Vaccine 26 (2008) 3480–3488
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/vaccine
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
Xiaoyan Zhana,1, Karen S. Sloboda,b,2, Sateesh Krishnamurthya,3, Laura E. Luquea, Toru Takimotoa,4,
Bart Jonesa, Sherri Surmana, Charles J. Russella,c, Allen Portnera,d, Julia L. Hurwitza,d,∗
aDepartment of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN 38105, United States
bDepartment of Pediatrics, University of Tennessee, Memphis, TN 38163, United States
cDepartment of Molecular Sciences, University of Tennessee, Memphis, TN 38163, United States
dDepartment of Pathology, University of Tennessee, Memphis, TN 38163, United States
a r t i c l ei n f o
Received 24 January 2008
Received in revised form 13 April 2008
Accepted 14 April 2008
Available online 1 May 2008
Respiratory syncytial virus
a b s t r a c t
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 cur-
rently no licensed vaccines for either the hPIV or RSV pathogens. Here we describe the testing of hPIV-3
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
of cotton rats resulted in protection against subsequent challenges with either homologous or heterolo-
gous 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
recombinant SeV vaccines to combat serious respiratory infections of children.
© 2008 Elsevier Ltd. All rights reserved.
tial virus (RSV) are the leading causes of viral pneumonia in infants
and children . Among the hPIVs, the hPIV-3 subtype causes the
most serious infections. In the United States, hPIV-3 epidemics
∗Corresponding author at: Department of Infectious Diseases, St. Jude Children’s
Research Hospital, 332 N. Lauderdale, Memphis, TN 38105, United States. Tel.: +1
901 495 2464; fax: +1 901 495 3099.
E-mail address: email@example.com (J.L. Hurwitz).
1Division of Infectious Diseases, Department of Medicine, Vanderbilt University
Medical Center, Nashville, TN, 37232, United States.
2Early Development, Novartis Vaccines and Diagnostics, 350 Massachusetts
Avenue, Cambridge, MA 02139, United States.
32645 NE Haskett PI, Mountain Home, ID 83647, United States.
4Department of Microbiology and Immunology, University of Rochester,
Rochester, NY 14642, United States.
occur annually during spring and summer months [1,2]. Approx-
imately 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 infec-
tion is generally most severe. Re-infection with hPIV-3 occurs
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 . 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 [5–7] and bovine PIV-3 . Chal-
lenges 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
0264-410X/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.
X. Zhan et al. / Vaccine 26 (2008) 3480–3488
Fig. 1. Production and testing of recombinant Sendai viruses. (A) The rSeV genome is shown with an engineered NotI site. (B) The hPIV-3 F gene, an SeV transcription
termination sequence and an SeV transcription initiation sequence were cloned into the NotI site to create rSeV-hPIV3-F. (C) The hPIV-3 HN gene, an SeV transcription
termination sequence and an SeV transcription initiation sequence were cloned into the NotI site to create rSeV-hPIV3-HN. (D and E) Immunoprecipitations were performed
to demonstrate F and HN expression by cells infected with rSeV-hPIV3-F and rSeV-hPIV3-HN. Experiments involved the infection of HEp-2 cells with recombinant SeV.
Sixteen hours later, cells were labeled with [35S] Promix. F protein (panel A) was immunoprecipitated with a polyclonal antibody specific for the cytoplasmic tail of hPIV-3 F.
HN protein (panel B) was immunoprecipitated with a polyclonal antibody specific for the cytoplasmic tail of hPIV-3 HN. Wild-type (wt) SeV and hPIV-3 were used as negative
and positive controls, respectively.
rendered certain vaccine candidates unacceptable. However, one
cold-adapted vaccine (HPIV3cp45) has met safety requirements
and may continue to advance [9–11]. 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 recom-
bine 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
because of its ability to prevent hPIV-1 infections in non-human
primates [14,15], its natural host range restriction  and its
safety profile in current clinical trials [16,17]. The hPIV-3 HN and F
membrane protein with known B-cell and T-cell immunogenicity
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
described SeV-based RSV vaccine [22,23] protects cotton rats from
challenges with three different respiratory viruses: hPIV-1, hPIV-3
2. Materials and methods
2.1. Construct design
Replication-competent recombinant SeVs were rescued using
a reverse genetics system, described previously [22–25]. The full-
length cDNA of SeV (Enders strain) was first cloned. To this end,
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 and a hepatitis delta virus
ribozyme sequence. As shown in Fig. 1A, a unique NotI site was
positioned in the non-coding region between the F and HN genes
For cloning of the hPIV-3 F and HN genes, LLC-MK2cells were
infected with the C243 strain of hPIV-3 (VR-93, American Type Cul-
ture Collection (ATCC), Rockland, MD) and viral RNA was extracted.
