Newcastle disease virus fusion protein expressed in a fowlpox virus recombinant confers protection in chickens

The nucleoprotein (NP) of Newcastle disease virus (NDV) functions primarily to encapsidate the virus genome for the purpose of RNA transcription, replication, and packaging. This conserved multifunctional protein is also efficient in inducing NDV-specific antibody in chickens. Here, we localized a conserved B-cell immunodominant epitope (IDE) spanning residues 447 to 455 and successfully generated a recombinant NDV lacking the IDE by reverse genetics. Despite deletion of NP residues 443 to 460 encompassing the NP-IDE, the mutant NDV propagated in embryonated specific-pathogen-free chicken eggs to a level comparable to that of the parent virus. In addition, a B-cell epitope of the S2 glycoprotein of murine hepatitis virus (MHV) was inserted in-frame to replace the NP-IDE. Recombinant viruses properly expressing the introduced MHV epitope were successfully generated, demonstrating that the NP-IDE not only is dispensable for virus replication but also can be replaced by foreign sequences. Chickens immunized with the hybrid recombinants produced specific antibodies against the S2 glycoprotein of MHV and completely lacked antibodies directed against the NP-IDE. These marked-NDV recombinants, in conjunction with a diagnostic test, enable serological differentiation of vaccinated animals from infected animals and may be useful tools in ND eradication programs. The identification of a mutation-permissive region on the NP gene allows a rational approach to the insertion of protective epitopes and may be relevant for the design of NDV-based cross-protective marker vaccines.The negative-strand RNA virus genome of Newcastle disease virus (NDV) contains six genes encoding six major structural proteins: nucleoprotein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN), and RNA-dependent RNA polymerase (L). The RNA together with NP, P, and L proteins forms the ribonucleoprotein complex (RNP), which serves as a template for RNA synthesis (15). The NP together with the polymerase proteins, P and L, plays an eminent role in encapsidating the RNA. Moreover, NP regulates transcription and replication of the viral genome by interacting with P alone, with P and L, or with itself (NP-NP interaction). For Sendai paramyxovirus, it was shown that a conserved N-terminal region of NP was involved in NP-RNA and NP-NP interaction (5), whereas the carboxyterminal domain was shown to be required for template function (8). Most of the NP is thus absolutely essential for virus replication due to multifold engagement of NP in the assembly and biologic activity of the RNP. In addition, NPs of negativestrand RNA viruses are highly immunogenic in nature and have been used as antigens for diagnostic purposes, including

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Newcastle Disease Virus (NDV) Marker Vaccine: an Immunodominant Epitope on the Nucleoprotein Gene of NDV Can Be Deleted or Replaced by a Foreign Epitope

Mebatsion¹,

Koolen²,

Vaan³

et al. 2002

“…For vP1071, which coexpressed the SIV K6W env and gag-pol components (17), an env expression cassette under the control of the vaccinia virus H6 (early/late) promoter (37) and a gag-pol expression cassette regulated by the vaccinia virus early and intermediate I3L promoters were inserted into the NYVAC tk and 14L loci, respectively (20). The expression of env and gag was confirmed by immunoprecipitation analysis of vP1071-infected Vero cells with a pooled serum sample from HIV-2-seropositive individuals, as described previously (45). Recombinant virus vP1241 was engineered by inserting a vaccinia virus H6-regulated human IL-2 (10) expression cassette into the NYVAC tk locus.…”

Section: Methodsmentioning

confidence: 99%

Recombinant Vaccine-Induced Protection against the Highly Pathogenic Simian Immunodeficiency Virus SIV_mac251: Dependence on Route of Challenge Exposure

Benson

Chougnet²,

Robert-Guroff

et al. 1998

140

Vaccine protection from infection and/or disease induced by highly pathogenic simian immunodeficiency virus (SIV) strain SIVmac251 in the rhesus macaque model is a challenging task. Thus far, the only approach that has been reported to protect a fraction of macaques from infection following intravenous challenge with SIVmac251 was the use of a live attenuated SIV vaccine. In the present study, the gag, pol, and env genes of SIVK6W were expressed in the NYVAC vector, a genetically engineered derivative of the vaccinia virus Copenhagen strain that displays a highly attenuated phenotype in humans. In addition, the genes for the α and β chains of interleukin-12 (IL-12), as well as the IL-2 gene, were expressed in separate NYVAC vectors and inoculated intramuscularly, in conjunction with or separate from the NYVAC-SIV vaccine, in 40 macaques. The overall cytotoxic T-lymphocyte (CTL) response was greater, at the expense of proliferative and humoral responses, in animals immunized with NYVAC-SIV and NYVAC–IL-12 than in animals immunized with the NYVAC-SIV vaccine alone. At the end of the immunization regimen, half of the animals were challenged with SIVmac251 by the intravenous route and the other half were exposed to SIVmac251 intrarectally. Significantly, five of the eleven vaccinees exposed mucosally to SIVmac251 showed a transient peak of viremia 1 week after viral challenge and subsequently appeared to clear viral infection. In contrast, all 12 animals inoculated intravenously became infected, but 5 to 6 months after viral challenge, 4 animals were able to control viral expression and appeared to progress to disease more slowly than control animals. Protection did not appear to be associated with any of the measured immunological parameters. Further modulation of immune responses by coadministration of NYVAC-cytokine recombinants did not appear to influence the outcome of viral challenge. The fact that the NYVAC-SIV recombinant vaccine appears to be effective per se in the animal model that best mirrors human AIDS supports the idea that the development of a highly attenuated poxvirus-based vaccine candidate can be a valuable approach to significantly decrease the spread of human immunodeficiency virus (HIV) infection by the mucosal route.

“…Immunoprecipitation experiments. Immunoprecipitation experiments were performed essentially as described by Taylor et al (57). The fusion protein was specifically precipitated by using a rabbit antiserum directed against a carboxy terminal fusion peptide (62).…”

Section: Methodsmentioning

confidence: 99%

Vaccinia virus recombinants expressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge

Taylor

Pincus

Tartaglia

et al. 1991

Self Cite

cDNA clones of the genes encoding either the hemagglutinin (HA) or fusion (F) proteins of the Edmonston strain of measles virus (MV) were expressed in vaccinia virus recombinants. Immunofluorescence analysis detected both proteins on the plasma membranes of unfixed cells as well as internally in fixed cells. Immunoprecipitation of metabolically radiolabeled infected-cell extracts by using specific sera demonstrated a 76-kDa HA polypeptide and gene products of 60, 44, and 23 kDa which correspond to a MV F precursor and cleavage products Fo, F1, and F2, respectively. Neither recombinant induced cell fusion of Vero cells when inoculated individually, but efficient cell fusion was readily observed upon coinfection of cells with both recombinants. Inoculation of dogs with the vaccinia virus-MV F recombinant (W-MVF) did not give rise to detectable MV-neutralizing antibody. Inoculation of dogs with the vaccinia virus-MV HA recombinant (W-MVHA) or coinoculation with both recombinants (W-MVF and W-MVHA) induced significant MVneutralizing titers that were increased following a booster inoculation. Inoculation of dogs with the vaccinia virus recombinants or with MV failed to induce canine distemper virus (CDV)-neutralizing antibodies. Upon challenge with a lethal dose of virulent CDV, signs of infection were observed in dogs inoculated with (VV-MVF). No symptoms of disease were observed in dogs that had been vaccinated with W-MVHA or with W-MVHA and W-MVF and then challenged with CDV. All dogs vaccinated with the recombinant viruses as well as those inoculated with MV or a vaccine strain of CDV survived CDV challenge.