instances as in pneumoviruses, an additional viral
protein is required to be coexpressed for efficient recovery
(Collins et al., 1995). While the use of VvT7 facilitated
the consistent recovery of recombinant RNA viruses, it
entailed several disadvantages, such as interference of
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Newcastle disease virus causes a highly contagious and
fatal disease affecting all species of birds. The disease can
vary from clinically inapparent to highly virulent forms,
depending on the virus strain and the host species. The
continuous spectrum of virulence displayed by NDV
strains enabled the grouping of them into three different
pathotypes: lentogenic, mesogenic, and velogenic (Alexander, 1997). Lentogenic strains do not usually cause disease in adult chickens and are widely used as live vaccines
in poultry industries in the United States and other countries. Viruses of intermediate virulence are termed mesogenic, while viruses that cause high mortality are termed
velogenic. The disease has a worldwide distribution and
remains a constant major threat to commercial poultry
production.
Newcastle disease virus is a member of the genus rubulavirus in the family Paramyxoviridae (Pringle, 1997). The
genome of NDV is a nonsegmented, single-stranded, negative-sense RNA of 15,186 nucleotides (Krishnamurthy
and Samal, 1998; Phillips et al., 1998; de Leeuw and Peeters, 1999; Nakaya, et al., 2001), which is the multiple of
six (Calain and Roux, 1993). The genomic RNA contains
a 3′ leader sequence of 55 nucleotides and a 5′ trailer
sequence of 114 nucleotides. The leader and trailer sequences are essential for virus transcription and replication, and they flank six structural genes in the order of 3′NP-P-M-F-HN-L-5′, which encode at least seven proteins
(Peeples, 1988; Steward, et al., 1993). The nucleocapsid
protein (NP) binds to the genomic RNA forming the nucleocapsid core, and the phosphoprotein (P) and the large
polymerase protein (L) are associated with the nucleocapsid core. Together, they form a tight functional ribonucleoprotein (RNP) complex, both in virions and infected cells. The matrix protein (M) forms the inner layer
of the envelope and is probably a driving force in viral
assembly. Two viral glycoproteins are present in the viral
envelope: hemagglutinin-neuraminidase protein (HN)
and fusion protein (F). The former is responsible for the
attachment of the virus to host cell receptor, whereas the
latter mediates fusion of the viral envelope with the hostcell membrane, enabling viral entry into the cell. Two
additional proteins, V and W, are produced by RNA editing (Curran et al., 1991) during P gene transcription, and
their functions remain unknown (Steward et al., 1993,
1995). At the beginning and end of each gene are conserved transcriptional control sequences known as genestart (3′-UGCCCAUCU/CU-5′) and gene-end (3′-AAU/
CUUUUUU-5′) signals. Between the gene boundaries are
intergenic regions, which vary in length from 1 to 47
nucleotides (Chambers et al., 1986; Krishnamurthy and
Samal, 1998). The intergenic sequences are not transcribed
into mRNA (Peeples, 1988). NDV follows the same general mode of transcription and replication as other nonsegmented negative-sense RNA viruses (Peeples, 1988;
Pringle, 1997; Lamb and Kolakofsky, 2001). Like other
nonsegmented negative-sense viruses (NSV), there is a
polar attenuation of transcription such that each downstream gene is transcribed less than its upstream neighbor
(Peeples, 1988; Nagai, 1999).
ANCILLARY SCIENTISTS SYMPOSIUM
RECOVERY SYSTEMS FOR NDV
The first recovery systems, based on a lentogenic vaccine strain (LaSota) of NDV, were reported simultaneously by two independent groups in 1999 (Peeters et al.,
1999; Römer-Oberdörfer et al., 1999). In the first reported
system, the full-length NDV cDNA from LaSota strain
(ATCC-VR699) was assembled in pOLTV5 transcription
vector containing a T7 DNA-dependent-RNA polymerase
promoter with two “G” residues, followed by two unique
restriction sites containing the full-length clone, the autocatalytic ribozyme from hepatitis delta virus, and the transcription termination signal from bacteriophage T7. Individual clones of the NDV transcriptase complex (NP, P,
and L) were cloned in a eukaryotic expression vector.
