A single dose intranasal combination panebolavirus vaccine

Ebolaviruses Ebola (EBOV), Sudan (SUDV) and Bundibugyo (BDBV) cause severe human disease, which may be accompanied by hemorrhagic syndrome, with high case fatality rates. Monovalent vaccines do not offer cross-protection against these viruses whose endemic areas overlap. Therefore, development of a panebolavirus vaccine is a priority. As a vaccine vector, human parainfluenza virus type 3 (HPIV3) has the advantages of needle-free administration and induction of both systemic and local mucosal antibody responses in the respiratory tract. We developed a HPIV3-vectored combination vaccine against EBOV, SUDV and BDBV. To minimize the anti-vector immunity, HPIV3 envelope genes were removed from the vaccine constructs which express only the ebolavirus envelope protein – glycoprotein (GP). A single intranasal vaccination of guinea pigs or ferrets with the trivalent combination vaccine elicited humoral responses to each of the targeted ebolaviruses, including binding and neutralizing antibodies, as well as Fc-mediated effector functions. This vaccine protected animals from death and disease caused by lethal challenges with EBOV, SUDV or BDBV. Notably, the combination vaccine elicited protection which was comparable to that induced by the monovalent vaccines, thus demonstrating the value of this combination trivalent vaccine.


INTRODUCTION
Viruses Ebola (EBOV), Sudan (SUDV) and Bundibugyo (BDBV), which cause a severe human disease, are members of the genus Ebolavirus of the family of Filoviridae.Because the endemic areas of these viruses in Africa overlap, it would be advantageous to have a vaccine which would protect against all pathogenic ebolaviruses.However, while these three viruses are related, monovalent vaccines do not offer effective cross-protection 1 .Because of that, the current SUDV outbreak in Uganda 2 cannot be controlled by any of the two approved vaccines against EBOV 3 .Therefore, a panebolavirus vaccine is highly desirable.The most feasible way to develop a panebolavirus vaccine is to use a combination approach.Still, several questions relevant for this approach must be answered.Would combination of three components of a polyvalent ebolavirus vaccine result in a sufficient immune response to protect against each of the three targeted ebolaviruses?Would this combination result in a skew of the immune response toward one targeted ebolavirus at the expense of the response against another virus?Is respiratory tract delivery feasible for combination ebolavirus vaccines?Human parainfluenza virus type 3 (HPIV3)-vectored vaccines against ebolaviruses are efficacious in small animal models [4][5][6] and in non-human primates [7][8][9] .The HPIV3 vaccine platform offers several advantages including the elicitation of mucosal humoral and cellular immunity in the respiratory tract, in addition to the systemic immune response, as well as a respiratory tract delivery that eliminates the need for trained medical personnel.However, pre-existing immunity against the vector is a concern since HPIV3 is a common pediatric pathogen that infects the respiratory tract.As HPIV3-specific neutralizing antibodies target the two HPIV3 envelope proteins expressed at the viral surface, hemagglutinin-neuraminidase (HN) and the fusion protein (F), a second generation of ebolavirus HPIV3-based vaccines was designed by substituting the native HPIV3 envelope proteins with the EBOV envelope glycoprotein (GP).This modification resulted in (1) high attenuation of the vaccine construct, (2) resistance of the vaccine particles to neutralizing HPIV3-specific antibodies, (3) elimination of the immune response to HPIV3 HN and F and, consequently, (4) enhanced targeting of the added EBOV GP antigen 10 .This second generation HPIV3-vectored EBOV GP vaccine demonstrated protection against lethal EBOV challenge in guinea pigs 6,10 and non-human primates 9 .
In this study, we investigated if a combination polyvalent strategy would offer protection against each pathogenic ebolavirus.We generated second generation HPIV3-vectored monovalent vaccines against SUDV and BDBV and demonstrated their protective efficacy in small animal models.We also used them, along with second generation HPIV3 EBOV vaccine, as a trivalent vaccine against EBOV, SUDV and BDBV, and tested the combination vaccine for immunogenicity and efficacy against lethal challenge with each of the three viruses in small animal models.Our findings demonstrate that a single immunization with the monovalent vaccines or the trivalent combination elicits homologous binding and neutralizing antibodies as well as Fc-dependent functions, and offers robust protection from death and disease caused by a lethal dose of each of the targeted viruses.

