Accepted Manuscript DNA vaccination regimes against Schmallenberg virus infection in IFNAR suggest two targets for immunization
−/−
mice
Hani Y. Boshra, Diego Charro, Gema Lorenzo, Isbene Sanchez, Beatriz Lazaro, Alejandro Brun, Nicola G.A. Abrescia PII:
S0166-3542(16)30545-9
DOI:
10.1016/j.antiviral.2017.02.013
Reference:
AVR 4015
To appear in:
Antiviral Research
Received Date: 27 September 2016 Revised Date:
4 February 2017
Accepted Date: 20 February 2017
Please cite this article as: Boshra, H.Y., Charro, D., Lorenzo, G., Sanchez, I., Lazaro, B., Brun, A., −/− Abrescia, N.G.A., DNA vaccination regimes against Schmallenberg virus infection in IFNAR mice suggest two targets for immunization, Antiviral Research (2017), doi: 10.1016/j.antiviral.2017.02.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
DNA Vaccination Regimes Against Schmallenberg Virus Infection in IFNAR-/- Mice Suggest Two Targets For Immunization
RI PT
Hani Y Boshraa, Diego Charroa, Gema Lorenzob, Isbene Sanchezc, Beatriz Lazaroc, Alejandro Brunb, Nicola GA Abresciaa,d, *
Structural Biology Unit, CIC bioGUNE, CIBERehd, Bizkaia Technology Park, 48160 Derio,
Spain.
Vacunek SL, Bizkaia Technology Park, 48160 Derio, Spain.
d
IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain.
TE D
c
INIA-CISA Valdeolmos, 28130 Madrid, Spain.
M AN U
b
SC
a
AC C
EP
*Corresponding author. E-mail address:
[email protected]
1
ACCEPTED MANUSCRIPT
ABSTRACT Schmallenberg virus (SBV) is an RNA virus of the Bunyaviridae family, genus Orthbunyavirus, that infects wild and livestock species of ruminants. While inactivated and
RI PT
attenuated vaccines have been shown to prevent SBV infection, little is known about their mode of immunity; specifically, which components of the virus are responsible for inducing immunological responses in the host.
SC
As previous DNA vaccination experiments on other bunyaviruses have found that glycoproteins, as well as modified (i.e. ubiquitinated) nucleoproteins (N) can confer immunity against virulent
M AN U
viral challenge, constructs encoding for fragments of SBV glycoproteins GN and GC, as well as ubiquitinated and non-ubiquitinated N were cloned in mammalian expression vectors, and vaccinated intramuscularly in IFNAR-/- mice. Upon viral challenge with virulent SBV, disease progression was monitored.
Both the ubiquitinated and non-ubiquitinated nucleoprotein
TE D
candidates elicited high titers of antibodies against SBV, but only the non-ubiquitinated candidate induced statistically significant protection of the vaccinated mice from viral challenge. Another construct encoding for a putative ectodomain of glycoprotein GC (segment aa. 678-947)
EP
also reduced the SBV-viremia in mice after SBV challenge.
When compared to other
experimental groups, both the nucleoprotein and GC-ectodomain vaccinated groups displayed
AC C
significantly reduced viremia, as well as exhibiting no clinical signs of SBV infection. These results show that both the nucleoprotein and the putative GC-ectodomain can serve as protective immunological targets against SBV infection, highlighting that viral glycoproteins, as well as nucleoproteins are potent targets in vaccination strategies against bunyaviruses.
2
ACCEPTED MANUSCRIPT
Keywords
AC C
EP
TE D
M AN U
SC
RI PT
Bunyaviridae, DNA vaccine, viral proteins, Schmallenberg virus, immunization.
3
ACCEPTED MANUSCRIPT
1. INTRODUCTION Schmallenberg virus (SBV) is a member of the Bunyaviridae family (genus Orthobunyaviridae).
Discovered in Germany in 2011 (Wernike et al., 2014;Gibbens,
RI PT
2012;Hoffmann et al., 2012), SBV has spread throughout the European continent, spanning from Ireland to Turkey (Yilmaz et al., 2014). Initially characterized in domesticated sheep, goat and cattle, further epidemiological studies have linked SBV to wild ruminants such as deer, mouflon
SC
(i.e. wild sheep) and bison (Larska et al., 2013a;Larska et al., 2014;Wernike et al., 2015b). In affected herds, SBV infection has been associated with diarrhea, fever and decreased milk
M AN U
production (Hoffmann et al., 2012;Wernike et al., 2013b;Peperkamp et al., 2015;LievaartPeterson et al., 2015). Although symptoms are generally mild and short-lived in adult ruminants, SBV infection has been linked to widespread abortions and developmental malformations in newborn domestic ruminant livestock (Bayrou et al., 2014).
TE D
SBV is an arthropod-borne pathogen that has been shown to be transmitted by biting midges (i.e. Culicoides) (Balenghien et al., 2014;Larska et al., 2013b;Larska et al., 2013c). Structural information on the whole virion is lacking and only the tridimensional crystal structure of the
EP
nucleoprotein is known (Dong et al., 2013;Ariza et al., 2013). Phylogenetic studies have shown the L, M and S-segments of the SBV genome to be most similar to viruses of the Simbu
AC C
serogroup of orthobunyaviruses, with viruses of the species Sathuperi virus being the closest relatives (Goller et al., 2012). Currently, inactivated vaccines against SBV are available for use in ruminant animals, and have been shown to be effective in inducing neutralizing antibodies and completely inhibiting viral replication upon experimental viral challenge (Hechinger et al., 2014;Wernike et al., 2013c). While effective, the cost-benefit ratio of the inactivated SBV vaccine may prevent its widespread
4
ACCEPTED MANUSCRIPT
use, possibly due to the fact that SBV outbreaks appear to be endemic, e.g. in Germany (Wernike et al., 2015c). Therefore, a more practical approach to SBV vaccination may involve the development of lower-cost subunit vaccines or DNA vaccines (Babiuk et al., 2014;Kutzler and
recombinant viral proteins to induce a potent immune response.