To generate infectious NDV, primary chicken embryo
fibroblast cells or QM 5 cells were infected with fowl
pox virus-T7 recombinant and cotransfected with the fulllength clone of NDV and the support plasmids. After
incubation for 3 to 6 d in medium containing 5% allantoic
fluid, the recombinant virus harvests from these cells
were amplified in 9-to-11-d-old embryonated specific
pathogen-free eggs. The use of allantoic fluid in the medium was to supply the necessary proteases for the cleavage of F protein. Creation of genetic tags in the form of
additional nucleotides after L gene or changing the F
cleavage site into a consensus cleavage site of virulent
strains did not affect recovery of infectious virus. The cotransfection protocol for generating recombinant NDV
(rNDV) was claimed to be efficient in that it can generate
several infective centers in infected monolayers (Peeters
et al., 1999).
The second system reported for recovery of a lentogenic
NDV from cloned cDNA essentially used the same strategy of assembling the full-length antigenomic expression
plasmid and support plasmids (Römer-Oberdörfer et al.,
1999). However, this system made use of BHK 21 cells,
clone BSR T7/5, stably expressing the T7 RNA polymer-
FIGURE 1. The reverse genetics protocol employed for generating
recombinant Newcastle disease viruses (NDV). The full-length antigenomic cDNA of NDV and the support plasmids encoding NP, P, and L
proteins were transfected into human expidermoid carcinoma (HEp-2)
cells that were preinfected with recombinant vaccinia virus (MVA), to
recover the recombinant virus. The recombinant virus was then amplified in embryonated chicken eggs post-transfection.
ase, as against the fowl pox virus-T7 vector in the previous
system. There were three “G” residues inserted in the
T7 promoter, preceding the first nucleotide of the leader
region of the cloned NDV to enhance T7 polymerasedriven transcription. The recombinant virus produced in
the cotransfected cells was amplified in embryonated
chicken eggs. This system also employed unique restriction sites as genetic tags to differentiate the wild type and
recombinant NDV. Although the biological properties of
the cloned virus were essentially similar to that of the
parental virus in both systems, the virulence of the rNDV
was lower than the wild-type virus in the first system.
This system, in principle, exploited a constitutive cellular
T7 expression system and a virus propagation step,
allowing amplification of a few primary infectious recombinant virions originating from single recovery events
and dispensed with the disadvantages associated with
the use of recombinant vaccinia virus (Römer-Oberdörfer
et al., 1999).
A third system was developed recently to recover a
lentogenic LaSota strain of NDV (Huang et al., 2001). This
system utilized a host-restricted recombinant vaccinia virus (MVA) and human epidermoid carcinoma (HEp-2)
cells for transfection, followed by amplification of the
rNDV in embryonated eggs. HEp-2 cells were used, as
these cells are host restricted for vaccinia virus and are
resistant to vaccinia virus induced cytopathic effects. To
circumvent the requirement of proteases for the cleavage
of F protein, medium-containing acetyl-trypsin was employed posttransfection (Figure 1). Although the method
of construction of the full-length antigenomic cDNA was
essentially similar to the other systems, a new cistron
to encode the chloramphenicol acetyl transferase (CAT)
reporter gene, flanked by NDV gene-start and gene-end
sequences, was incorporated before the NP open-reading
frame to assess the expression of a foreign gene in the
rNDV. The biological properties and virulence of the
rNDV was reported to be similar to that of the parental
strain.
The only system available for the recovery of recombinant mesogenic NDV was described by Krishnamurthy
et al. (2000). This system utilized the vaccinia virus recom-
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vaccinia virus in differentiating cytopathic effects due to
the recombinant NSV, the necessity to purify the recovered virus from vaccinia virus (Schnell et al., 1994; Collins
et al., 1995; Whelan et al., 1995), and RNA recombination
induced by vaccinia virus during recovery (Garcin et al.,
1995). To circumvent these difficulties, host-restricted
vaccinia virus vectors, such as vaccinia virus Ankara
(MVA) strain (He et al., 1997; Leyrer et al., 1998) or cell
lines constitutively expressing T7 polymerase are being
currently used (Radecke et al., 1995; Schneider et al., 1997;
Buchholz et al., 1999; Finke and Conzelmann, 1999;
Römer-Oberdörfer et al., 1999; Harty et al., 2001). The
authenticity of the recovery system for NSV from cloned
cDNA is now well established (Conzelmann, 1998; Roberts and Rose, 1998). Recovery systems for recombinant
NSV are now based on intracytoplasmic reconstitution
of the RNP complex, which represents the template for
the viral polymerase and is the prerequisite for initiating
an infectious cycle (Palese et al., 1996; Conzelmann, 1998;
Roberts and Rose, 1998).
