Veterinary medicine considers prophylactic vaccines, in combination with strict bio-security, to be the most cost-effective tools for preventing viral infections. In general, veterinary viral vaccines aim to protect susceptible host animals from fatal infectious diseases by inducing a rapid and long-lasting immune response and by preventing the spread of such diseases among populations by reducing viral shedding from infected animals. Such vaccines must be cost-effective, stable, and easy to administer. Live attenuated virus and/or inactivated virus vaccines have been used for decades to prevent viral infectious diseases; however, many vaccines do not satisfy the requirements for an “ideal vaccine” in the field due to limited effectiveness and/or side effects. In addition, no vaccines are available for some infectious diseases.

Advances in recombinant DNA technology have made it possible to design new innovative genetically engineered vaccines with improved safety profiles and greater protective efficacy. These “next generation” vaccines include DNA vaccines, subunit or virus-like particle vaccines, genetically modified marker vaccines, and virus vectored vaccines. Of these, the latter are thought to be a promising tool for developing polyvalent or antigen delivery vaccines that express foreign antigen(s) derived from pathogens of economic importance from a veterinary and human perspective.

Many viruses have been used to develop virus vectored vaccines, which provide effective protective immunity against foreign antigens. The number of vectored vaccines licensed for veterinary and human use has increased over time. Initially, vectored vaccines were based on DNA viruses such as herpesviruses, animal poxviruses, and adenoviruses [1]. Currently, with advances in reverse genetics approaches, many RNA viruses have been adapted for use as vectored vaccines [2]. Such vaccine vectors include both positive-sense RNA viruses (picornaviruses, coronaviruses, and flaviviruses) and negative-sense RNA viruses (paramyxoviruses and orthomyxoviruses) [2]. In particular, Newcastle disease virus (NDV), a paramyxovirus that infects birds, is used as an important vaccine vector for development of bivalent vaccines against pathogens of economic importance to the poultry industry. Numerous studies demonstrate that NDV is a promising vector for delivery of protective antigens derived from pathogens infecting mammals and humans; indeed, NDV vectors are safer and more efficient than conventional whole virus vaccines [3]. This article reviews recent developments in the field of NDV-vectored vaccines and the potential use of such vaccines in veterinary and human medicine.

Virulent NDV, the causative agent of Newcastle disease (ND), results in devastating economic losses to the poultry industry worldwide, particularly in Asia, the Middle East, and Africa [4]. To control ND, poultry flocks are subjected to prophylactic vaccination with live attenuated and/or killed NDV vaccines [5].

Gallinaceous birds (e.g., chickens, quail, and turkeys) are highly susceptible to NDV, although the virus infects most bird species. The clinical severity in chickens varies greatly, depending on the virulence of the NDV isolate. Based on their effects in chickens, isolates of NDV are classified into at least four pathotypes: velogenic (a highly pathogenic form), mesogenic (a moderately pathogenic form), lentogenic (a low pathogenic form), and avirulent (a subclinical form) [6]. Lentogenic (e.g., LaSota and B1) and avirulent (e.g., VG/GA and V4) viruses are widely used as vaccines to prevent ND in poultry.

NDV is an avian paramyxovirus type 1 virus belonging to the genus Avulavirus within the family Paramyxoviridae [7]. The virus contains a nonsegmented, negative-sense, single-stranded RNA genome of 15,186 to 15,198 nucleotides in length, which encodes (3' to 5') six structural proteins: nucleocapsid (N) protein, phosphor (P) protein, matrix (M) protein, fusion (F) protein, hemagglutinin-neuraminidase (HN) protein, and RNA-dependent RNA polymerase (L) protein [8]. The length of the NDV genome follows the “rule of six,” which allows efficient virus replication because the N protein binds effectively to six nucleotides [910].

Fig. 1 shows a schematic diagram of NDV morphology. The NDV F and HN glycoproteins are the major structural proteins and comprise a bilayer lipid membrane (envelope) that plays an important role in virus attachment, entry, and release. The M protein resides beneath the envelope and is involved in assembly and budding. The N protein binds to the genomic RNA to form the nucleocapsid (NC) core, and the P and L proteins associate with the NC core to form the ribonucleoprotein (RNP) complex, which is essential for virus replication and transcription [9]. Two nonstructural proteins, V and W, can be produced by RNA editing of the P gene: one (V) or two (W) G residues are inserted at a specific location within the mRNA encoding the P gene [9]. The V protein is an interferon (IFN) antagonist [11].

The life cycle of NDV occurs entirely in the cytoplasm. First, NDV enters the cell cytoplasm via viral attachment and subsequent membrane fusion, which are mediated by the HN and F proteins, respectively. The viral NC or RNP complex is then released into the cytoplasm, whereupon the RNP complex transcribes the viral RNA to produce viral mRNAs, which are then translated into viral proteins. Genome replication occurs when sufficient viral proteins (especially the N protein) are synthesized. The full-length anti-genomic RNA (plus-strand) serves as the template for synthesis of the viral RNA genome (minus-strand). The newly formed genomic RNAs are then encapsidated by the N protein before associating with the polymerase complex. All viral components are then transported to the cell membrane, where they are assembled into progeny virus particles: the progeny acquire an envelope by budding under the direction of the M protein before being released from the cell surface by a process of detachment (elution), which is regulated by the HN protein (which has neuraminidase activity).

