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
Introduction
The human parainfluenza viruses (hPIVs) and respiratory syncytial virus (RSV) are the leading causes of viral pneumonia in infants and children [1]. Among the hPIVs, the hPIV-3 subtype causes the most serious infections. In the United States, hPIV-3 epidemics occur annually during spring and summer months [1], [2]. Approximately 62% of humans are infected with hPIV-3 by age 1, more than 90% by age 2, and almost 100% by age 4 [3], [4].
Clinical observations have indicated that the first hPIV-3 infection is generally most severe. Re-infection with hPIV-3 occurs throughout life, but tends to result in more mild disease and is associated only infrequently with serious lower respiratory tract illness. The more mild disease is likely attributed to the larger airways of infected individuals and to the memory T-cell and B-cell activities elicited by first infections [1]. The production of an effective hPIV-3 vaccine is clearly desired as a means to combat the more serious infections of younger individuals.
Previous efforts to develop hPIV-3 vaccines have included studies of cold-adapted viruses [5], [6], [7] and bovine PIV-3 [8]. Challenges facing the advancement of cold-adapted vaccines have concerned the safety of vaccinated infants and their close contacts. In early studies, the frequency of adverse events and transmission rendered certain vaccine candidates unacceptable. However, one cold-adapted vaccine (HPIV3cp45) has met safety requirements and may continue to advance [9], [10], [11]. The main challenge facing the bovine PIV-3 strategy has been its limited antigenic relation to human PIV-3. The vaccine has appeared to be safe in humans, but has not generated protective immune responses. Researchers hope to remedy this situation by producing vaccines that recombine the hPIV-3 hemagglutinin-neuraminidase (HN) and fusion (F) genes with the bovine PIV-3 backbone [12], [13].
Here, we describe a new strategy for the development of hPIV-3 vaccines: the use of reverse genetics to create Sendai virus (SeV)-based vectors that express the hPIV-3 genes HN and F. SeV (mouse PIV-1) was chosen as the delivery vehicle for these vaccines, because of its ability to prevent hPIV-1 infections in non-human primates [14], [15], its natural host range restriction [16] and its safety profile in current clinical trials [16], [17]. The hPIV-3 HN and F genes were selected as target antigens because each encodes a viral membrane protein with known B-cell and T-cell immunogenicity [18], [19], [20], [21].
In this report, we show that the SeV-based hPIV-3 vaccines not only elicit robust immune responses, but also mediate protection against homologous and heterologous hPIV-3 infections in a cotton rat model. Further, we show that a vaccine formulated by mixing one of these candidate SeV-based hPIV-3 vaccines with a previously described SeV-based RSV vaccine [22], [23] protects cotton rats from challenges with three different respiratory viruses: hPIV-1, hPIV-3 and RSV.
Section snippets
Construct design
Replication-competent recombinant SeVs were rescued using a reverse genetics system, described previously [22], [23], [24], [25]. The full-length cDNA of SeV (Enders strain) was first cloned. To this end, Enders SeV RNA was extracted from purified stock virus and reverse transcription (RT)-PCR was performed. PCR products of each gene were cloned into pTF1 and then cloned into pUC19 to construct the full genome SeV Enders cDNA (pSV(E)). The SeV genome in this clone was straddled by a T7 promoter
Human PIV-3 F and HN proteins are expressed by cells infected with recombinant SeV vaccines
Recombinant SeVs were prepared by the insertion of hPIV-3 F or HN genes between the SeV F and HN genes of the full SeV Enders genome (Fig. 1, panels A–C). The viruses rSeV-hPIV3-F and rSeV-hPIV3-HN were subsequently rescued and sequenced (demonstrating precise maintenance of passenger gene sequences). To examine expression of passenger genes by new viruses, we infected Hep-2 cells with the recombinant SeVs and performed radio-immunoprecipitation experiments. As shown in Fig. 1 (panels D and E),
Discussion
This report describes two new recombinant SeV vaccines that express the hPIV-3 F (rSeV-hPIV3-F) and HN (rSeV-hPIV3-HN) proteins, respectively. We initiated studies by demonstrating PIV-3 protein expression by cells infected with the recombinant SeVs. We then employed a cotton rat model to show that each candidate vaccine elicited neutralizing B-cell and T-cell activities and protected animals against homologous and heterologous hPIV-3 challenges. These hPIV-3 results confirmed and supplemented
Acknowledgements
We thank Dr. Greg Prince (Virion Systems) for providing cotton rat antibody reagents. We thank Robert Sealy and Ruth Ann Scroggs for expert technical assistance. We thank Sharon Naron for critical editorial review. This work was supported by NIH NIAID grant P01 AI054955, NIH NCI grant P30-CA21765, and the American–Lebanese Syrian Associated Charities (ALSAC). We thank Dr. R. Hayden (St. Jude Children's Research Hospital, Memphis, TN) and the American Type Culture Collection (ATCC, Rockville,
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- 1
Division of Infectious Diseases, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, 37232, United States.
- 2
Early Development, Novartis Vaccines and Diagnostics, 350 Massachusetts Avenue, Cambridge, MA 02139, United States.
- 3
2645 NE Haskett PI, Mountain Home, ID 83647, United States.
- 4
Department of Microbiology and Immunology, University of Rochester, Rochester, NY 14642, United States.