RI PT
Weiner, 2008). However, the efficacy of such as strategy depends on the ability of individual
Previously, the use of DNA vaccination against components of Rift Valley fever virus (RVFV)
SC
with particular emphasis on studying the protective role of glycoproteins (encoded by the M segment), as well as the nucleoprotein was successfully evaluated (Lagerqvist et al., 2009;Boshra
M AN U
et al., 2011a). Using interferon α/β knockout mice (IFNAR-/-), it was demonstrated that the glycoprotein-encoding segment of RVFV could confer complete protection against viral challenge (Lagerqvist et al., 2009;Lorenzo et al., 2010); and that nucleoprotein, when expressed as a fusion protein with ubiquitin, can also confer near-complete protection against RVFV
TE D
infection (Boshra et al., 2011b). In the case of SBV, monoclonal antibodies generated in mice inoculated with whole virus demonstrated a high degree of antigenicity against the N-terminal portion of glycoprotein C (GC; aa 468-702 of the M-segment), as well as the nucleoprotein
EP
(Wernike et al., 2015a), with the former being associated with neutralizing activity (Roman-Sosa et al., 2016). However, whether or not these antigenic sites are sufficiently immunogenic to
AC C
induce a complete protective immune response in vivo is not known. Over the past two decades, IFNAR-/- have been used as animals models for the study of bunyaviral infection and immunity (Lorenzo et al., 2010;Boshra et al., 2011b;Hefti et al., 1999;Pavlovic et al., 2000;Schuh et al., 1999;Proenca-Modena et al., 2015;Oestereich et al., 2014;Zivcec et al., 2013).
Recently,
IFNAR-/- mice were also shown to be susceptible to SBV infection (Wernike et al., 2012;Ponsart et al., 2014;Sailleau et al., 2013). Although, unlike with RVFV, clinical signs were not as evident
5
ACCEPTED MANUSCRIPT
following infection with virulent strains of the virus. While IFNAR-/- mice infected with RVFV resulted in complete mortality, IFNAR-/- infected with virulent SBV displayed primarily decreased weight loss, ataxia, apathy and limited mortality (Kraatz et al., 2015). This model has
RI PT
already been used to validate deletion mutants of SBV for their potential use as attenuated strains for vaccination (i.e. mutants lacking NSm and/or NSs) (Kraatz et al., 2015).
Here, we report on the use of DNA vaccination, encoding for different components of
SC
SBV. These include: a putative ectodomain of glycoprotein N (GN) (aa 23-181), and two putative ectodomain segments of glycoprotein C (aa 468-1403), GC-ecto1 (aa 678-947) and GC-ecto2 (aa
M AN U
866-1323) as well as ubiquitinated and non-ubiquitinated forms of the nucleoprotein (N). We show that, when expressed separately, cDNAs encoding for the GC–ecto1 as well as the nucleoprotein N can render mice asymptomatic following SBV challenge. These results demonstrate that individual components of SBV used here are sufficient to induce protective
AC C
EP
infection in ruminants.
TE D
immunity; and serve as a basis for the creation of subunit vaccines against Schmallenberg virus
6
ACCEPTED MANUSCRIPT
2. MATERIALS AND METHODS 2.1
Virus and mice
RI PT
The BH80/11-4 strain of SBV was amplified once in cultured BHK cells in order to obtain sufficient titers for subsequent challenge studies. The mice used were Sv/129 IFN α/β -/- (B & K Universal Ltd, UK). All experiments were performed in the BSL3 facilities of INIA-CISA
SC
(Madrid), adhering to institutional and ethical guidelines for animal care and experimentation. Approval for the containment, vaccination, viral challenge and euthanasia were obtained prior to
M AN U
all in vivo experiments being performed. Throughout all steps of experimentation, the mice were monitored by staff veterinarians, and all animals that showed significant signs of morbidity were sacrificed by cervical dislocation. Construction of DNA vaccines
TE D
2.2
Five DNA-vaccines were designed based on different components of Schmallenberg virus (Fig. 1A), using pCMV-based expression vectors (pOPIN vector suite; Oxford Protein Production
EP
Facility UK). The vectors either lacked a secretory signal (pOPINE for N and ubiquitinated-N), or possessed a secretory signal (pOPING for GN-ecto, GC-ecto1, GC-ecto2). An empty vector was
AC C
used as a negative control and all constructs possessed a carboxy-terminal histidine tag for in vitro confirmation of expression (Fig. 1B). All viral genes were synthesized (Genscript, USA), based on the Schmallenberg virus isolate Bovine Schmallenberg virus BH80/Germany/2011. PCR amplification steps were performed using Fusion HF DNA polymerase (New England Biolabs, USA). The nucleoprotein alone as well as the ubiquitinated construct, was expressed as the complete 233-amino acid protein (Genbank accession number H2AM13). The modified Ubiquitin gene 7
ACCEPTED MANUSCRIPT
was designed as a 76-amino acid fusion protein, expressed at the N-terminus of the nucleoprotein, as previously described (Rodriguez et al., 1997;Rodriguez et al., 2001). Three glycoprotein constructs were also generated based on the same viral isolate. Primary
RI PT
sequences for GN and GC glycoproteins were also submitted to TMpred software (Krogh et al., 2001) for prediction of transmembrane helices and to Phyre2 protein fold recognition server (Kelley and Sternberg, 2009) to grasp possible structural homology with available sequences of
SC
known structure. The first, GN-ecto, was designed based on the putative soluble ectodomain of glycoprotein N (amino acids 23-181 of the M-polyprotein). Similarly, the segments GC-ecto1
M AN U
(aa: 678-947 of the M-polyprotein) and GC-ecto2 (aa: 866-1323 of the M-polyprotein) were designed, cloned and expressed, as putatively soluble globular portions. Interestingly, the GCecto2 resulted to encompass the highly confident (96.2%) homology model (aa 953-1297) (Fig. 1A, inset) that Phyre2 returned in our recent sequences resubmission, as being similar to the
(Halldorsson et al., 2016)].
