Sendai virus recombinant vaccine expressing hPIV-3 HN or F elicits protective immunity and combines with a second recombinant to prevent hPIV-1, hPIV-3 and RSV infections

Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, TN 38105, United States.
Vaccine (Impact Factor: 3.62). 07/2008; 26(27-28):3480-8. DOI: 10.1016/j.vaccine.2008.04.022
Source: PubMed


The human parainfluenza viruses (hPIVs) and respiratory syncytial virus (RSV) are the leading causes of serious respiratory illness in the human pediatric population. Despite decades of research, there are currently no licensed vaccines for either the hPIV or RSV pathogens. Here we describe the testing of hPIV-3 and RSV candidate vaccines using Sendai virus (SeV, murine PIV-1) as a vector. SeV was selected as the vaccine backbone, because it has been shown to elicit robust and durable immune activities in animal studies, and has already advanced to human safety trials as a xenogenic vaccine for hPIV-1. Two new SeV-based hPIV-3 vaccine candidates were first generated by inserting either the fusion (F) gene or hemagglutinin-neuraminidase (HN) gene from hPIV-3 into SeV. The resultant rSeV-hPIV3-F and rSeV-hPIV3-HN vaccines expressed their inserted hPIV-3 genes upon infection. The inoculation of either vaccine into cotton rats elicited binding and neutralizing antibody activities, as well as interferon-gamma-producing T cells. Vaccination of cotton rats resulted in protection against subsequent challenges with either homologous or heterologous hPIV-3. Furthermore, vaccination of cotton rats with a mixture of rSeV-hPIV3-HN and a previously described recombinant SeV expressing the F protein of RSV resulted in protection against three different challenge viruses: hPIV-3, hPIV-1 and RSV. Results encourage the continued development of the candidate recombinant SeV vaccines to combat serious respiratory infections of children.

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    • "The full genome cDNA of rSeV (pSeV(E), strain Enders) [39] was mutated at the non-coding regions between M and F, F and HN, and HN and L to include unique restriction sites for FseI, NotI, and AscI, respectively. The SeV HN and F genes in pSeV(E) were replaced with those of hPIV1 HN and F cDNAs produced by PCR using primers containing appropriate restriction sites. "
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    ABSTRACT: Interactions between viral glycoproteins, matrix protein and nucleocapsid sustain assembly of parainfluenza viruses at the plasma membrane. Although the protein interactions required for virion formation are considered to be highly specific, virions lacking envelope glycoprotein(s) can be produced, thus the molecular interactions driving viral assembly and production are still unclear. Sendai virus (SeV) and human parainfluenza virus type 1 (hPIV1) are highly similar in structure, however, the cytoplasmic tail sequences of the envelope glycoproteins (HN and F) are relatively less conserved. To unveil the specific role of the envelope glycoproteins in viral assembly, we created chimeric SeVs whose HN (rSeVhHN) or HN and F (rSeVh(HN+F)) were replaced with those of hPIV1. rSeVhHN grew as efficiently as wt SeV or hPIV1, suggesting that the sequence difference in HN does not have a significant impact on SeV replication and virion production. In sharp contrast, the growth of rSeVh(HN+F) was significantly impaired compared to rSeVhHN. rSeVh(HN+Fstail) which expresses a chimeric hPIV1 F with the SeV cytoplasmic tail sequence grew similar to wt SeV or rSeVhHN. Further analysis indicated that the F cytoplasmic tail plays a critical role in cell surface expression/accumulation of HN and F, as well as NP and M association at the plasma membrane. Trafficking of nucelocapsids in infected cells was not significantly affected by the origin of F, suggesting that F cytoplasmic tail is not involved in intracellular movement. These results demonstrate the role of the F cytoplasmic tail in accumulation of structural components at the plasma membrane assembly sites.
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    • "Since Sendai virus is a murine parainfluenza virus (PIV) with certain homologies to human PIV, it was tested as xenotropic vaccine in African Green monkeys and humans without any significant adverse reactions [34] [35]. Recombinant SeV vector carrying human PIV was also tested in rats [36] [37]. Further, recombinant SeV vaccine for human immunodeficiency virus (HIV) infection is going to be tested in humans ( "
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    • "The full genome cDNA of rSeVLeGFP (pSeVLeGFP) was constructed as follows. The cDNA containing the L protein C-terminus was subcloned from pSeV(E) [57] to plasmid pTF1 [58] and an FseI site was created at the end of the L coding region using the QuikChange Mutagenesis Kit (Stratagene). The eGFP gene was amplified by PCR from plasmid pEGFP-N1 (Clontech) using primers containing FseI sites flanking the gene, and was inserted into the L gene fragment in pTF1 at the FseI site. "
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    ABSTRACT: Paramyxoviruses are assembled at the plasma membrane budding sites after synthesis of all the structural components in the cytoplasm. Although viral ribonuclocapsid (vRNP) is an essential component of infectious virions, the process of vRNP translocation to assembly sites is poorly understood. To analyze real-time trafficking of vRNPs in live infected cells, we created a recombinant Sendai virus (SeV), rSeVLeGFP, which expresses L protein fused to enhanced green fluorescent protein (eGFP). The rSeVLeGFP showed similar growth kinetics compared to wt SeV, and newly synthesized LeGFP could be detected as early as 8 h postinfection. The majority of LeGFP co-localized with other components of vRNPs, NP and P proteins, suggesting the fluorescent signals of LeGFP represent the locations of vRNPs. Analysis of LeGFP movement using time-lapse digital video microscopy revealed directional and saltatory movement of LeGFP along microtubules. Treatment of the cells with nocodazole restricted vRNP movement and reduced progeny virion production without affecting viral protein synthesis, suggesting the role of microtubules in vRNP trafficking and virus assembly. Further study with an electron microscope showed close association of vRNPs with intracellular vesicles present in infected cells. In addition, the vRNPs co-localized with Rab11a protein, which is known to regulate the recycling endocytosis pathway and Golgi-to-plasma membrane trafficking. Simultaneous movement between LeGFP and Rab11a was also observed in infected cells, which constitutively express mRFP-tagged Rab11a. Involvement of recycling endosomes in vRNP translocation was also suggested by the fact that vRNPs move concomitantly with recycling transferrin labeled with Alexa 594. Collectively, our results strongly suggest a previously unrecognized involvement of the intracellular vesicular trafficking pathway in vRNP translocation and provide new insights into the transport of viral structural components to the assembly sites of enveloped viruses.
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