Development of second-generation HPIV3-vectored ebolavirus vaccine constructs
The second generation HPIV3-vectored EBOV vaccine was developed previously as follows.The entire F and HN genes were removed from the HPIV3 full-length clone.A transcriptional cassette for expression of EBOV GP was constructed by flanking its open reading frame with HPIV3specific gene-start and gene-end transcriptional signals upstream and downstream, respectively.
The cassette was inserted in the full-length clone between the P and M genes resulting in the subsequent recovery of the replication-competent yet highly attenuated vaccine construct HPIV3/ΔF-HN/EboGP 10 .Here, we replaced the ORF of EBOV GP with that of SUDV or BDBV in the full-length clone to generate HPIV3/ΔF-HN/SUDV-GP and HPIV3/ΔF-HN/BDBV-GP vectored vaccine constructs, respectively (Fig. 1a).The resulting replication-competent chimeric viruses were recovered as previously described 4 .As expected, the constructs were deficient in HPIV3 glycoproteins F and HN, so that the only glycoprotein expressed on the viral surface was the SUDV GP or BDBV GP (Fig. 1b).The constructs were propagated in LLC-MK2 cells, and the expected genomic sequences were confirmed by sequence analysis.Protein expression of the GP antigens by the individual vaccine constructs and the trivalent combination (HPIV3/ΔF-HN/Trivalent) was assessed by western blot analysis of LLC-MK2 cells infected with the vaccine viruses (Fig. 1c).SUDV GP protein was expressed at comparable levels in infected cells with the monovalent SUDV vaccine or HPIV3/ΔF-HN/Trivalent, while EBOV GP and BDBV GP expression was higher in cells infected with the monovalent vaccine constructs than HPIV3/ΔF-HN/Trivalent.

The individual vaccines and their combination induce robust ebolavirus-binding and neutralizing antibody responses
The immunogenicity and the protective efficacy of the vaccines were evaluated in the guinea pig model against EBOV and SUDV and in the ferret model against BDBV (Fig. 1d).On day 0, groups of Dunkin-Hartley guinea pigs were vaccinated by a single intranasal dose of the individual EBOV or SUDV monovalent vaccines at 4x10 5 PFU per animal, or HPIV3/ΔF-HN/Trivalent at 4x10 5 PFU of the EBOV, SUDV and BDBV individual vaccines resulting in a total vaccine dose of 1.2x10 6 PFU per animal.The control group received 4x10 5 PFU of wild-type HPIV3.On day 33, all guinea pigs were challenged intraperitoneally with lethal doses of 10 3 PFU of guinea pig-adapted EBOV 11 or guinea pig-adapted SUDV 12 .The two monovalent vaccines and HPIV3/ΔF-HN/Trivalent elicited IgG binding to the homologous EBOV GP or SUDV GP (Fig. 2a).HPIV3/ΔF-HN/Trivalent elicited EBOV and SUDV GP-binding antibodies at lower level compared to the respective monovalent vaccines, but the differences were not statistically significant.IgA-specific homologous antibody titers resulted in similar profiles (Fig. 2b).Serum samples from the EBOV and SUDV monovalent vaccine-immunized guinea pigs effectively neutralized EBOV (isolate Mayinga) 13 and SUDV (strain Gulu) 14 , respectively (Fig. 2c).The immune sera from HPIV3/ΔF-HN/Trivalent vaccinated animals also elicited antibodies neutralizing SUDV, but not EBOV or BDBV (strain Uganda) 15 (Fig. 2c).This distinct neutralization profile may be due to the different antigen expression level in HPIV3/ΔF-HN/Trivalent compared to the monovalent vaccines (Fig. 1c).
Groups of ferrets were vaccinated on day 0 by a single intranasal dose of the BDBV monovalent vaccine at 1x10 7 PFU per animal or with the combination of EBOV, SUDV and BDBV individual vaccines in the HPIV3/ΔF-HN/Trivalent at 1x10 7 PFU per vaccine for a total vaccine dose of 3x10 7 PFU per ferret (Fig. 1d).The control group received 1x10 7 PFU of wild-type HPIV3.On day 33, ferrets were challenged intramuscularly with a lethal dose of 10 3 PFU BDBV (strain Uganda).The HPIV3/ΔF-HN/Trivalent elicited IgG binding antibodies to EBOV GP, SUDV GP and BDBV GP while the monovalent BDBV vaccine induced high IgG binding titers to BDBV GP and low IgG binding antibody titers to EBOV GP (Fig. 3a).The trivalent and the BDBV monovalent vaccines also elicited IgA binding antibodies to SUDV GP and BDBV GP but not EBOV GP (Fig. 3b).
Trivalent vaccine-induced ADNP responses specific for BDBV GP were significantly higher compared to EBOV and SUDV monovalent vaccine-induced responses in guinea pigs (Fig. 4b).
While monovalent vaccine-induced ADCP responses were not significant in guinea pigs (Fig. 4c), monovalent BDBV vaccine elicited significantly higher ADCP responses than HPIV3/ΔF-HN/Trivalent in vaccinated ferrets (Fig. 5b).The strongest vaccine-induced Fc-dependent responses were ADCD in both guinea pigs and ferrets (Fig. 4d, 5c).Monovalent SUDV elicited significantly higher SUDV GP-specific ADCD responses in guinea pigs compared to EBOVvaccinated and mock-vaccinated animals (Fig. 4d) and HPIV3/ΔF-HN/Trivalent induced higher responses than monovalent BDBV vaccine and mock vaccine in ferrets (Fig. 5c).In addition, HPIV3/ΔF-HN/Trivalent elicited BDBV GP-specific ADCD responses in immunized guinea pigs (Fig. 4d), and BDBV monovalent vaccine induced higher ADCD responses in ferrets compared to HPIV3/ΔF-HN/Trivalent and mock vaccine (Fig. 5c).Taken together, these results show that one immunization with the trivalent or monovalent vaccines induced detectable levels of ADCD which was more pronounced in animals vaccinated with the trivalent vaccine, while ADNP and ADCP were not detected in most of the animals.