TE D
severe fever with thrombocytopenia syndrome (SFTS) virus GC glycoprotein [PDB ID 5G47;
In all three glycoprotein constructs, an ATG codon was cloned to the 5'-end of the gene. For a
In vitro expression of DNA vaccine constructs
AC C
2.3
EP
negative control, an empty pOPING construct was used.
In order to verify the expression of all of the DNA vaccine candidates, each construct was transiently transfected in human embryonic kidney (HEK) 293T cells adapting protocols previously described (Aricescu et al., 2006). Briefly, 1 µg of each construct was incubated with 2 µg of polyethylenimine (PEI) in a volume of 100 µl Dulbecco’s Modified Essential Medium (DMEM) for 30 minutes, followed by incubation for 2 hours in 106 cells in a total volume of 500
8
ACCEPTED MANUSCRIPT
µl of DMEM. The cells were then supplemented with 2 mL of DMEM/10 % FBS and incubated at 37oC for 24 hours. Following transfection, the cells were washed with PBS, and lysed using 100 µl RIA buffer (150mM NaCl, 1% Triton X-100, 0.1% SDS, 50mM Tris–Cl pH 8.0).
RI PT
Western blotting of lysates was then performed using mouse anti-Histidine antibodies (GE Biosciences, USA) and HRP-conjugated anti-mouse IgG antibodies, and detected using enhanced chemiluminescence.
SC
In the case of the pOPINE constructs, protein expression was also tested in bacteria, as this vector is a shuttle vector. For both N and Ub-N constructs, 100 ng of each plasmid was used to
M AN U
transform E.coli BL21 cells. Bacterial isolates were then cultured in 10 mL LB-Ampicillin (100 µg/mL) and induced with IPTG for 20 hours at 20oC. 10 µl of each culture was then run on 12% SDS PAGE, followed by Western blotting, and detection by enhanced chemiluminescence. DNA immunization and SBV viral challenge
TE D
2.4
Six groups of IFNAR-/- mice were inoculated three times, intramuscularly, at two week intervals. Each group of mice (n=5) were vaccinated with each of the DNA vaccine constructs previously
EP
described (i.e. N, Ub-N, GN-ecto, GC-ecto1, GC-ecto2 and the negative control plasmids). One mouse in the N-group was found to be underweight prior to the experiment, and was not used for
AC C
the subsequent challenge experiments. For all inoculations, the plasmids were purified using a GeneJet Maxiprep Endo-Free plasmid purification kit (Thermo Scientific, USA), and the purified plasmid was resuspended in sterile PBS at a concentration of 500 µg/mL, where 100 µL of plasmid was injected during each vaccination step (for a vaccination dose of 50 µg). Two weeks following the final DNA vaccination, all mice were challenged with subcutaneous injections of 108 TCID50 SBV (strain BH80/11-4), resuspended in 100 µl of DMEM. The changes in weights
9
ACCEPTED MANUSCRIPT
were analyzed for statistical significance using a one-way ANOVA test with Origin software (OriginLab, Northamton, MA). Serological detection of SBV-specific IgG
RI PT
2.5
Ten days following the third vaccination, blood samples were collected from each mouse, and serum was collected following coagulation. ELISA assay was performed on the plates coated
SC
with enriched SBV antigen. SBV antigen was obtained from collecting supernatant from BHK infected cells (MOI=0.01) after three days, and pelleted on a 20% sucrose cushion (in Tris-
M AN U
Buffered Saline pH 8.0). The sucrose cushion was then dialysed in TBS buffer pH 8.0. The antigenic extract (measured at TCID50=108/mL) was then diluted 1/1000 and 50 µL was added to each well of the ELISA plate. The sera, previously inactivated at 56 oC for 1 hour, were added starting at a 1/100 dilution in PBS and serially diluted to 12800. The plates were incubated at
TE D
37oC for 1 hour, followed by 3 washes with PBS/0.1% Tween. 50 µL of HRP conjugated antimouse IgG (Sigma, USA) was then added to each well incubated for another hour at 37oC. Following three more washes with PBS/0.1% Tween, the plates were developed with 1-Step
EP
Ultra TMB substrate solution (Fisher Scientific, USA) for 30 minutes, stopped with 0.2M sulfuric acid and measured at a wavelength of 450 nm. Statistical significance was determined
2.6
AC C
using a one-way ANOVA test.
Detection and quantification of viremia by real-time RT-PCR
During the viral challenge described in 2.3, 100 µL of blood was collected from each mouse 3 and 6 dpi. RNA was extracted from 50 µL of each sample using the Paramagnetic Beads RNA Extraction Kit (Life River, Shanghai ZJ Biotech, China) according to the manufacturer’s instructions, and eluted in 50 µL. The presence of SBV genomic RNA was detected by using the 10
ACCEPTED MANUSCRIPT
Monodose SBV dtec-RT-qPCR kit (Genetic PCR Solutions, Elche, Spain) using 5 µL of the extracted total RNA samples. Real-time RT-PCR was performed on an ABI7500 Real-time PCR system (Applied Biosystems, USA) using the FAM channel. The thermal profile used was as
o
C for 10 seconds and 60 oC for 60 seconds. Cell proliferation assay
SC
2.7
RI PT
follows: 50 oC for 10 min and 95 oC for 60 seconds, followed by 40 cycles of the following: 95
A cell proliferation assay was performed from cells extracted from the spleens of mice following
M AN U
vaccination. A separate DNA vaccination experiment in IFNAR-/- mice was performed, but only for vaccination groups N, Ub-N, Gc-ecto1 and the negative control plasmids. Furthermore, the IFNAR-/- mice were only vaccinated twice. One week following the final vaccination, all mice were sacrificed, and splenocytes from individual mice were isolated in a manner similar to that
TE D
previously described (Lorenzo et al., 2010). Following isolation, a concentration of 106 splenocytes/ml from each mouse were stained with 5µM of CellTrace CFSE cell proliferation reagent in DMSO, according to the manufacturer’s instructions (Molecular Probes, USA). Next,
EP
cells were resuspended to a final concentration of 4x106 cells/ml in RPMI 1640 medium supplemented with 10% foetal calf serum and seeded in 96 well plates pre-coated with 500 ng
AC C
per well of anti-CD3e (BD-Biosciences) in triplicates in the presence of partially purified SBV extract (i.e. SBV antigen, prepared as described in section 2.5). Phytohemagglutinin (5µg/ml) was used as a positive control, and medium without antigen was used as a negative control. Plates were incubated three days at 37 oC in the presence of 5% CO2. After 72 hours, cells were washed, stained for the surface markers PE- CD8a and APC- CD4 (BD Biosciences, USA), and fixed. Data were acquired by FACS analysis on a FACSscalibur (Becton Dickinson) and were analyzed with FlowJo (Tree Star 7.6.5 software). The fluorescence of CFSE staining was 11
ACCEPTED MANUSCRIPT
measured to determine the proportion of divided cells among CD8+ cell subsets. The initial generation, and each of the following generations were used by the software to calculate the
2.8
Virus neutralization titration (VNT)
RI PT
number of dividing cells.