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HUANG ET AL.
binant (MVA) and HEp-2 cells for transfection. The fulllength clone of the mesogenic strain Beaudette C and the
support plasmids (N, P, and L) from the same strain were
used for transfection. Two unique restriction sites were
introduced as genetic markers in the full-length cDNA
clone. An additional transcriptional unit encoding the
CAT reporter gene was placed between the HN and L
genes. The growth of the rNDV expressing the CAT gene
was delayed and the virus was attenuated. The CAT reporter gene was stably expressed for several passages in
cell culture.
EXPRESSION OF FOREIGN GENES
FIGURE 2. Comparison of chloramphenicol acetyl transferase (CAT)
expression by rBC/CAT and rLaSota/CAT viruses at the sixth passage
in DF1 (chicken embryo fibroblast cell line). The CAT gene was inserted
between HN and L genes in rBC/CAT, while it was expressed from
the 3′ proximal position in rLaSota/CAT. Equal amounts of cell lysates
were analyzed for acetylation of [14C]-chloramphenicol as visualized
by thin-layer chromatography. The relative CAT activity was quantified
by densitometry.
of the foreign gene. To address these concerns, we introduced the CAT gene into the most 3′-proximal locus of
NDV strain LaSota and recovered the recombinant virus
(Huang et al., 2001). With foreign gene inserted upstream
of NP, a higher-level expression of the inserted gene was
obtained (Figure 2). Moreover, the growth characteristics
of this recombinant virus in cell culture and in vivo was
similar to that of the wild-type virus (Figure 3). These
results indicate that NDV can be engineered to express
9
Virus titer (log pfu/ml)
8
7
6
5
4
LaSota
3
rLaSota
2
rLaSota/CAT
1
0
0
8
16
24
32
40
Time Post-infection (h)
48
56
FIGURE 3. Multistep growth curve of rLaSota in DF1 cells. The
chicken embryo fibroblast cells were infected with 0.005 PFU per cell
with three replicates per virus. Samples were collected at every 8 h for
56 h. The virus in the supernatant was titrated by a plaque-forming
assay in DF1 cells. The mean virus titer + standard deviation is shown
for different time points.
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The modular nature of NSV genomes facilitates engineering additional genes with ease. The construction of
viral genomes expressing foreign genes depends on the
addition of the foreign gene flanked by appropriate transcription start and stop signals, which are recognized
by the viral RNA-dependent-RNA polymerase. However,
the level of expression of foreign genes is determined
by the site of insertion within the genome, due to polar
attenuation of transcription (Roberts and Rose, 1998). A
variety of reporter genes as additional transcriptional
units have been inserted in appropriate genome positions
of several NSV, and their applications explored (Collins
et al., 1991; Bukreyev et al., 1996; Mebatsion, et al., 1996;
Schnell et al., 1996; Hasan et al., 1997). Genes encoding
other viral antigens have been investigated in vesicular
stomatitis virus (VSV) (Kretzschmar et el., 1997; Roberts
et al., 1998), rinderpest virus (Baron et al., 1999), and NDV
(Krishnamurthy et al., 2000; Huang et al., 2001; Nakaya
et al., 2001). Chimeric viruses with replacement of genes,
such as human parainfluenza virus 3 (HPIV3) and measles virus or respiratory syncytial virus and HPIV 3 recombinants have been developed to function as dual vaccines (Durbin et al., 2000; Schmidt et al., 2001). Moreover,
genes that encode proteins with known antiviral activities
or with known immunoregulatory functions could be inserted into the viral genome, which may attenuate the
virus but augment immunogenicity. For respiratory syncytial virus (RSV), insertion of the interferon-γ gene attenuated the virus while inducing a vigorous humoral immune response (Bukreyev et al., 1999).
We reported the recovery of a recombinant NDV strain
Beaudette C expressing CAT reporter gene between the
HN and L genes (Krishnamurthy et al., 2000). The inserted
CAT gene was maintained very stably and expressed
well for serials of passage in cell culture. However, the
recombinant virus was associated with reduced plaque
size, slower replication kinetics, and more than 100-fold
decrease in yield compared to the wild-type virus. Pathogenicity studies also suggested that the recombinant virus
was attenuated, most probably due to the disruption of
NP:P:L ratio, which seems to be critical to viral replication. Although the results showed that the recombinant
NDV could be used as a vaccine vector, they raised concerns about the level of expression of foreign gene and
growth retardation of the virulent strain after insertion
ANCILLARY SCIENTISTS SYMPOSIUM
ROLE OF NDV AS A VACCINE VECTOR
The use of a viral vector for immunization has the
general advantage that the foreign antigens are expressed
naturally in the context of an infected cell, thereby inducing cellular, as well as humoral, immune responses.