The hPIV-3 F and HN genes were amplified by RT-PCR (Titan One
Tube System; Roche). The PCR forward primer included a NotI site
and the reverse primer included an SeV transcription termination
signal, an intergenic (IG) sequence CTT, a transcription initiation
X. Zhan et al. / Vaccine 26 (2008) 3480–3488
signal, and a second NotI site (Fig. 1B and C). The hPIV-3 F and HN
cDNAs were digested with NotI and ligated into the NotI site of
2.2. Virus rescue
To rescue the recombinant viruses, we infected 293 T cells
with a UV-inactivated, T7 RNA polymerase-expressing recombi-
nant vaccinia virus (vTF7.3 ) for 1h at 37◦C at an MOI of 3. Cells
hPIV-3 F or HN gene (1?g) and three supporting T7-driven plas-
mids expressing the NP, P, or L gene of SeV (1?g pTF1SVNP, 1?g
pTF1SVP, and 0.1?g pTF1SVL, respectively) in the presence of lipo-
fectamine (Life Technologies, Grand Island, NY). Cells were then
incubated for 40h. Cell lysates were prepared and inoculated into
10-day-old embryonated chicken eggs. Allantoic fluids were har-
eggs to prepare vaccine stocks. Recovered viruses were designated
rSeV-hPIV3-F (SeV expressing hPIV-3 F protein) or rSeV-hPIV3-HN
(SeV expressing hPIV-3 HN protein).
2.3. Immunoprecipitation of F and HN proteins from infected cells
To confirm that the recombinant vectors expressed hPIV-3 F or
HN proteins, we examined lysates of rSeV-hPIV3-F or rSeV-hPIV3-
HN infected Hep-2 cells by radio-immunoprecipitation. Briefly,
Hep-2 cells were infected at an MOI of 5 with rSeV-hPIV3-F, rSeV-
hPIV3-HN or wild-type SeV, and incubated at 33◦C in DMEM,
10% fetal calf serum (FCS) and 1% l-glutamine. Sixteen hours
post-infection, the cells were washed twice with PBS contain-
ing 0.1g/l calcium and magnesium (PBS+). Cells were maintained
in culture for 30min in methionine- and cysteine-free medium
and then labeled for 15min with 100?Ci [35S]Promix (Amersham
Pharmacia Biotech) in 1ml of DMEM lacking methionine and cys-
teine and containing 20mM HEPES buffer (pH 7.3). The cells were
washed once with PBS+ and chased with 3ml of DMEM contain-
ing 2mM methionine, 2mM cysteine, and 20mM HEPES buffer
(pH 7.3) for 180min. Samples were lysed with ice-cold radio-
immunoprecipitation assay (RIPA) buffer containing 0.15M NaCl,
9.25mg/ml iodoacetamide, 1.7mg/ml aprotinin, 10mM phenyl-
methylsulfonyl fluoride. The lysate was centrifuged at 67,000×g in
an Optima TLX ultracentrifuge (Beckman Coulter). The supernatant
was incubated overnight (18–22h) at 4◦C with 25?l rabbit anti-
hPIV-3 F or anti-hPIV-3 HN tail peptide polyclonal antibody (1:40
dilution of rabbit sera, Harlan Bioproducts for Science, Madison,
WI). Immune complexes were adsorbed to protein A-Sepharose Cl-
containing 0.15M NaCl, and once with 50mM Tris buffer (0.25mM
tein bands were visualized using a Typhoon 9200 phosphoimager
2.4. Animals, inoculations and challenges
Groups of five adult female cotton rats (Sigmodon hispidus;
Harlan Sprague Dawley, Indianapolis, IN) were intranasally inoc-
ulated with rSeV-hPIV3-F (2×106plaque-forming units (PFU)),
rSeV-hPIV3-HN (2×106PFU), a mixture of 1×106rSeV-hPIV3-F
and 1×106rSeV-hPIV3-HN, or a mixture of 2×106rSeV-hPIV3-
HN and 2×106rSeV-RSV-F (a previously described recombinant
SeV expressing the full-length RSV F protein ). Control animal
5–10 days, mediastinal lymph nodes (MLNs) were collected for T-
cell assays. Serum samples were taken 4 weeks post-inoculation
and challenges were performed on week five. The intranasal chal-
lenge doses were 2×106PFU/cotton rat of homologous hPIV-3
(strain C243) or 1.5×106PFU/cotton rat of heterologous hPIV-3
(8-94, kindly provided by Dr. R. Hayden, Clinical Virology, St. Jude
Children’s Research Hospital). Lungs were harvested 3 days post-
challenge for virus measurements. In some experiments, animals
were challenged intranasally with either hPIV-1(C35 from ATCC,
2×106PFU/cotton rat) or RSV (strain A2, 1.5×106PFU/cotton rat).
2.5. Enzyme-linked immunosorbent assay (ELISA)
hPIV-3 stock was prepared from culture supernatants by concen-
in disruption buffer (0.5% Triton X-100, 0.6M KCl, 0.05M Tris pH
7.8), diluted with PBS (1:3000) and coated on 96-well ELISA plates.
Lysates of wild-type SeV were plated as controls. After overnight
incubation, plates were blocked with PBS containing 3% bovine
serum albumin (BSA, Sigma, St. Louis, MO). Serum samples from
vaccinated and control animals were serially diluted and incubated
on plates for 2h at 37◦C. Plates were then washed and incubated
with rabbit anti-cotton rat antibody (kindly provided by Dr. Greg
Prince, Virion Systems, Rockville, MD) for 30min at room tempera-
ture. After further washing, plates were incubated with anti-rabbit
IgG–horseradish peroxidase conjugate (diluted 1:3000 in PBS/1%
BSA, Bio-Rad, Hercules, CA, Cat# 170-6515) for 30min at room
temperature, washed again, and incubated with 2,2?-azino-bis-(3-
ethylbenzthiazolinesulfonic acid) (ABTS, Southern Biotechnology
Associates, Inc., Birmingham, AL). Absorbance was read at 405nm.