VNTs against the virus isolate used for challenge infection were performed as previously
AC C
EP
TE D
M AN U
SC
described (Wernike et al., 2013a) starting at a 1:10 dilution. All sera were tested in triplicates.
12
ACCEPTED MANUSCRIPT
3. RESULTS 3.1
Expression of DNA vaccine constructs in vitro
RI PT
In order to verify the expression of all the cloned SBV genes prior to DNA vaccination studies against SBV, all of the constructs were transfected in HEK293 cells; and expression was detected by Western blotting against the C-terminal histidine tags. All genes encoding for the
SC
SBV proteins had specific bands corresponding to molecular weights that approximate their theoretical molecular weights (Fig. 1B) although, as expected, their degree of expression varied.
M AN U
In the case of N, a single, specific band corresponding to approximately 27 kDa was observed. However, the ubiquitinated nucleoprotein (Ub-N) also exhibited a comparably similar band, with a faint band corresponding to approximately 35 kDa, as indicated by the two arrowheads in the Ub-N lane. In order to confirm that Ub-N is initially expressed as a fusion protein, BL21 E.coli cells were transformed with both N and Ub-N, followed by induction with IPTG. Protein
TE D
expression was then evaluated using western blotting. As seen in Fig. 1B left, both proteins were expressed at sizes corresponding to their theoretical values.
EP
The construct encoding for the soluble N-terminal domain of glycoprotein N (GN-ecto) expressed a band of approximately 20 kDa, slightly higher than the theoretical molecular weight, possibly
AC C
due to mammalian post-translational modifications (i.e. glycosylation). The constructs encoding for both segments of glycoprotein C (GC-ecto1 and GC-ecto2, respectively) were also found to express protein products slightly higher than their predicted molecular weights, migrating at approximately 37 (versus 31.3 kDa) and 58 kDa (versus 51.5 kDa), respectively. These results demonstrated that candidate DNA vaccines for the individual SBV proteins are expressed in a mammalian cell system.
13
ACCEPTED MANUSCRIPT
3.2
Generation of SBV-specific antibodies following vaccination
Ten days following the third vaccination with the SBV DNA constructs, serum was collected
RI PT
from the vaccinated mice, and SBV-specific IgG was measured using ELISA. As seen in Fig. 2A, both the Ub-N (yellow line) and N- (grey line) vaccinated mice exhibited the highest degree of SBV-specific antibodies.
Vaccinated IFNAR-/- mice display varying degrees of protection following SBV challenge
SC
3.3
M AN U
Following three vaccinations with the candidate DNA vaccine constructs the IFNAR-/- mice were subjected to viral challenge with virulent SBV, which was administered subcutaneously (see Materials and Methods). The clinical signs primarily associated with weight loss were measured for two weeks after virus challenge. The average change in weight is shown in Fig. 3A, where both the N and GC-ecto1 groups showed continued weight gain (relative to weights measured
TE D
prior to viral challenge) throughout the entire experiment, with mice averaging 104% and 106% of pre-challenge weight. We also noted a qualitative difference between the overall appearance of these two groups, with mice in the N and GC-ecto1 groups showing greater activity and
EP
appetite relative to the other groups. This was in contrast to the other vaccinated groups (i.e. Ub-
AC C
N, GC-ecto2, GN-ecto and negative control), which exhibited continued weight loss until 7 dpi, followed by an eventual average weight gain between 7 and 15 dpi. It should be noted that in the case of GC-ecto1 and Ub-N, one mouse from each group had to be euthanized due to excessive morbidity. Aside from weight loss and overall physical activity, no other clinical signs were observed in the negative control mice.
14
ACCEPTED MANUSCRIPT
A detailed analysis of the weight changes of individual mice is shown in Fig. 3B, where the mean weights of all groups are shown until 7 dpi. While the masses of individual mice varied considerably, only N and GC-ecto1 had average body masses above 100% throughout the study.
challenge out of the six experimental groups used in this study. Quantification of SBV viremia following challenge
SC
3.4
RI PT
These results demonstrate that only N and GC-ecto1 prevented weight loss following SBV
Real-time RT-PCR was performed on mouse blood samples 3- and 6-dpi. As seen in Fig. 4, all
M AN U
mice at all time points had viral loads of less than 6 x103 copies/mL at 3 dpi. While GC-ecto1 and N had the lowest viral loads at 3 dpi (2.0 x103 and 1.5 x103 copies/mL, respectively), all groups had average viral loads below 1.0 x104. However, at 6 dpi, only GC-ecto1 and N had average viral loads of less than 1 x104, with GC-ecto1 and N groups having average viral loads of 2.8
TE D
x103 and 1.7 x103 copies/mL, respectively. It should also be noted that in the GC-ecto1 group, 3 out of the 5 mice had no detectable viral loads. In the case of N, only one mouse had a nondetectable viral load. All other mice in all of the other groups had measurable levels of SBV
Virus neutralizing titers of vaccinated IFNAR-/- mice
AC C
3.5
EP
viremia.