A single dose of the monovalent and trivalent vaccines protects animals from disease and death caused by ebolaviruses
Four weeks after vaccination, guinea pigs were challenged with guinea pig-adapted EBOV (strain Mayinga) 11 or SUDV (strain Boneface) 12 , while ferrets were challenged with wild type BDBV strain Uganda 16 (Fig. 6).In the EBOV challenge study, all guinea pigs vaccinated with the EBOV monovalent vaccine and 80% of animals vaccinated with HPIV3/ΔF-HN/Trivalent survived (Fig. 6a) while all control animals succumbed to the disease.Only two animals in the combination group showed signs of disease (Fig. 6b) and displayed loss of weight (Fig. 6c) but no vaccinated animals had viremia during the entire observation period (Fig. 6d).In the SUDV challenge study, all guinea pigs vaccinated with the combination vaccine survived, and one animal vaccinated with the SUDV monovalent vaccine did not (Fig. 6a), while all control animals succumbed to the disease.One animal in each of the monovalent and the combination groups showed signs of disease (Fig. 6b) and displayed temporary loss of weight (Fig. 6c) but none of the vaccinated animals had viremia (Fig. 6d).In the BDBV challenge study, all ferrets vaccinated with the monovalent BDBV vaccine, or the combination survived (Fig. 6a).All control animals had viremia and displayed signs of disease (Fig. 6b,d), they all reach moribund status and were euthanized (Fig. 6a).In contrast, none of the BDBV vaccinated ferrets showed any loss of weight or other signs of the disease during the observation period and none had any viremia (Fig. 6b-d).Taken together, our data show that a single dose of the monovalent vaccines and HPIV3/ΔF-HN/Trivalent offer robust protection from death and disease caused by EBOV, SUDV and BDBV.