2.6. Neutralization assays
To conduct neutralization assays, we mixed serially diluted sera
with approximately 10 TCID50hPIV-3 per well in DMEM (Cambrex
Bio Science Walkersville, Inc., Walkersville, MD) for 1h at 37◦C.
Viruses were either homologous (C243) or heterologous (St. Jude
Children’s Research Hospital isolates 4-04, 5-97 and 8-94, named
mixtures were then added to wells (6 wells per sample in 24-
well plates) of LLC-MK2cell monolayers, which were incubated for
1h (33◦C, 5% CO2) and then fed with DMEM supplemented with
glutamine, antibiotics and 5% FCS. After 4 days of culture (33◦C,
5% CO2), supernatants (100?l) from test wells were mixed with
100?l of 0.5% fresh Turkey red blood cells in round-bottomed 96-
well plates and incubated at 4◦C for 30min. Hemagglutination was
scored as positive or negative for each well and the percent neu-
tralization was calculated as the reduction in frequency of positive
wells for test versus control samples.
2.7. IFN-? ELISPOT assays
For analyses of hPIV-3-specific T-cell responses, overlapping
peptides (derived from the hPIV-3 F and HN sequences) were pre-
pared by the Hartwell Center for Bioinformatics and Biotechnology
at St. Jude Children’s Research Hospital. Peptides were generally 15
amino acids in length and were initiated at intervals of 10 amino
prepared with 10 peptides per pool for use in the ELISPOT assay.
The ELISPOT assay was conducted by incubating 3.3?g/ml
goat anti-cotton rat IFN-? antibody (R&D Systems, Minneapo-
lis, MN) in multiscreen-hemagglutinin filtration plates (Millipore,
Bedford, MA) overnight at 4◦C. After washing, the plates were
blocked for at least 1h at 37◦C with complete tumor medium
X. Zhan et al. / Vaccine 26 (2008) 3480–3488
(CTM [27,28], a modified Eagle’s medium (Invitrogen, Grand
Island, NY) supplemented with 10% FCS, dextrose (500?g/ml),
glutamine (2mM), 2-mercaptoethanol (3×10−5M), essential and
non-essential amino acids, sodium pyruvate, sodium bicarbonate,
and antibiotics). Mediastinal lymph node (MLN) cells were har-
vested from cotton rats 5–10 days after vaccination. Fresh cells
were suspended in CTM and added to plates at 0.25–1×106cells
per well containing individual peptide pools. The final concentra-
tion of each peptide was approximately 10?M. Positive control
wells received 4?g/ml Con A (Sigma–Aldrich, St. Louis, MO) rather
than peptides. The plates were incubated for 48h at 37◦C and
washed four times with PBS and four times with PBS wash buffer
(PBS with 0.05% Tween 20). Biotinylated goat anti-cotton rat IFN-
? antibody (R&D Systems, Minneapolis, MN) was diluted in PBS
(containing 0.05% Tween 20 and 1% FCS) and was added to wells
for at least 2h. After additional washing, streptavidin-conjugated
alkaline phosphatase (Cat# D0396, DAKO, Copenhagen, Denmark)
diluted 1:500 in PBS wash buffer was added. One hour later, plates
were rinsed with wash buffer and water. The IFN-? spots were
blue tetrazolium alkaline phosphatase substrate (Sigma–Aldrich).
Spots were counted with an Axioplan 2 microscope and software
(Carl Zeiss, Munich–Hallbergmoos, Germany).
2.8. Virus challenge assay
Three days after intranasal viral challenge, cotton rats were sac-
Briefly, lungs were homogenized on ice with a mechanical Dounce
homogenizer (PowerGen125 PCR Tissue Homogenizing kit; Fisher
Scientific) to yield 5ml of homogenate in PBS. Homogenates were
centrifuged (1500×g, 10min) and supernatants were collected.
For hPIV-1 detection, LLC-MK2 cells were grown to conflu-
ency in 6-well plates in complete medium (MEM, 0.2% NaHCO3,
2mM glutamine, and 50?g/ml gentamicin) with 5% FCS. Plates
were washed once with PBS/calcium/magnesium. 100?l of seri-
ally diluted supernatants were inoculated into wells. After 1h at
33◦C, 5% CO2, the cells were overlaid with 4ml per well of com-
plete medium supplemented with vitamins, amino acids, 0.15%
BSA, 5?g/ml acetylated trypsin (Sigma), and 0.9% agarose (elec-
trophoresis grade, BRL, Gaithersberg, MD). After the agarose was
set, plates were inverted and incubated at 33◦C in a 5% CO2incuba-
tor. 5 days later, plates received a second overlay (3ml), similar to
the first, but with 5% FCS instead of BSA, 0.0035% neutral red, and
plaques were counted.
were added to wells (in 24-well plates) of LLC-MK2cell mono-
layers. Cultures were incubated for 1h (33◦C, 5% CO2) and then
fed with DMEM supplemented with glutamine, antibiotics and 5%
FCS. After 4 days incubation (33◦C, 5% CO2), 100?l supernatants
were removed from hPIV-3-infected wells for hemagglutination
assays with Turkey red blood cells. TCID50were calculated using
the Reed–Meunch formula.