Following the viral challenge experiments, the mode of immunity of the vaccine constructs was analyzed. In order to assess the extent of humoral immunity induced by the DNA vaccine candidates, VNTs were performed on mice that were vaccinated twice with either the N, Ub-N, GC-ecto1 or negative control constructs (see Materials and Methods). With dilutions starting at 1/10, none of the vaccinated groups had detectable levels of neutralizing titers (figure not shown). 15
ACCEPTED MANUSCRIPT
3.6
Cell proliferation of isolated splenocytes vaccinated IFNAR-/- mice
For the T-cell assays, 4 groups of 5 IFNAR-/- mice were vaccinated twice using N, Ub-N, GC-
RI PT
ecto1 or the negative control plasmid. This vaccination strategy was a consequence of the fact that we found both N and GC-ecto1 were highly protective against virulent SBV challenge (neither of which involved the generation of neutralizing antibodies), thus we wanted to find out
SC
if cell-mediated immunity could be induced with less DNA. One week following the second vaccination, the mice were sacrificed, and individual spleens were removed in order to measure
M AN U
the T-cell proliferative response. For each mouse, the degree of cellular proliferation was evaluated by measuring the degree of cell division by either CD4+ or CD8+ T-cells (as determined by flow cytometry - see Materials and Methods). The degree of cellular proliferation was indexed as the number of proportion of SBV antigen-stimulated cells that divided (as seen by decreased CFSE signals) relative to the proportion of cells that divided without SBV antigen.
TE D
As seen in Fig. 5, mice in the Gc-ecto1 group had the highest degree of cell proliferation, with a 45% increase in CD8+ cellular proliferation, with a p-value of less than 0.0001. The N-group also had a 6.5% increase in CD8+ proliferation, with a p-value of 0.2, while the negative control
EP
group had approximately 1% proliferation. The Ub-N group had no statistically significant level
AC C
of proliferation. When CD4+ levels were analyzed, no significant differences in cellular proliferation were observed (Figure not shown).
16
ACCEPTED MANUSCRIPT
4. DISCUSSION Evaluating the efficacy of DNA vaccines has multiple advantages. Firstly, the benefits of DNA vaccination over conventional inactivated/attenuated vaccine strains have been previously
RI PT
described (Babiuk et al., 2014;Kutzler and Weiner, 2008). Briefly, lower cost of production, the lack of a required refrigeration/cold storage chain and the ability to store dehydrated DNA for prolonged periods of time enables for a cost-effective and practical method of vaccination.
SC
Furthermore, the use of DNA vaccine candidates encoding for different ORFs of the SBV genome enables for further immunological study of the virus; in particular, which components of
M AN U
the virus are responsible for protective immunity in the host. To date, little is known of the host immune response following SBV infection. While SBV virulence factors have been located to the GC (Varela et al., 2016) and NSs (Varela et al., 2016;Varela et al., 2013) components of the viral genome, there does not appear to be any data directly linking CD8+ T-cell immunity
TE D
following SBV infection.
The use of IFNAR-/- mice as a model for SBV infection was previously found to display clinical signs that were far more pathogenic than SBV infection in ruminant animals (i.e. hemorrhagic
EP
fever, death), with the most common clinical sign being weight loss. (Wernike et al., 2012). In this work, we aimed to validate five DNA vaccine candidates against SBV using a similarly
AC C
aged IFNAR-/- model that differed only in the genetic background from previous studies (i.e. our Sv/129 mice model versus the C57BL/6 mice used elsewhere). To induce disease progression Sv/129 mice required higher viral titers, 108 TCID50, then the 103 or 104 TCID50 used in C57BL/6 mice (Wernike et al., 2012;Kraatz et al., 2015). Despite the difference in inoculum titers, the induction of weight loss at approximately 4 dpi was similarly observed, followed by recovery (i.e. weight gain) at 10 dpi in the surviving mice.
17
ACCEPTED MANUSCRIPT
Two promising targets have been identified on the basis that an effective DNA vaccine candidate should be able to prevent any significant weight loss, and limit viremia relative to unvaccinated mice. These DNA constructs, both capable to individually protect mice from SBV-induced
RI PT
weight loss, encode for two different components of the virus: (i) the segment aa 678-947 of the glycoprotein GC (GC-ecto1) and (ii) the full-length nucleoprotein. Furthermore, when the presence of the virus was measured, both constructs were found to significantly limit viremia at 3
SC
and 6 dpi.