DISCUSSION
Combination polyvalent vaccine formulation is an effective approach to provide broad protection against ebolaviruses, as we previously demonstrated with the first generation HPIV3-vectored EBOV vaccine 17 .However, the first generation HPIV3-vectored vaccines are partially sensitive to HPIV3-neutralzing antibodies 4,5 , which are present in a significant part of the adult human population (reviewed in ref. 18 ).In contrast, the second-generation vaccine vector, which lacks the HPIV3 envelope proteins HN and F, is resistant to HPIV3-specific neutralizing antibodies 10 .We developed second-generation vaccines against SUDV and BDBV and tested them, along with the similar vaccine against EBOV, for protection against EBOV and SUDV in guinea pigs and against BDBV in ferrets.Our data show that a single immunization with the monovalent vaccines or HPIV3/ΔF-HN/Trivalent elicited homologous binding and neutralizing antibodies as well as modest levels of Fc-dependent responses, mostly ADCD.Importantly, monovalent and combination vaccines offered a robust protection from death and disease caused by each of the three lethal ebolavirus challenges.
EBOV infection routes include transmission through fomites, biological fluid droplets and contact with mucosal surface of the respiratory tract (reviewed in ref. 19 ).Therefore, a strong local immune response in the respiratory tract would be beneficial.IgA plays an important immunological role in the mucosa, where it can interact with pathogens before they establish a systemic infection.
While we did not test IgA responses in the respiratory tract, serum IgA responses to acute respiratory tract infection can be an indirect measure of a mucosal immune response 20,21 .We previously demonstrated that the first generation HPIV3-vestored vaccine against EBOV does induce mucosal EBOV-specific IgA response in the respiratory tract of vaccinated non-human primates 8 .We also demonstrated that systemic EBOV-specific IgA induced by this vaccine in non-human primates is one of the most important correlates of protection 9 .Therefore, the detected IgA antibody response is likely to provide an additional layer of protection against ebolavirus exposure through the respiratory tract.
Immunization with a combination of antigenically related vaccines can skew immune responses and change the epitope hierarchy as shown with multivalent vaccines against polio or dengue viruses, resulting in an imbalanced response to individual vaccine components [22][23][24][25] .Multivalent approaches have been tested with VSV-or HPIV3-based ebolavirus vaccines and have also shown antigenic bias.A trivalent VSV-vectored vaccine with EBOV, SUDV and Marburg virus (a filovirus from a heterologous genus) antigens protected macaques from death caused by each of the three viruses, but animals exposed to SUDV showed signs of disease before recovering 26 , which likely was due to a more dominant response to the EBOV and Marburg virus vaccine components.In our study, mammalian cells infected with the monovalent vaccines expressed high levels of GP antigens but cells infected with the combination vaccine expressed high levels of SUDV GP only.We observed a similar expression pattern when these antigens were expressed by HPIV3/Trivalent based on the first generation HPIV3 vector 17 .This discrepancy in expression is likely to be the cause for the lower EBOV-and BDBV-specific binding and neutralizing antibodies induced in animals immunized with the vaccine combination compared to their monovalent counterparts.However, the ADCD was generally higher for the combination vaccine as compared to the monovalent vaccines.Notably, despite these expression level differences, the vaccine protection efficacy between the monovalent and the combination vaccine was comparable thus suggesting that the protection was likely mediated both by virus neutralization and ADCD.This is consistent with our previous study with several EBOV vaccines which demonstrated correlation of survival with ADCD, in addition to some other parameters 9 .However, we cannot rule out the role of T cell immunity in the protection, which was not evaluated in the present study due to the limited availability of reagents specific for immune cells of guinea pigs, and especially ferrets.Despite this limitation, our data show that one dose of the combination vaccine offers robust protection against lethal challenge with EBOV, SUDV and BDBV, which is an important feature for vaccines to be deployed in emergency situations to fight ebolavirus outbreaks.in 200 µl phosphate buffered saline via the intranasal route (100 µl in each nostril).On days -1