For RSV measurements, serially diluted supernatants from lung
homogenates were inoculated on Hep-2 cell monolayers in 12-well
plates; after 1h at 37◦C and 5% CO2, the wells were overlaid with
and 0.75% methylcellulose. After incubation for 5–6 days at 37◦C
and 5% CO2, the methylcellulose was removed, cells were fixed
with formalin phosphate, and the plates were stained with hema-
toxylin and eosin for enumeration of plaques. For each virus, the
total pulmonary burden per cotton rat was scored.
3.1. 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
To examine expression of passenger genes by new viruses, we
infected Hep-2 cells with the recombinant SeVs and performed
D and E), both of the hPIV-3 proteins were expressed.
3.2. Sendai virus vaccines expressing hPIV-3 F or HN induce
hPIV-3-specific binding and neutralizing antibodies in a cotton rat
To study the immunogenicity of the SeV-based vaccines, we
inoculated groups of 5 cotton rats intranasally. Each cotton rat
received 2×106PFU rSeV-hPIV3-F or rSeV-hPIV3-HN, or received
1×106PFU of each of the two vaccines in a mixture. Unmodified
SeV and PBS were used as controls. Blood was collected 4 weeks
later for measurement of hPIV-3-specific antibodies by ELISA. Sera
Fig. 2. Recombinant SeV immunizations elicit hPIV-3-specific antibodies. Groups
of 5 cotton rats were inoculated with 2×106PFU rSeV-hPIV3-F, rSeV-hPIV3-HN, a
combination of the two vaccines (each at 1×106PFU), unmodified SeV or PBS. Sera
collected 4 weeks later were pooled, serially diluted, and tested by ELISA for hPIV-3-
specific antibody activity. Absorbance values are shown with standard error bars (5
replicates per test). Individual sera were also tested and yielded similar results. (A)
ELISAs were conducted with serially diluted samples on plates coated with hPIV-3
lysate. (B) ELISAs were conducted with serially diluted samples on plates coated
with wild-type SeV lysate.
X. Zhan et al. / Vaccine 26 (2008) 3480–3488
Recombinant SeV vaccines elicit PIV-3-specific antibodies with cross-reactive neu-
Imm.Serum dilution Percent neutralization of hPIV-3 isolates
Tested viruses were the homologous C243 (ATCC) and the heterologous 4-04, 5-97
and 8-94 (kindly provided by Dr. Randy Hayden). Sera were mixed with virus for
1h and then inoculated onto LLC-MK2cell monolayers. Neutralization is scored as
the percent reduction of wells with hemagglutination activity. Imm.=immunogen.
1:500 and 1:5000) for testing. All cotton rats immunized with the
single or mixed recombinant vaccines showed high serum anti-
hPIV-3 antibody activity (Fig. 2A). Responses were not improved
by use of the mixed vaccine versus rSeV-hPIV3-HN. Sera from indi-
vidual cotton rats were also tested and yielded similar results (data
not shown). Interestingly, cotton rats inoculated with unmodified
wild-type SeV had a weak antibody reaction toward hPIV-3. This
cross-reactive response was not surprising as PIV-1 and PIV-3, both
respiroviruses, have sequence and antigenic similarities [19,29,30].
Sera from all groups of cotton rats were also tested for antibody
responses to the SeV backbone by ELISA with SeV lysate as the tar-
get antigen (Fig. 2B). SeV-specific antibody activity was induced by
both the recombinant and wild-type viruses.
Having identified PIV-3-specific antibodies, we next investi-
gated neutralizing activity. Serum samples taken 4 weeks after
inoculation were highly efficient at neutralizing the homologous
C243 hPIV-3 isolate in tissue culture (Table 1, column 3). Results
showed that the rSeV-hPIV3-HN vaccine elicited higher responses
than the rSeV-hPIV3-F vaccine. In the former case, neutralization
was evident at serum dilutions of >1000 (neutralizing activity was
reduced at a serum dilution of 1:4096, data not shown). Again,
the mixed vaccine was not superior to rSeV-hPIV3-HN. We also
observed that sera from animals immunized with wild-type SeV
were able to neutralize the infectious hPIV-3, but this activity was
only evident at a serum dilution of 1:16.
To characterize further the vaccines, we tested neutralizing
activities toward heterologous hPIV-3 isolates. For these tests, we
used viruses that had been isolated from several different yearly
outbreaks of hPIV-3 (viruses 4-04, 5-97 and 8-94 were obtained
from the Clinical Pathology Department of St. Jude Children’s
Research Hospital). As shown in Table 1 (columns 4–6), all of these
viruses were neutralized by sera from vaccinated animals. Again,
tralization results (positive scores were evident at serum dilutions
>1000). The mixed vaccine did not enhance neutralizing antibody
activity compared to rSeV-PIV3-HN. The results with heterologous
viruses demonstrated the potent cross-neutralizing capability of
antibodies elicited by recombinant SeV hPIV-3 vaccines.
3.3. Both rSeV-hPIV3-F and rSeV-hPIV3-HN elicit hPIV3-specific
Intranasal inoculation with SeV is well known for its capac-
ity to elicit B-cell and T-cell responses within the lung and local
draining lymph nodes [14,20,31–33]. Accordingly, we conducted
IFN-?-ELISPOT assays to identify vaccine-induced, hPIV-3-specific
T-cell responses. For this study, MLN were harvested from cotton
hPIV3-HN or wild-type SeV. T cells were then isolated from the
draining MLN for testing against pooled peptides representing HN
and F proteins (see Fig. 3, panels A and B, for peptide locations).