Since SBV's discovery nearly five years ago, multiple vaccine candidates have been described
M AN U
and validated; these include inactivated vaccines (which are currently on the market), as well as recombinant Nsm and NSs-deleted SBV strains (Kraatz et al., 2015). However, until now, little was known about the ability of individual components of SBV in inducing a protective immune response. Thus GC-ecto1 and N could be used as individual DNA vaccines; and the possibility of
TE D
using these targets in other forms of subunit vaccines can provide an alternative strategy in the development of antiviral measures against SBV. Furthermore, the availability of multiple vaccine targets would enable for validation of vaccines against heterologous challenge. This
EP
might be of particular interest when considering the hypervariable region associated with the Nterminus of GC. Previously, sequencing of two SBV genomes from two lambs within a single
AC C
flock identified a 1200-nucleotide region of the M-segment that could undergo rapid variation within intraherd populations (Coupeau et al., 2013). Interestingly, this region includes the segment of the protective GC-ecto1 DNA vaccine target, which has been described in this work. While this may suggest that the nucleoprotein, previously proven to be far more conserved (Fischer et al., 2013) would make for a more preferable construct for widespread vaccination programs, GC-ecto1 should still be considered. It should be noted that conflicting information
18
ACCEPTED MANUSCRIPT
pertaining to the GC’s
hypervariable region (HVR) has been proposed when phylogenetic
studies from a 2011-12 outbreak in cattle demonstrated a high degree of conservation of the HVR following SBV outbreaks (Coupeau et al., 2013). Therefore, field studies using both
be beneficial in preventing future SBV outbreaks
RI PT
vaccine candidates would be an important step in determining the extent of which target would
Along with a DNA construct encoding for SBV nucleoprotein, an ubiquitinated fusion construct
SC
based on SBV N was also evaluated. Such a vaccine candidate was inspired by the detected increased antigenicity of RVFV nucleoprotein when a similar strategy was used (Boshra et al.,
M AN U
2011a). Constructs encoding for ubiquitinated RVFV N showed near complete protection when challenged with RVFV in IFNAR-/- mice contrary to the limited protection offered by RVFV N (Boshra et al., 2011b). In the case of SBV, the opposite effect was observed - N showed to be the most potent DNA vaccine construct in protecting against virulent SBV challenge, while its
TE D
ubiquitinated counterpart appeared to increase pathogenicity following challenge (even relative to the negative control plasmid). Although both constructs generated high titers of SBV-specific antibodies, mice vaccinated with Ub-N showed the highest degree of weight loss, as well as the
EP
highest degree of viremia post-infection. These results appear to be corroborated when cellular immunity was analyzed. While GC-ecto1 and N had measurable levels of CD8+ T-cell
AC C
proliferation, the Ub-N group appeared to have a level of cellular proliferation comparable to the negative control group, suggesting an insignificant or deleterious effect of this construct on SBVspecific immunity. Furthermore, as no observable CD4+ cellular proliferation was observed, these experiments indicate that SBV-mediated cellular immunity is primarily achieved through CD8+ T-cell activation. Also, while GC-ecto1 and N had comparable levels of immune protection, N did not have a comparable level of cellular proliferation; and given that no
19
ACCEPTED MANUSCRIPT
vaccinated groups generated any neutralizing antibodies, one would have expected the nucleoprotein to have a greater level of cellular proliferation than observed. The fact that GCecto1 gave a significantly higher level of CD8+ T cells activation may be attributed to (i) the
RI PT
lack of sufficient antigen presenting cells (APCs) from the isolated splenocytes; (ii) the lack of free cytosolic nucleoprotein (as would normally occur in an SBV-infected cells), or; (iii) the possibility of the existence of another nucleoprotein activated T-cell population that was not
SC
present in the spleen. Moreover, and contrary to other bunyaviruses such as RVFV where neutralizing antibody responses were observed following DNA vaccination regimes with a
M AN U
construct encoding for the M-segment glycoproteins (Lagerqvist et al., 2009;Lorenzo et al., 2010;Spik et al., 2006), the SBV constructs used in this study failed to induce neutralizing antibodies (including those that provided protectively immunity following viral challenge). The IFNAR-/- mouse model used in this study demonstrates that SBV virulence occurs as early as 3
TE D
dpi, becoming most extensive at 6 dpi as observed by the significant weight loss and viremia in the negative control mice. Weight-loss and viremia were minimal in mice vaccinated with either GC-ecto1 or N, correlating with the increased levels of CD8+ T-cell proliferation observed in
EP
vitro. Considering that no neutralizing antibodies were observed, it appears as though both GCecto1 and N were able to induce protection entirely through cell-mediated immunity. This
AC C
suggests that prior to viral challenge, both groups had stimulated, SBV-specific CD8+ T-cells; and that following SBV challenge, both vaccinated groups were able to reduce virus replication within the first 3 days, thereby mitigating viremia and clinical signs. A further finding of our study was that the ubiquitin did not augment nucleoprotein-mediated immunity, an outcome seen also in other studies with DNA vaccines encoding for pseudorabies virus glycorproteins (Gravier et al., 2007), as well as a tandemly fused BTT-cell epitope of foot-
20
ACCEPTED MANUSCRIPT
and-mouse disease virus (Borrego et al., 2006). Contrarily, ubiquitin has been shown to increase protection against RVFV (Boshra et al., 2011b), mycobacterial protein MPT64 (Wang et al., 2004), porcine reproductive respiratory syndrome virus protein GP5 and lymphocytic
RI PT
chloriomeningitis virus nucleoprotein (Rodriguez et al., 1997). Nevertheless, it is worth noting that such a contrast has never been observed within the same viral family (i.e. Bunyaviridae). While these results, along with the previous results from RVFV vaccination suggest that
SC
nucleoprotein-based vaccines may be promising candidates against bunyaviruses, the need to use immunostimulatory molecules/self-adjuvants is still an empirical strategy. Ultimately,
M AN U
nucleoprotein-targeting could still be a promising vaccine strategy against other pathogenic bunyaviruses such as Crimean-Congo Hemorrhagic fever virus (CCHFV), Hantavirus, or severe fever with thrombocytopenia syndrome virus (SFTS).
In conclusion, we have shown that both SBV GC-ecto1 domain and nucleocapsid N
TE D
protein represent viable candidates for DNA vaccination; and that these two constructs represent two individual targets for eliciting a protective immune response. Furthermore, the results obtained in these studies serve to offer new avenues for vaccine development and immune
AC C
EP
targeting in other bunyaviruses.
21
ACCEPTED MANUSCRIPT
ACKNOWLEDGEMENTS We would like to thank Martin Beer and Kerstin Wernike for kindly providing reagents, help in performing VNT experiments and for the critical reading of the manuscript. We are grateful to
RI PT
the Oxford Protein Production Facility UK at the Research Complex at Harwell for the pOPIN vector suite and to Fernando Rodriguez at Centre de Recerca en Sanitat Animal IRTA-CReSA (Spain) for supplying the Ubiquitin vector. We would also like to thank Stavros Azinas at CIC
M AN U
FUNDING INFORMATION
SC
bioGUNE for help in the statistical analysis of the quantitative data.
This work was supported by the Marie Sklodowska Curie Actions programme number MSCAIF-EF-ST-660155 (HB and NGAA), the Spanish MINECO/FEDER BFU2015-64541-R
AC C
EP
TE D
(NGAA).
22
ACCEPTED MANUSCRIPT
REFERENCES
RI PT
Aricescu, A.R., Lu, W., Jones, E.Y., 2006. A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr. D Biol. Crystallogr. 62, 1243-1250. doi: S0907444906029799 [pii].