Therefore, the recombinant viral-vectored vaccines hold
many promises for the future. In the poultry industry, so
far, fowl pox virus, herpesvirus of turkeys, Marek’s disease virus, adenovirus, and members of retrovirus family
have been used most extensively as expression vectors
(Dodds et al., 1999; Jackwood, 1999). These recombinant
viruses demonstrated protective efficacy against a variety
of avian diseases. However, their practical usage still
needs to be evaluated, particularly with regard to factors,
such as safety, efficacy, and cost of production (Yokoyama
et al., 1997; Jackwood, 1999).
Certain characteristics of NDV suggest that rNDV expressing a foreign viral protein would be very good vaccine candidates. NDV grows to very high titers in many
cell lines and eggs, and it elicits strong humoral and
cellular immune responses in vivo. NDV naturally infects
via respiratory and alimentary tract mucosal surfaces, so
it is especially useful to deliver protective antigens of
respiratory disease pathogens. In addition, commercially
available live NDV vaccines are widely used in the United
States and most other countries. Vaccines based on live
NDV recombinant would also have advantages over
other live recombinant vaccine vectors. First, the foreign
protein is expressed with only a few NDV proteins. In
contrast, pox and herpes virus vectors express a large
number of additional proteins from their large-size genomes. For the generation of specific immune responses
in vaccine applications, it would be advantageous to have
only a limited number of proteins expressed. Second,
NDV replicates in the cytoplasm of the infected cells without a DNA phase, which eliminates the problem of integration of viral genome into the host cell DNA. The virus
does not undergo detectable genetic recombination,
which would make this expression vector much more
stable and safe.
The availability of the reverse genetics system would
greatly enhance the development of NDV as a better vaccine and vaccine vector. One great promise is the generation of novel deletion mutants. NDV encodes nonessential
proteins, V and W, by way of RNA editing during the
transcription of its P gene. The main function of these
accessory proteins is to facilitate virus replication in vivo,
by acting as an interferon antagonist (Z. Huang et al.,
unpublished data). Deletion of these proteins would render the virus more attenuated but still immunogenic;
therefore, it could be used for in ovo vaccination (Mebatsion et al., 2001).
Another application of reverse genetics is to develop
a more effective and better NDV vaccine. The current
NDV vaccines utilize naturally occurring lentogenic
strains, such as the Hitchner B1 and LaSota. As encountered with any other live attenuated vaccines, the current
NDV vaccines may still cause disease due to reversion
to virulence. A highly stable and efficacious vaccine,
therefore, becomes a desired objective. The introduction
of the reverse genetics system to the molecular biology
of NDV has opened new horizons in the development
of stable NDV vaccines. For example, mutations can be
introduced into a vaccine strain at the F cleavage site to
prevent its reversion to virulent virus, thus making the
vaccine more stable. Lentogenic vaccine strains of NDV
with rearranged gene order may provide a rational strategy for vaccine development, since the recombinant virus
would be more immunogenic and more attenuated at the
same time, as shown in the case of vesicular stomatitis
virus (Wertz et al., 1998). A recombinant chimeric NDV
vaccine that allows serological differentiation between
vaccinated and infected animals has been generated (Peeters, et al., 2001). In this marker virus, the HN gene has
been replaced by a hybrid HN gene with immunogenic
globular domain of HN from avian paramyxovirus type
4; thus, it induces a protective immune response against
NDV by eliciting antibodies against the F protein but
can be differentiated from NDV infection on the basis
of different antibody profiles against their HN proteins.
These examples show that viral attenuation through specific mutations has obvious practical significance in vaccine development. Armed with the knowledge of basic
studies on the virus, genetically stable, safe, and effective
NDV vaccines can be generated.