Data showed that both rSeV-hPIV3-F and rSeV-hPIV3-HN recom-
binants induced virus-specific T cells able to produce IFN-? (Fig. 3,
panels C and D). The MLN from cotton rats that had received only
unmodified SeV were also tested for T-cell responses toward the
hPIV-3 F and HN proteins (Fig. 3, panels E and F). In SeV-primed
animals, a significant response was demonstrated toward hPIV-3 F,
reflecting sequence similarities between the F proteins of SeV and
3.4. rSeV-hPIV3-F and rSeV-hPIV3-HN vaccines confer complete
protection against homologous and heterologous hPIV-3
challenges in the cotton rat model
Having identified the induction of binding and neutralizing
antibodies and T-cell responses toward hPIV-3, we next assessed
protection from hPIV-3 challenge. Five weeks after inocula-
tions with rSeV-hPIV3-F, rSeV-hPIV3-HN, a mixture of the two
constructs, wild-type SeV or PBS, cotton rats were challenged
intranasally with the homologous hPIV-3 C243 strain at a dose of
2×106PFU/animal. Three days after challenge, animals were sacri-
ficed and lungs were collected for determination of hPIV-3 burden.
As shown in Fig. 4A, animals vaccinated with either or both of the
recombinant SeV vaccines were completely protected from hPIV-3
challenge. Wild-type SeV conferred partial protection as compared
to the PBS inoculation. The results clearly demonstrated the effi-
To investigate further the protective capacities of the hPIV-3
constructs, we challenged vaccinated animals with a heterologous
hPIV-3 isolate. Again, experiments were performed with groups of
animals (five cotton rats per group) immunized with rSeV-hPIV3-
F or rSeV-hPIV3-HN either independently or in combination. As
shown in Fig. 4B, all of the recombinant vaccines provided protec-
3.5. A mixed rSeV-RSV-F and rSeV-hPIV3-HN vaccine confers
protection against RSV, hPIV-3 and hPIV-1 challenges in the cotton
Finally, we tested whether a mixture of vaccines could be used
showed that the unmodified SeV protected against hPIV-1  and
that recombinants expressing either RSV G or F protected against
RSV [22,23,25]. To determine whether a mixture of vaccines could
be used to protect against hPIV-1, hPIV-3 and RSV, we combined
two constructs expressing RSV and hPIV-3 antigens in a single vac-
cine formulation. Specifically, we chose to combine the rSeV-RSV-F
and rSeV-hPIV3-HN viruses. The rSeV-RSV-F vector was chosen
X. Zhan et al. / Vaccine 26 (2008) 3480–3488
Fig. 3. T cells respond to peptide pools representing hPIV-3 F and HN sequences. The synthesized peptides were generally 15 amino acids in length and were initiated at
bold and underlined sequences. T-cell tests involved the isolation of MLN from cotton rats 10 days after animal inoculation with rSeV-hPIV3-F (Panel C), rSeV-hPIV3-HN
(panel D), or unmodified SeV (panels E and F) to assess T-cell function with the IFN-? ELISpot assay. MLN were combined from each group (≥3 animals per group) and tested
against each of 6 peptide pools or against no peptide. Cells plated per well were 0.5×106for panels C and D, and 1×106for panels E and F. Values are the mean±standard
rather than rSeV-RSV-G in this study, because of the greater con-
servation of RSV F sequences in nature  and because RSV G
has been reported to be associated with enhanced inflammation
and eosinophilia in some mouse studies [35–37]). The rSeV-PIV3-
HN construct was chosen over rSeV-PIV3-F because of the greater
nizations (Table 1).
Recombinants were mixed so that each virus was included at
a dose of 2×106PFU per inoculation. Groups of cotton rats then
received the mixed vaccine, the wild-type SeV or PBS. Five weeks
after vaccination, the groups were challenged with RSV, hPIV-3 or
ulated with the mixed vaccine were successfully protected against
all three pathogens. The wild-type SeV also conferred protection
against hPIV-1 and partial protection against hPIV-3. Together,
three different viruses in the cotton rat model.
This report describes two new recombinant SeV vaccines that
express the hPIV-3 F (rSeV-hPIV3-F) and HN (rSeV-hPIV3-HN) pro-
teins, respectively. We initiated studies by demonstrating PIV-3
then employed a cotton rat model to show that each candidate vac-
cine 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 our previous
studies showing that SeV recombinants expressing either RSV G or
RSV F could protect against RSV [22,23,25,38].
We also tested a mixture of two recombinants (the rSeV-RSV-F
and rSeV-hPIV3-HN constructs) expressing RSV and PIV-3 proteins
in cotton rats. The mixed candidate vaccine completely protected
cotton rats against challenge with three different pathogens: hPIV-
1, hPIV-3 and RSV. The full protection against HPIV-3 and RSV
was dependent on the presence of PIV-3 HN and RSV F genes,
respectively. However, SeV alone was sufficient to protect com-
pletely against hPIV-1 (as previously demonstrated ) and
partially against hPIV-3.