SC
Ariza, A., Tanner, S.J., Walter, C.T., Dent, K.C., Shepherd, D.A., Wu, W., Matthews, S.V., Hiscox, J.A., Green, T.J., Luo, M., Elliott, R.M., Fooks, A.R., Ashcroft, A.E., Stonehouse, N.J., Ranson, N.A., Barr, J.N., Edwards, T.A., 2013. Nucleocapsid protein structures from orthobunyaviruses reveal insight into ribonucleoprotein architecture and RNA polymerization. Nucleic Acids Res. 41, 5912-5926. doi: 10.1093/nar/gkt268 [doi].
M AN U
Babiuk, S., Boshra, H., Babiuk, L., 2014. DNA Vaccines, in: Caplan, M. (Ed.), Reference Module in Biomedical Sciences-Diagnosis and Treatment of Viral Diseases . Elsevier Inc, pp. 1-2,-5. Balenghien, T., Pages, N., Goffredo, M., Carpenter, S., Augot, D., Jacquier, E., Talavera, S., Monaco, F., Depaquit, J., Grillet, C., Pujols, J., Satta, G., Kasbari, M., Setier-Rio, M.L., Izzo, F., Alkan, C., Delecolle, J.C., Quaglia, M., Charrel, R., Polci, A., Breard, E., Federici, V., CetreSossah, C., Garros, C., 2014. The emergence of Schmallenberg virus across Culicoides communities and ecosystems in Europe. Prev. Vet. Med. 116, 360-369. doi: 10.1016/j.prevetmed.2014.03.007 [doi].
TE D
Bayrou, C., Garigliany, M.M., Sarlet, M., Sartelet, A., Cassart, D., Desmecht, D., 2014. Natural intrauterine infection with Schmallenberg virus in malformed newborn calves. Emerg. Infect. Dis. 20, 1327-1330. doi: 10.3201/eid2008.121890 [doi].
EP
Borrego, B., Fernandez-Pacheco, P., Ganges, L., Domenech, N., Fernandez-Borges, N., Sobrino, F., Rodriguez, F., 2006. DNA vaccines expressing B and T cell epitopes can protect mice from FMDV infection in the absence of specific humoral responses. Vaccine 24, 3889-99. doi: S0264410X(06)00211-8 [pii] 10.1016/j.vaccine.2006.02.028.
AC C
Boshra, H., Lorenzo, G., Busquets, N., Brun, A., 2011a. Rift valley fever: recent insights into pathogenesis and prevention. J. Virol. 85, 6098-6105. doi: 10.1128/JVI.02641-10. Boshra, H., Lorenzo, G., Rodriguez, F., Brun, A., 2011b. A DNA vaccine encoding ubiquitinated Rift Valley fever virus nucleoprotein provides consistent immunity and protects IFNAR(-/-) mice upon lethal virus challenge. Vaccine 29, 4469-4475. doi: 10.1016/j.vaccine.2011.04.043. Coupeau, D., Claine, F., Wiggers, L., Kirschvink, N., Muylkens, B., 2013. In vivo and in vitro identification of a hypervariable region in Schmallenberg virus. J. Gen. Virol. 94, 1168-1174. doi: 10.1099/vir.0.051821-0 [doi].
23
ACCEPTED MANUSCRIPT
Dong, H., Li, P., Elliott, R.M., Dong, C., 2013. Structure of Schmallenberg orthobunyavirus nucleoprotein suggests a novel mechanism of genome encapsidation. J. Virol. 87, 5593-5601. doi: 10.1128/JVI.00223-13 [doi].
RI PT
Fischer, M., Hoffmann, B., Goller, K.V., Hoper, D., Wernike, K., Beer, M., 2013. A mutation 'hot spot' in the Schmallenberg virus M segment. J. Gen. Virol. 94, 1161-1167. doi: 10.1099/vir.0.049908-0 [doi]. Gibbens, N., 2012. Schmallenberg virus: a novel viral disease in northern Europe. Vet. Rec. 170, 58. doi: 10.1136/vr.e292 [doi].
SC
Goller, K.V., Hoper, D., Schirrmeier, H., Mettenleiter, T.C., Beer, M., 2012. Schmallenberg virus as possible ancestor of Shamonda virus. Emerg. Infect. Dis. 18, 1644-1646. doi: 10.3201/eid1810.120835 [doi].
M AN U
Gravier, R., Dory, D., Rodriguez, F., Bougeard, S., Beven, V., Cariolet, R., Jestin, A., 2007. Immune and protective abilities of ubiquitinated and non-ubiquitinated pseudorabies virus glycoproteins. Acta Virol 51, 35-45. Halldorsson, S., Behrens, A.J., Harlos, K., Huiskonen, J.T., Elliott, R.M., Crispin, M., Brennan, B., Bowden, T.A., 2016. Structure of a phleboviral envelope glycoprotein reveals a consolidated model of membrane fusion. Proc. Natl. Acad. Sci. U. S. A. 113, 7154-7159. doi: 10.1073/pnas.1603827113 [doi].
TE D
Hechinger, S., Wernike, K., Beer, M., 2014. Single immunization with an inactivated vaccine protects sheep from Schmallenberg virus infection. Vet. Res. 45, 79-014-0079-6. doi: 10.1186/s13567-014-0079-6 [doi].
EP
Hefti, H.P., Frese, M., Landis, H., Di Paolo, C., Aguzzi, A., Haller, O., Pavlovic, J., 1999. Human MxA protein protects mice lacking a functional alpha/beta interferon system against La crosse virus and other lethal viral infections. J. Virol. 73, 6984-6991.
AC C
Hoffmann, B., Scheuch, M., Hoper, D., Jungblut, R., Holsteg, M., Schirrmeier, H., Eschbaumer, M., Goller, K.V., Wernike, K., Fischer, M., Breithaupt, A., Mettenleiter, T.C., Beer, M., 2012. Novel orthobunyavirus in Cattle, Europe, 2011. Emerg. Infect. Dis. 18, 469-472. doi: 10.3201/eid1803.111905 [doi]. Kelley, L.A., Sternberg, M.J., 2009. Protein structure prediction on the Web: a case study using the Phyre server. Nat. Protoc. 4, 363-371. doi: 10.1038/nprot.2009.2 [doi]. Kraatz, F., Wernike, K., Hechinger, S., Konig, P., Granzow, H., Reimann, I., Beer, M., 2015. Deletion mutants of Schmallenberg virus are avirulent and protect from virus challenge. J. Virol. 89, 1825-1837. doi: 10.1128/JVI.02729-14 [doi].