Because NDV is a negative-sense single-stranded RNA virus, a functional viral RNP complex is required for viral transcription and replication. Thus, the reverse genetics technology used to generate recombinant NDV (rNDV) requires a full-length complementary DNA (cDNA) clone and three helper plasmids encoding the NDV N, P, and L proteins under the transcriptional control of a T7 RNA polymerase promoter [812]. Once simultaneously transfected into permissive cells, the three helper plasmids express the N, P, and L proteins, which then bind to genomic or anti-genomic RNA to form the RNP complex.

In general, reverse genetics approaches used to rescue rNDV are based on one of three different systems: (1) a chicken fibroblast cell (or QM5 cell line) and fowl pox virus-T7 recombinant system [13]; (2) a T7 RNA polymerase expressed BSR T7/5 (baby hamster kidney-21 cell clone) system [14]; or (3) a human epidermoid carcinoma (HEp-2) cell and a host-restricted recombinant vaccinia virus (modified vaccinia virus Ankara [MVA]) system [15]. Fig. 1 depicts the reverse genetics system used in our own laboratory. Briefly, HEp-2 cells are infected with MVA and then cotransfected with a NDV cDNA clone (LaSota backbone) and three helper plasmids (N, P, and L). The recovered viruses are purified on HEp-2 cells in a plaque assay, and clones are selected and passaged several times in specific pathogen free (SPF) embryonated chicken eggs.

Insertion of foreign genes into the full-length cDNA clone is a pivotal step for generating rNDV expressing foreign antigens. Manipulating cDNA clones to express foreign antigens must take several factors into account. The foreign gene of interest should be synthesized as a transcription unit, which comprises a gene start (GS) sequence, the foreign gene, and a gene end sequence (Fig. 2). The transcription unit is then inserted into a noncoding intergenic region of the NDV genome [8]. The number of nucleotides that make up the rNDV genome must follow the “rule of six” to ensure integrity of the genome and efficient virus replication. The insertion site is one of the most important factors affecting efficient expression of the foreign gene. In general, genes closer to the 3' terminus are transcribed more abundantly than downstream genes (the 3' to 5' transcription gradient) due to imperfect restart of transcription at the GS. In other words, the foreign gene is expressed more abundantly and more efficiently when placed closer to the 3' end of the genome [8]. While the foreign gene can be placed at any intergenic site within the NDV genome, the P/M intergenic site is preferred to ensure optimal expression of foreign genes [1617]. The length of the inserted foreign gene also affects efficient expression of the foreign antigen. Incorporating a foreign gene increases the size of a genome and the number of transcriptional units within that genome, which often leads to retarded growth of rNDV in permissive cells [8]. A single NDV vector can accommodate foreign genes up to 4.5 kb in length with a good degree of stability and express at least three different foreign genes [818].

NDV vaccine strains infect many mammals but are very safe due to restricted host tropism. Nevertheless, NDV is a strong stimulator of humoral and cellular immune responses at both the local and systemic levels; it also has an adjuvant effect [8]. Importantly, NDV is antigenically distinct from known paramyxoviruses that infect mammals; thus it is unlikely to be affected by pre-existing immunity in mammals [8]. NDV does not establish persistent infection in animals and humans because it replicates only in the cytoplasm [8]. In addition, the rule of six means that genetic changes (e.g., insertions, deletions, and recombinations) affecting the integrity of the genome are rare [8]. Therefore, NDV vaccine strains are potential virus vectors that can be used to develop antigen delivery vaccines for use in animals and/or humans.

For several decades, the global poultry industry has used live attenuated or killed vaccines to control and prevent ND. Conventional NDV vaccines are highly immunogenic and induce protective immunity in poultry. Importantly, live attenuated NDV vaccines can be administrated in drinking water, eye drops, and sprays, and even via in ovo injection. This makes it possible to vaccinate poultry (especially young birds) on a large scale. NDV vaccines can be combined with other poultry vaccines (i.e., used as polyvalent vaccines) to control economically important poultry diseases. The production of NDV vaccines is highly cost-effective since they grow to very high titers in embryonated SPF chicken eggs and in cell culture. This makes NDV vaccine strains attractive as viral vectors for the development of polyvalent vaccines against pathogens that could devastate the poultry industry [12].

NDV vaccine strains show promise as a base from which to develop effective vaccines against pathogens that infect animals and humans. Most NDV-vectored vaccines used for poultry are bivalent and provide protective efficacy against virulent NDVs and several foreign pathogens. NDV-vectored poultry vaccines have been developed to provide protection against HPAIV (A/H5 and A/H7), IBDV, ILTV, IBV, and aMPV. Safe NDV-vectored vaccines have been developed as antigen delivery vaccines for veterinary and human use. Such vaccines express the foreign target antigen and induce robust immune responses at both the local and systemic level as shown with NDV-vectored veterinary vaccines in cattle/sheep (e.g., BHV-1, BEFV, RVFV, and VSV), dogs/cats (e.g., CDV and RV), pigs (e.g., NiV), and horses (e.g., WNV). NDV-vectored human vaccines currently under development aim to provide protection against HIV, HPIV-3, and RSV, newly emerging zoonotic viruses (e.g., HPAIV A/H5, SARS-CoV, EBOV, and NiV), and noncultivable human viruses (e.g., human papillomavirus, hepatitis C virus, and NoV). A primary vaccination by an NDV-vectored vaccine expressing a foreign protein can be effective but the efficacy of an updated or new vaccine based on the NDV vector may be reduced by pre-existing NDV antibodies, which could limit the continuous use of NDV vector vaccines in human.

In conclusion, NDV vaccine strains are attractive vectors that can be used to develop effective vaccines against pathogens that infect animals and/or humans; such vaccines are safe and efficient, and provide high levels of protective immunity, although there is a risk that previous vaccinations can reduce the efficacy of the vectored vaccine.