Recent studies have demonstrated the potential of NDV
as a vaccine vector (Krishnnamurthy et al., 2000; Huang
et al., 2001; Nakaya et al., 2001). The results of these
studies have shown that the expression levels of foreign
proteins are quite high and the foreign genes are very
stable after many passages in vitro and in vivo. It was
also demonstrated that when inserted into the most 3′proximal locus of the viral genome, the expression level
of the foreign gene was higher. Moreover, the replication
and pathogenesis of the recombinant virus was not modified significantly. It would be very appealing to construct
a rNDV to deliver the immunogenic proteins of other
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foreign protein stably and manipulated for use as a vaccine vector.
A full-length cDNA clone of NDV vaccine strain Hitchner B1, expressing an influenza virus hemagglutinin protein, was recently constructed (Nakaya et al., 2001). In this
report, the HA protein open-reading frame was inserted
between the P and M genes. It was demonstrated that
the rescued virus stably expressed the HA protein on the
surface of infected cells and that a significant amount of
the expressed protein was incorporated into the virions.
The maximal titer of the recombinant virus was approximately 20-fold lower than that of the wild-type virus;
however, it induced a strong protective antibody response against influenza virus in mice. As vaccination
against NDV is a common practice in the poultry industry, the results obtained here suggested that the recombinant rNDV vaccine strain expressing an avian influenza
virus HA could have a practical use as a combined vaccine
against both avian influenza viruses and NDV.
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HUANG ET AL.
in poultry and beyond through successful recombinant
vaccines but may also be exploited for gene therapy and
cancer treatment.
ACKNOWLEDGMENTS
We thank Daniel D. Rockemann and Peter Savage for
excellent technical assistance. This work was supported
by U.S. Department of Agriculture NRI Grant # 200235204-11601.
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respiratory viruses, such as the spike protein of infectious
bronchitis virus (IBV), due to the following considerations: both NDV and IBV infect the respiratory tract; in
practice, NDV vaccines are already routinely administered as combination products containing live IBV vaccines, and the current IBV vaccine itself contributes to
generation of new variants due to recombination and
mutations. Therefore, NDV-vectored IBV vaccine will
eliminate the generation of new variants and will aid in
the control of IBV. rNDV can also be used for the expression of protective antigens of avian pneumovirus, since
avian pneumovirus causes severe economic losses and,
currently, no effective vaccines are available. It is also
highly possible to use NDV to express protective immunogens of other avian viral pathogens as polyvalent vaccines. Other genes coding for cytokines or other immunoregulatory proteins or both can also be inserted into the
recombinant virus, thus, making the virus more attenuated but still highly immunogenic.
Besides being a vector for polyvalent vaccines, NDV
can be engineered as a surrogate virus in which the viral
envelope can be completely replaced with other viral envelope proteins or by chimeric envelope proteins (Collins
et al., 1999). Since the surrogate virus may be used to
express other virus envelope or cellular membrane proteins to manipulate host range or cell tropism, it has a
great potential for gene therapy or treatment of cancer
or to prevent diseases for which no effective vaccines are
available. By expressing the human coreceptor complex
for HIV-1, the engineered Rhabdoviruses deliver genes
only to HIV-1-infected cells, not to the normal bystander
cells, as shown for rabies virus and vesicular stomatitis
virus (Mebatsion et al., 1995, 1997; Schnell et al., 1997).
NDV strains can infect many different types of cells, even
nondividing cells, such as neurons, and certain strains of
NDV can selectively kill human tumor cells (Reichard et
al., 1992). Therefore, NDV would be a better choice for
targeted gene expression and somatic gene therapy. Now,
there is an increasing interest in the use of NDV for cancer
therapy (Nelson, 1999), due to its unique properties: it
binds specifically and replicates selectively in tumor cells.
Thus, NDV may be exploited to treat cancer by the temporal expression of anti-angiogenic factors or the induction
of immune responses to tumor-specific antigens.
In conclusion, vaccination strategies have undergone a
radical change in recent years. Virus vectors have been
widely studied for vaccination and for use as a system
of gene transfer into the living body. The use of biotechnology to create recombinant viral-vectored vaccines
holds many promises for the future. With the advent of
the reverse genetics system, the manipulation of NDV is
as easy as that of other DNA and positive-sense RNA
viruses. The modular nature of transcription, undetectable rate of recombination, and the lack of DNA phase
in the replication cycle render NDV as a suitable substrate
for the rational design of live attenuated vaccines and
vaccine vectors. A deeper understanding of NDV molecular biology may not only be subsequently applied to the
control of Newcastle disease and other related diseases
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