The precise contributions of B-cell and T-cell activities to pro-
tection against the hPIVs and RSV were not dissected in the current
study, but will be a topic of future research. It is likely that both
B-cells and T cells contributed to the successful outcome. PIV-
specific antibodies are known to provide a first line of defense
against virus infection by preventing virus entry into target host
cells [31,32,39], while T cells may provide back-up mechanisms by
secreting cytokines and lysing virus-infected cells . Research in
a mouse model has shown that antibodies or T cells activated by
an SeV-based RSV F vaccine can each reduce viral load by approx-
imately 2 logs or more after an RSV challenge . Today’s most
efficacious, licensed vaccines generally elicit a combination of B-
cell and T-cell responses [42–46]. Vaccines that elicit only T-cell
X. Zhan et al. / Vaccine 26 (2008) 3480–3488
Fig. 4. Recombinant viruses confer protection against challenges with either
homologous or heterologous hPIV-3. Five weeks after immunizations with rSeV-
hPIV3-F, rSeV-hPIV3-HN, or a 1:1 mixture of the two vaccines, at a final dose of
2×106PFU/cotton rat, animals were challenged with the homologous virus (C243,
2×106PFU/cotton rat, panel A) or a heterologous virus (8-94, 1.5×106PFU/cotton
rat, panel B). Pulmonary virus loads were measured on day 3 post-challenge. Each
symbol represents the pulmonary titer (TCID50) in an individual cotton rat. Control
animals received wild-type SeV (2×106PFU/cotton rat) or PBS instead of recombi-
activity in the absence of antibody have often failed to provide
complete protection in pre-clinical and clinical studies [47–49].
Results in the present report encourage further testing of both
unmodified and recombinant SeV vaccines . In 1952, when SeV
was first isolated in Sendai Japan, it was thought to be the etio-
logic agent of a human respiratory disease, but this conclusion was
discounted by leaders in the field in later years . Five decades
have passed since the discovery of SeV, and there remains no evi-
dence of SeV-related infection or disease in humans. SeV therefore
holds great clinical appeal, particularly because the vaccine can be
) without needle sticks.
Our own clinical trials have thus far shown unmodified SeV to
be safe in adults . No serious vaccine-related adverse events
have been observed in any of our study participants. Live SeV was
not isolated from these volunteers after vaccination, a reflection
of pre-existing hPIV-specific immune responses in adults. Clinical
trials in a younger volunteer population are ongoing.
The absence of serious vaccine-related adverse events is pre-
dicted in humans, because SeV is a mouse pathogen and is
host-range restricted. This is in part due to the unique sensitivity
of SeV to the innate immune activities elicited by human inter-
feron . As a demonstration of viral sensitivity, Bousse et al.
showed that unlike hPIV-1, SeV could not overcome IFN-mediated
growth suppression in human lung cells. As further suggestion of
SeV safety and efficacy in primates, we and others showed that the
virus caused no disease in either African Green Monkeys (AGM)
or chimpanzees, yet conferred complete protection against hPIV-1
Fig. 5. Mixed rSeV-hPIV3-HN and rSeV-RSV-F recombinant SeV vaccines con-
fer protection against challenges with hPIV-1, hPIV-3 and RSV. Five weeks after
immunizations with a vaccine mixture (2×106PFU rSeV-RSV-F and 2×106PFU
rSeV-hPIV3-HN per cotton rat), groups of 5 animals were challenged with RSV (A2,
1.5×106PFU/cotton rat, top panel), hPIV-3 (C243, 2×106PFU/cotton rat, middle
panel) or hPIV-1 (C35, 2×106PFU/cotton rat, bottom panel). Cotton rats immu-
nized with wild-type SeV (2×106PFU/cotton rat) or PBS instead of recombinant
vaccines were used as controls. Viral loads were measured in animal lungs on day 3
post-challenge. RSV and hPIV-1 viral loads were measured as PFU/cotton rat, while
hPIV-3 viral loads were measured as TCID50/cotton rat. Each symbol represents an
(in AGM, protection was superior to that conferred by hPIV-1 itself
[14,15]). Also, when the peak growth of SeV in the lower respira-
tory tract (LRT) of chimpanzees was determined, it was found to
be less than that of bPIV-3 (a vaccine that appears to be safe in
human infants ). Specifically, the peak tracheal lavage fluid titer
in chimpanzees was 103after intratracheal inoculation with 104
TCID50of bPIV-3, but was less than 103after intranasal and intra-
as great [15,51]). Each of the points stated above encourage the fur-
X. Zhan et al. / Vaccine 26 (2008) 3480–3488
ther testing of SeV in clinical trials, the completion of which will
fully define vaccine safety in humans.
In conclusion, we have demonstrated that two new SeV con-
structs are capable of conferring complete protection against
hPIV-3 in a cotton rat model. We have also shown that a dual vac-
Such a dual vaccine may eventually protect human infants from
three serious respiratory pathogens; the xenogenic SeV backbone
may protect from hPIV-1 while the passenger genes may protect
from both hPIV-3 and RSV. Clearly, a single intranasal inoculation
that can target multiple respiratory pathogens would offer great
benefit in clinical pediatrics.
We thank Dr. Greg Prince (Virion Systems) for providing cot-
ton 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 Hospi-
tal, Memphis, TN) and the American Type Culture Collection
(ATCC, Rockville, MD) for providing virus isolates used in this
 Karron RA, Collins PL. Parainfluenza viruses. In: Knipe DM, Howley PM, Griffin
DE, Lamb RA, Martin MA, Roizman B, et al., editors. Fields virology. Lippincott
Williams and Wilkins; 2007. p. 1497–526.