METHODS
(one day prior the first vaccine inoculation) and 28 retro-orbital blood collections were performed.
On day 33, vaccinated and control animals were exposed to the targeted dose of 10 3 PFU of guinea pig-adapted EBOV strain Mayinga or guinea-pig adapted SUDV strain Boneface delivered by IP injection.Animals were monitored up to three times daily for weight loss and signs of disease.Animals that had reached the moribund state (which was defined as reaching one of the following conditions: failure to move upon stimulation, inability to reach food or water, greater than 20% body weight loss, or paralysis) were euthanized.Retro-orbital blood collections were performed from surviving animals at days 3, 6, 9, 13, and 28 post challenge.All remaining guinea pigs were euthanized at 28 days post challenge.
Twelve-week-old female ferrets were acquired from Marshall BioResources (North Rose, NY).
For blood collections and vaccine inoculations, animals were anesthetized with 5% isoflurane.On day 0, ferrets were inoculated with 1x10 7 PFU of each of the monovalent vaccine constructs or a mixture of the three constructs at 1x10 7 of each construct resulting in the total dose of 3x10 7 total PFU in 1.0 ml phosphate buffered saline via the intranasal route (500 µl in each nostril).On day -1 (one day prior to the vaccine inoculation) and on day 28, blood collections were performed.On day 33, vaccinated and control animals were exposed to the targeted dose of 10 3 PFU of BDBV strain Uganda by IM injection.Animals were monitored up to three times daily for weight loss and signs of disease.Animals that had reached moribund state (which was defined as one of the following conditions: failure to move upon stimulation, visible ecchymosis, respiratory rate >80 beats per minute, open mouth breathing, or severe dehydration evidenced by sunken eyes, vertebrae, dorsal pelvis and/or ribs prominent) were euthanized.Blood collections were performed from surviving animals on days 3, 6, 9, 13, and 28 post challenge.All remaining ferrets were euthanized 28 days post BDBV challenge.The control group had two ferrets, and three control ferrets from a previous study 17 were added as historic controls to reach N = 5.For these historic controls, IgG ELISA binding data, virus neutralization data and challenge data were taken from the original study, which was performed in an identical to this study manner (as described in the next subsections), while the Fc effector assays in the current study were run with sera from the three historic control serum samples analyzed in parallel with the current samples.
Enzyme linked immunosorbent assays (ELISA).Sera collected from animals were tested for their ability to bind GP of the three ebolaviruses by ELISA.For the guinea pig sera, ELISA plates were coated overnight at room temperature with 8 ng of recombinant EBOV GP, BDBV GP or SUDV GP (His-tagged glycoproteins minus the transmembrane domain produced in Sf9 insect cells, IBT Bioservices) diluted in PBS.Plates were washed five times in PBS with 0.1% Tween 20 and blocked with 3% dry milk in PBS for 1 h at 37°C.After blocking, four-fold dilutions of guinea pig sera were prepared in blocking buffer and incubated on the GP coated plates for 1 h at 37°C.
Plates were washed as above and incubated with a 1:5,000 dilution of horseradish peroxidaseconjugated goat anti-guinea pig antibody (Jackson ImmunoResearch, West Grove, PA) for 1 h at 37°C.Plates were washed as above, developed using the SureBlue ELISA substrate system (KPL, Gaithersburg, MD), and color intensity was measured on BioTek Synergy HT microplate reader (Winooski, VT) at 630 nm.
For the ferret sera, ELISA plates were coated overnight at room temperature with 6.25 ng of recombinant EBOV GP, BDBV GP or SUDV GP (IBT Bioservices) diluted in PBS.Plates were washed five times in PBS with 0.1% tween 20 and blocked for 1 h at 37°C with PBS containing 5% dry milk and 2.5% BSA.After blocking, four-fold dilutions of ferret sera were prepared in blocking buffer and incubated on the GP coated plates for 1 h at 37°C.Plates were washed as above and incubated with a 1:10,000 dilution of horseradish peroxidase-conjugated goat antiferret IgG antibody (Abcam, Cambridge, MA) for 1 h at 37°C.Plates were washed as above, developed using the SureBlue ELISA substrate system (KPL), and color intensity was measured on BioTek Synergy HT microplate reader (Winooski, VT) at 630 nm.
Plaque reduction assay.Sera collected from animals were tested for virus neutralizing capabilities against EBOV strain Mayinga, BDBV strain Uganda and SUDV strain Gulu.Briefly, 10-fold diluted sera were further diluted in a 2-fold serial fashion.Fifty µl of each serum dilutions were mixed with 200 PFU of virus in 50 µl.The serum/virus mixtures were incubated for 1 h at 37°C.Fifty µl (100 PFU) of the mixtures were then added to Vero E6 cell monolayers in flat-bottom 96-well plates and incubated for 1 h at 37°C.Serum/virus mixtures were removed from the cells, which were then covered with 1% methylcellulose in MEM with 2% FBS (Gibco, Grand Island, NY).Cells were fixed in 10% formalin according to approved standard operating procedure, removed from the BSL-4 laboratory, and plaques were visualized by immunostaining and counted.Briefly, EBOV plaques were stained with goat anti-EBOV serum at dilution 1:2,000 and horse radish peroxidase (HRP) conjugated bovine anti-goat IgG at dilution 1:2,000 (Jackson ImmunoResearch, West Grove, PA, # 805-035-180).BDBV and SUDV plaques were stained with 1 µg/ml human mAb BDBV52 or BDBV43 27 , respectively and HRP-conjugated goat anti-human IgG at dilution 1:2,000 (KPL Gaithersburg, MD, # 474-1002).

