Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 87 (10), 5959-69

Influence of Antigen Insertion Site and Vector Dose on Immunogenicity and Protective Capacity in Sendai Virus-Based Human Parainfluenza Virus Type 3 Vaccines

Affiliations

Influence of Antigen Insertion Site and Vector Dose on Immunogenicity and Protective Capacity in Sendai Virus-Based Human Parainfluenza Virus Type 3 Vaccines

John N Mason et al. J Virol.

Abstract

Recombinant Sendai virus (rSeV) was used as a live, attenuated vaccine vector for intranasal inoculation and mucosal expression of the hemagglutinin-neuraminidase (HN) surface glycoprotein of human parainfluenza virus type 3 (HPIV3). Two vaccine candidates rSeV-HPIV3HN(P-M) and rSeV-HPIV3(F-HN) were constructed in which the HPIV3 HN open reading frame and an additional gene junction was inserted in the P-M and F-HN gene junctions of rSeV, respectively. The rSeV-HPIV3HN(P-M) virus was attenuated compared to rSeV-HPIV3(F-HN) in LLC-MK2 cells, and yet both vaccine candidates grew to similar extents in NHBE cells and in the respiratory tracts of cotton rats. These results suggest that in vitro vector growth in NHBE cells more accurately predicts virus yield in cotton rats than does growth in LLC-MK2 cells. Both vaccine vectors elicited high levels of serum neutralizing antibodies and conferred protection from HPIV3 challenge in cotton rats. Compared to vaccination with a high dose (2,000,000 PFU), intranasal inoculation with a low dose (200 PFU) resulted in a 10-fold decrease in vector growth in the nasal cavity and trachea and a 50-fold decrease in the lungs. However, low-dose vaccination resulted in only modest decreases in anti-HPIV3 antibodies in sera and was sufficient to confer complete protection from HPIV3 challenge. Varying the HPIV3 antigen insertion site and vector dose allowed fine-tuning of the in vivo growth and immunogenicity of rSeV-based vaccines, but all four vaccination strategies tested resulted in complete protection from HPIV3 challenge. These results highlight the versatility of the rSeV platform for developing intranasally administered respiratory virus vaccines.