 Durbin AP, Karron RA. Progress in the development of respiratory syncytial
virus and parainfluenza virus vaccines. Clin Infect Dis 2003;37(December 15
 Glezen WP, Frank AL, Taber LH, Kasel JA. Parainfluenza virus type 3: sea-
sonality and risk of infection and reinfection in young children. J Infect Dis
 Lee MS, Greenberg DP, Yeh SH, Yogev R, Reisinger KS, Ward JI, et al. Antibody
responses to bovine parainfluenza virus type 3 (PIV3) vaccination and human
PIV3 infection in young infants. J Infect Dis 2001;184(October 1 (7)):909–13.
 Hall SL, Stokes A, Tierney EL, London WT, Belshe RB, Newman FC, et al. Cold-
passaged human parainfluenza type 3 viruses contain ts and non-ts mutations
leading to attenuation in rhesus monkeys. Virus Res 1992;22:173–84.
 Belshe RB, Karron RA, Newman FK, Anderson EL, Nugent SL, Steinhoff M, et al.
in children. J Clin Micro 1992;30:2064–70.
 Hall SL, Sarris CM, Tierney EL, London WT, Murphy BR. A cold-adapted mutant
of parainfluenza virus type 3 is attenuated and protective in chimpanzees. J
Infect Dis 1993;167(April 4):958–62.
of a live attenuated bovine parainfluenza type 3 vaccine in two- to six-month-
old infants. Pediatr Infect Dis J 1996;15(August (8)):650–4.
 Ranjit R, Galinski MS, Heminway BR, Meyer K, Newman FK, Belshe RB.
Temperature-sensitive phenotype of the human parainfluenza virus type 3
candidate vaccine strain (cp45) correlates with a defect in the L gene. J Virol
 Skiadopoulos MH, Tatem JM, Surman SR, Mitcho Y, Wu S-L, Elkins WR, et al. The
recombinant chimeric human parainfluenza virus type 1 vaccine candidate,
rHPIV3-1cp45, is attenuated, immunogenic, and protective in African green
monkeys. Vaccine 2002;20:1846–52.
 Madhi SA, Cutland C, Zhu Y, Hackell JG, Newman F, Blackburn N, et al. Trans-
missibility, infectivity and immunogenicity of a live human parainfluenza type
3 virus vaccine (HPIV3cp45) among susceptible infants and toddlers. Vaccine
2006;24(March 20 (13)):2432–9.
 Haller AA, Miller T, Mitiku M, Coelingh K. Expression of the surface glyco-
proteins of human parainfluenza virus type 3 by bovine parainfluenza virus
type 3, a novel attenuated virus vaccine vector. J Virol 2000;74(December
 Pennathur S, Haller AA, MacPhail M, Rizzi T, Kaderi S, Fernandes F, et al.
Evaluation of attenuation, immunogenicity and efficacy of a bovine parain-
PIV-3 vaccine vector in rhesus monkeys. J Gen Virol 2003;84(December (Pt
 Hurwitz JL, Soike KF, Sangster MY, Portner A, Sealy RE, Dawson DH, et al.
Intranasal Sendai virus vaccine protects African green monkeys from infection
with human parainfluenza virus-type one. Vaccine 1997;15:533–40.
 Skiadopoulos MH, Surman SR, Riggs JM, Elkins WR, St Claire M, Nishio M, et al.
Sendai virus, a murine parainfluenza virus type 1, replicates to a level similar to
human PIV1 in the upper and lower respiratory tract of African green monkeys
and chimpanzees. Virology 2002;297(May 25 (1)):153–60.
virus type 1 but not Sendai virus replicates in human respiratory cells despite
IFN treatment. Virus Res 2006;121(October (1)):23–32.
 Slobod KS, Shenep JL, Lujan-Zilbermann J, Allison K, Brown B, Scroggs RA,
et al. Safety and immunogenicity of intranasal murine parainfluenza virus
type 1 (Sendai virus) in healthy human adults. Vaccine 2004;22(August 13
 Smith FS, Portner A, Leggiadro RJ, Turner EV, Hurwitz JL. Age-related develop-
ment of human memory T-helper and B-cell responses toward parainfluenza
virus type-1. Virology 1994;205(December (2)):453–61.
 Dave VP, Allan JE, Slobod KS, Smith SF, Ryan K, Powell U, et al. Viral cross-
type 1-specific cytotoxic T-cells. Virology 1994;199:376–83.
 Sealy R, Surman S, Hurwitz JL, Coleclough C. Antibody response to influenza
Immunology 2003;108(April (4)):431–9.
 Wyatt LS, Shors ST, Murphy BR, Moss B. Development of a replication-deficient
tion in an animal model. Vaccine 1996;14(October (15)):1451–8.
 Zhan X, Hurwitz JL, Krishnamurthy S, Takimoto T, Boyd K, Scroggs RA, et al.
Respiratory syncytial virus (RSV) fusion protein expressed by recombinant
Sendai virus elicits B-cell and T-cell responses in cotton rats and confers pro-
tection against RSV subtypes A and B. Vaccine 2007;25(December 17 (52)):
 Takimoto T, Hurwitz JL, Zhan X, Krishnamurthy S, Prouser C, Brown B, et al.
virus. Viral Immunol 2005;18(2):255–66.