Antibody-mediated neutrophil phagocytosis (ADNP).
Recombinant GP proteins were biotinylated and coupled to 1 µm FITC + Neutravidin beads (Life Technologies).Briefly, GPs were biotinylated using Sulfo-NHS-LC-LC biotin (ThermoFisher Scientific), and excess biotin was removed using a Zeba desalting column (ThermoFisher Scientific).Biotinylated GPs were incubated with Neutravidin-coated fluorescent beads (ThermoFisher Scientific) at 4°C overnight (1 µg biotinylated protein per 1 µl of beads).Beads were washed twice with 0.1% BSA in PBS and resuspended in 100 µl of 0.1% BSA in PBS per 1 µl of coupled beads.Sera from vaccinated animals were diluted 1:100 in cell culture medium and incubated with GP-coated beads for 2 h at 37°C.Freshly isolated neutrophils from donor blood were added at a concentration of 5.0 x 10 4 cells per well and incubated for 1 h at 37°C.Cells were stained at 1:100 with CD66b (Pacific Blue, Clone G10F5; Biolegend cat # 305111), fixed with 4% paraformaldehyde, and analyzed by flow cytometry on a Sartorius iQue flow cytometer, and a minimum of 30,000 events were recorded and analyzed.Neutrophils were defined as SSC-A high CD66b + .The phagocytic score was determined using the following formula: (percentage of FITC + cells) x (median fluorescent intensity (MFI) of the FITC + cells)/10,000.

Antibody-dependent cellular phagocytosis (ADCP) by human monocytes. Recombinant
GPs were biotinylated and coupled to 1 µm FITC + Neutravidin beads.Serum samples from vaccinated animals were diluted 1:500 in culture medium and incubated with GP-coated beads for 2 h at 37°C followed by addition of THP-1 human monocytic cells for 18 h.Cells were fixed with 4% paraformaldehyde and analyzed on a Sartorius iQue flow cytometer, and a minimum of 10,000 events were recorded and analyzed.The phagocytic score was determined as described above.
Antibody-mediated complement deposition (ADCD).Recombinant GPs were biotinylated and coupled to 1 µm red fluorescent Neutravidin beads.Serum samples from vaccinated animals were diluted 1:10, 1:100, and 1:200 in culture medium and incubated with GP-coated beads for 2 h at 37°C followed by the addition of reconstituted guinea pig complement (Cedarlane Labs, Burlington, Canada) diluted in gelatin veronal buffer containing magnesium and calcium (Boston Bioproducts, Milford, MA) and incubation for 20 min at 37°C.Beads were washed twice with Vaccine

Supplementary Files
This is a list of supplementary les associated with this preprint.Click to download.

SupplFigsv16.pdf
Generation of the vaccine constructs.The vaccine constructs were based on the following GP sequences: EBOV -Ebola virus/H.sapiens-tc/COD/1976/Yambuku-Mayinga(/200706291/Butalya-811249 (KU182911.1),and SUDV -Sudan virus/H.sapienstc/UGA/2000/Gulu-200011676(MH121163.1).The construction of full-length DNA clone for the second generation EBOV vaccine (HPIV3/ΔF-HN/EboGP) was previously described 10 .To generate the second generation HPIV3-vectored BDBV and SUDV full-length clones, the EBOV GP ORF were replaced with that of SUDV and BDBV, which were taken from the full-length clones of the first generation HPIV3-vectored SUDV and BDBV described previously 17 .The inserts were confirmed by sequence analysis.Viruses were recovered by transfection into BSR-T7 cells with a subsequent 48 h long incubation at 32°C and 3-5 passages in LLC-MK2 cells (5 day incubation at 32°C).To reach viral stock titers sufficiently high for intranasal immunization of ferrets, viral stocks were concentrated with Centricon Plus 70 devices (EMD Millipore, Burlington, MA) according to the manufacturer's instructions.
constructs and study design.a. Vaccine candidates were designed by inserting the GP gene of EBOV, SUDV or BDBV between the P and M genes of HPIV3.b.The F and HN HPIV3 genes were removed so that the lovirus glycoprotein of interest is expressed as the sole transmembrane envelope protein in the vaccine constructs.c.Expression of lovirus GP proteins by cells infected with the monovalent or trivalent vaccine constructs evaluated by Western blotting.Puri ed GP proteins were used as positive controls, and actin or GAPDH were used as loading controls.The experiment was performed independently twice with essentially similar results.D. Guinea pigs and ferrets were vaccinated intranasally on day 0 with the monovalent vaccines or with the trivalent combination, and the control group received the empty HPIV3 vector.The lovirus challenge occurred on day 33, via the intraperitoneal route for the guinea pig model and via the intramuscular route for the ferret model.

Figure 6 Protection
Figure 6