Figures

Fig 1
Fig 1
Generation of recombinant Sendai virus (rSeV)-based human parainfluenza virus 3 (HPIV3) vaccine candidates. (A) rSeVs that contain the HPIV3 HN gene and an additional gene junction inserted into the P-M or F-HN gene junctions were generated. rSeV-HPIV3HN(P-M) was generated in the present study and rSeV-HPIV3HN(F-HN) was generated previously (15). rSeV-WT, reverse genetics wild-type virus, was used as a control virus. (B) To generate the HPIV3 HN gene insert, the open reading frame of the HPIV3 HN gene and an additional intergenic junction was cloned with flanking NotI restriction sites (15). The intergenic junction includes the described SeV gene end, intergenic, and gene start sequences. (C) A unique NotI restriction site had been cloned previously into the P-M or F-HN intergenic junction of SeV cDNA plasmids (15, 19). The HPIV3 HN gene cassette in panel B was cloned into the two cDNA plasmids in panel C to rescue the rSeV vaccine candidates depicted in panel A.
Fig 2
Fig 2
Genetic stability of rSeV-HPIV3HN vectors. To assess the genetic stability of rSeV-HPIV3HN(P-M) and rSeV-HPIV3HN(F-HN), the viruses were serially passaged eight times in chicken eggs and clones from each were selected by plaque purification. Each clone was propagated in LLC-MK2 cells, and cell supernatants were collected for analysis by RT-PCR. Shown are 1,700- and 2,240-bp fragments generated from RT-PCR of rSeV-HPIV3HN(P-M) and rSeV-HPIV3HN(F-HN), respectively. Fragments confirm the existence of the inserted HPIV3 HN gene in each clone and were generated using a forward primer specific to the 5′ region of the inserted HPIV3 HN gene and a reverse primer specific to the SeV genome plasmid downstream of the insertion site. The sequences of clones were confirmed to be correct and complete by RT-PCR and sequencing.
Fig 3
Fig 3
Expression of HPIV3 HN, SeV F, and SeV HN proteins. (A) Total expression of viral surface glycoproteins by radioimmunoprecipitation. LLC-MK2 cells infected with HPIV3, rSeV-WT (SeV), rSeV-HPIV3HN(P-M) (P-M), or rSeV-HPIV3HN(F-HN) (F-HN) were radiolabeled, immunoprecipitated with polyclonal antibodies raised against cytoplasmic tail peptides, and analyzed by SDS-PAGE. (B to D) Quantitation of protein expression levels by densitometry. SDS-PAGE gels were exposed to film, and the intensities of bands representing the proteins HPIV3 HN (B), SeV F (C), and SeV HN (D) were measured by densitometry. The values of each band were normalized to 100% corresponding to expression after infection with wild-type virus and 0% for mock-infected cells. In panel A, the results of one of two experiments are shown, and panels B to D include the cumulative results from two independent experiments. Error bars represent two standard deviations.
Fig 4
Fig 4
Replication kinetics of rSeVs in vitro. In multiple-step growth curves, confluent cells were infected with 0.01 PFU/cell of rSeV-WT (○), rSeV-HPIV3HN(P-M) (■), or rSeV-HPIV3HN(F-HN) (Δ). Conditions include virus growth in LLC-MK2 cells at 37°C (A), LLC-MK2 cells at 33°C (B), and NHBE cells at 33°C (C). Cell supernatants were collected at the indicated times after infection, and virus yield was measured by plaque titration in LLC-MK2 cells. Error bars represent two standard deviations.
Fig 5
Fig 5
Growth of rSeV viruses in cotton rats after vaccination. Virus titers of rSeV-WT (WT), rSeV-HPIV3HN(P-M) (P-M), and rSeV-HPIV3HN(F-HN) (F-HN) were determined after 3 days of infection in cotton rat tissues collected from homogenates of nasal turbinates (A), trachea (B), and lungs (C). Each symbol represents the total load of virus (PFU) from the entire nasal turbinate, trachea, or lungs of an individual cotton rat. Animals were intranasally inoculated at either a low dose (2 × 102 PFU) or a high dose (2 × 106 PFU). ND, not detected (limit of detection is 100 PFU).
Fig 6
Fig 6
Binding antibody responses to rSeV-HPIV3HN vaccines. Cotton rats were intranasally inoculated with either 2 × 102 PFU or 2 × 106 PFU of rSeV-WT (SeV), rSeV-HPIV3HN(P-M) (P-M), rSeV-HPIV3HN(F-HN) (F-HN), or HPIV3. Mock-infected animals were intranasally administered PBS. Serum samples were collected 4 weeks after vaccination and were tested for HPIV3 (A) or SeV (B) specific binding antibodies by ELISA on virus-coated plates. Reciprocal endpoint dilutions are shown. Groups were compared using an unpaired, two-tailed Student t test with a 95% confidence level (*, P < 0.05; ***, P < 0.001). Although not shown for simplicity, HPIV3-binding antibody responses to SeV (A) and SeV-binding antibody responses to HPIV3 (B) were significantly lower than those of the other experimental groups (P < 0.0001).
Fig 7
Fig 7
Neutralizing antibody responses toward rSeV-HPIV3HN vaccines. Cotton rats were intranasally inoculated and sera were collected after 4 weeks, as described in Fig. 6. Serum samples were tested for neutralization activity against the following isolates of HPIV3: C243 from the ATCC (A) or clinical isolates 8-94, 5-97, and 4-04 from St. Jude Children's Research Hospital (B to D, respectively). The average neutralization values of five animals from one of two experiments are shown. Error bars represent two standard deviations. Groups were compared using an unpaired, two-tailed Student t test with a 95% confidence level (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Although not shown for simplicity, HPIV3-neutralizing antibody responses elicited by SeV were significantly lower than those elicited by HPIV3 and the rSeV-HPIV3HN vaccines (P < 0.0001).
Fig 8
Fig 8
Protection from HPIV3 challenge conferred by vaccination with rSeV-HPIV3HN vaccines. Cotton rats were intranasally inoculated as described in Fig. 6. At 5 weeks after immunization with either 2 × 102 PFU or 2 × 106 PFU of virus, the animals were challenged with 2 × 106 PFU of HPIV3 (strain C243). Tissues from the nasal turbinates (A), trachea (B), and lungs (C) were collected 3 days after challenge and the amount of HPIV3 challenge virus growth was measured by plaque titration in LLC-MK2 cells. Each symbol represents the total load of HPIV3 virus (PFU) from the entire nasal turbinate, trachea, or lungs of an individual cotton rat. Results from two experiments are shown (n = 10 for the P-M and F-HN groups). ND, not detected (limit of detection is 100 PFU).

Similar articles

See all similar articles

Cited by 4 articles

Publication types

MeSH terms

LinkOut - more resources

Feedback