 Bousse T, Matrosovich T, Portner A, Kato A, Nagai Y, Takimoto T. The long non-
coding region of the human parainfluenza virus type 1 f gene contributes to the
read-through transcription at the m–f gene junction. J Virol 2002;76(August
 Takimoto T, Hurwitz JL, Coleclough C, Prouser C, Krishnamurthy S, Zhan X, et
al. Recombinant Sendai virus expressing the G glycoprotein of respiratory syn-
cytial virus (RSV) elicits immune protection against RSV. J Virol 2004;78(June
based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA
polymerase. Proc Natl Acad Sci USA 1986;83(November (21)):8122–6.
 Kappler JW, Skidmore B, White J, Marrack P. Antigen-inducible, H-2-restricted,
interleukin-2-producing T cell hybridomas. Lack of independent antigen and
H-2 recognition. J Exp Med 1981;153(May 1 (5)):1198–214.
 Woodland DL, Happ MP, Bill J, Palmer E. Requirement for cotolerogenic gene
products in the clonal deletion of I–E reactive cells. Science 1990;247:964–7.
 Crennell S, Takimoto T, Portner A, Taylor G. Crystal structure of the mul-
tifunctional paramyxovirus hemagglutinin-neuraminidase. Nat Struct Biol
 Morrison T, Portner A. Structure, function and intracellular processing of the
glycoproteins of paramyxoviridae. In: Kingsbury DW, editor. The paramyx-
oviruses. New York, NY: Plenum Press; 1991. p. 347–82.
forming cell response to Sendai virus infection of mice in different anatomical
compartments. Virology 1995;207:287–91.
 Hyland L, Sangster M, Sealy R, Coleclough C. Respiratory virus infection of mice
provokes a permanent humoral immune response. J Virol 1994;68:6083–6.
 Kast WM, Bronkhorst AM, De Waal LP, Melief CJM. Cooperation between
cytotoxic and helper T lymphocytes in protection against lethal Sendai virus
infection. J Exp Med 1986;164:723–38.
 Olmsted RA, Elango N, Prince GA, Murphy BR, Johnson PR, Moss B, et al.
Expression of the F glycoprotein of respiratory syncytial virus by a recombi-
nant vaccinia virus: comparison of the individual contributions of the F and
G glycoproteins to host immunity. Proc Natl Acad Sci USA 1986;83(October
 Hancock GE, Speelman DJ, Heers K, Bortell E, Smith J, Cosco C. Generation of
atypical pulmonary inflammatory responses in BALB/c mice after immuniza-
tion with the native attachment (G) glycoprotein of respiratory syncytial virus.
J Virol 1996;70(November (11)):7783–91.
 Johnson TR, Johnson JE, Roberts SR, Wertz GW, Parker RA, Graham BS. Prim-
ing with secreted glycoprotein G of respiratory syncytial virus (RSV) augments
interleukin-5 production and tissue eosinophilia after RSV challenge. J Virol
by a specific amino acid sequence of the attachment (G) protein of respiratory
syncytial virus. J Exp Med 1998;188(November 16 (10)):1967–72.
 Hurwitz JL. Development of recombinant Sendai virus vaccines for prevention
Dis J; in press.
system in health and disease. 6th ed. New York, NY: Garland Publishing;
 Kast WM, Roux L, Curren J, Blom HJ, Voordouw AC, Meloen RH, et al. Protec-
tion against lethal Sendai virus infection by in vivo priming of virus-specific
3488 Download full-text
X. Zhan et al. / Vaccine 26 (2008) 3480–3488
cytotoxic T lymphocytes with a free synthetic peptide. Proc Natl Acad Sci USA
1991;88(March 15 (6)):2283–7.
 Voges B, Vallbracht S, Zimmer G, Bossow S, Neubert WJ, Richter K, et al. Recom-
binant Sendai virus induces T cell immunity against respiratory syncytial virus
that is protective in the absence of antibodies. Cell Immunol 2007;247(June
 ZuckermanJN. Protectiveefficacy,
safety of hepatitis B vaccines. J Med Virol 2006;78(February (2)):169–
 Webby RJ, Sandbulte MR. Influenza vaccines. Front Biosci; in press.
 Griffin DE, Pan CH, Moss WJ. Measles vaccines. Front Biosci 2008;13:
 Arvin AM, Greenberg HB. New viral vaccines. Virology 2006;344(January 5
 Ada G. The importance of vaccination. Front Biosci 2007;12:1278–90.
 News in Brief. HIV vaccine failure prompts Merck to halt trial. Nature
2007;449(September 27 (7161)):390.
 Cohen J. AIDS vaccines. HIV dodges one-two punch. Science 2004;305(Septem-
ber 10 (5690)):1545–7.
 Andreansky S, Liu H, Adler H, Koszinowski UH, Efstathiou S, Doherty PC. The
limits of protection by “memory” T cells in Ig−/−mice persistently infected
with a gamma-herpesvirus. Proc Natl Acad Sci USA 2004;101(February 17
 McCormick J, Tubman R. Readmission with respiratory syncytial virus (RSV)
infection among graduates from a neonatal intensive care unit. Pediatr Pul-
monol 2002;34(October (4)):262–6.
 Wyke Coelingh KL, Winter CC, Tierney EL, London WT, Murphy BR. Attenuation
of bovine parainfluenza virus type 3 in nonhuman primates and its ability to
confer immunity to human parainfluenza virus type 3 challenge. J Infect Dis