24
ACCEPTED MANUSCRIPT
Krogh, A., Larsson, B., von Heijne, G., Sonnhammer, E.L., 2001. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305, 567-580. doi: 10.1006/jmbi.2000.4315 [doi].
RI PT
Kutzler, M.A., Weiner, D.B., 2008. DNA vaccines: ready for prime time? Nat. Rev. Genet. 9, 776-788. doi: 10.1038/nrg2432 [doi]. Lagerqvist, N., Naslund, J., Lundkvist, A., Bouloy, M., Ahlm, C., Bucht, G., 2009. Characterisation of immune responses and protective efficacy in mice after immunisation with Rift Valley Fever virus cDNA constructs. Virol J 6, 6. doi: 1743-422X-6-6 [pii] 10.1186/1743422X-6-6.
SC
Larska, M., Krzysiak, M.K., Kesik-Maliszewska, J., Rola, J., 2014. Cross-sectional study of Schmallenberg virus seroprevalence in wild ruminants in Poland at the end of the vector season of 2013. BMC Vet. Res. 10, 967-014-0307-3. doi: 10.1186/s12917-014-0307-3 [doi].
M AN U
Larska, M., Krzysiak, M., Smreczak, M., Polak, M.P., Zmudzinski, J.F., 2013a. First detection of Schmallenberg virus in elk (Alces alces) indicating infection of wildlife in Bialowieza National Park in Poland. Vet. J. 198, 279-281. doi: 10.1016/j.tvjl.2013.08.013 [doi].
TE D
Larska, M., Lechowski, L., Grochowska, M., Zmudzinski, J.F., 2013b. Detection of the Schmallenberg virus in nulliparous Culicoides obsoletus/scoticus complex and C. punctatus--the possibility of transovarial virus transmission in the midge population and of a new vector. Vet. Microbiol. 166, 467-473. doi: 10.1016/j.vetmic.2013.07.015 [doi]. Larska, M., Polak, M.P., Grochowska, M., Lechowski, L., Zwiazek, J.S., Zmudzinski, J.F., 2013c. First report of Schmallenberg virus infection in cattle and midges in Poland. Transbound Emerg. Dis. 60, 97-101. doi: 10.1111/tbed.12057 [doi].
EP
Lievaart-Peterson, K., Luttikholt, S., Peperkamp, K., Van den Brom, R., Vellema, P., 2015. Schmallenberg disease in sheep or goats: Past, present and future. Vet. Microbiol. 181, 147-153. doi: 10.1016/j.vetmic.2015.08.005 [doi].
AC C
Lorenzo, G., Martin-Folgar, R., Hevia, E., Boshra, H., Brun, A., 2010. Protection against lethal Rift Valley fever virus (RVFV) infection in transgenic IFNAR(-/-) mice induced by different DNA vaccination regimens. Vaccine 28, 2937-44. doi: S0264-410X(10)00186-6 [pii] 10.1016/j.vaccine.2010.02.018. Oestereich, L., Rieger, T., Neumann, M., Bernreuther, C., Lehmann, M., Krasemann, S., Wurr, S., Emmerich, P., de Lamballerie, X., Olschlager, S., Gunther, S., 2014. Evaluation of antiviral efficacy of ribavirin, arbidol, and T-705 (favipiravir) in a mouse model for Crimean-Congo hemorrhagic fever. PLoS Negl Trop. Dis. 8, e2804. doi: 10.1371/journal.pntd.0002804 [doi]. Pavlovic, J., Schultz, J., Hefti, H.P., Schuh, T., Molling, K., 2000. DNA vaccination against La Crosse virus. Intervirology 43, 312-321. doi: 53999 [pii].
25
ACCEPTED MANUSCRIPT
Peperkamp, N.H., Luttikholt, S.J., Dijkman, R., Vos, J.H., Junker, K., Greijdanus, S., Roumen, M.P., van Garderen, E., Meertens, N., van Maanen, C., Lievaart, K., van Wuyckhuise, L., Wouda, W., 2015. Ovine and Bovine Congenital Abnormalities Associated With Intrauterine Infection With Schmallenberg Virus. Vet. Pathol. 52, 1057-1066. doi: 10.1177/0300985814560231 [doi].
RI PT
Ponsart, C., Pozzi, N., Breard, E., Catinot, V., Viard, G., Sailleau, C., Viarouge, C., Gouzil, J., Beer, M., Zientara, S., Vitour, D., 2014. Evidence of excretion of Schmallenberg virus in bull semen. Vet. Res. 45, 37-9716-45-37. doi: 10.1186/1297-9716-45-37 [doi].
SC
Proenca-Modena, J.L., Sesti-Costa, R., Pinto, A.K., Richner, J.M., Lazear, H.M., Lucas, T., Hyde, J.L., Diamond, M.S., 2015. Oropouche virus infection and pathogenesis are restricted by MAVS, IRF-3, IRF-7, and type I interferon signaling pathways in nonmyeloid cells. J. Virol. 89, 4720-4737. doi: 10.1128/JVI.00077-15 [doi].
M AN U
Rodriguez, F., Harkins, S., Redwine, J.M., de Pereda, J.M., Whitton, J.L., 2001. CD4(+) T cells induced by a DNA vaccine: immunological consequences of epitope-specific lysosomal targeting. J Virol 75, 10421-30. doi: 10.1128/JVI.75.21.10421-10430.2001. Rodriguez, F., Zhang, J., Whitton, J.L., 1997. DNA immunization: ubiquitination of a viral protein enhances cytotoxic T-lymphocyte induction and antiviral protection but abrogates antibody induction. J Virol 71, 8497-503.
TE D
Roman-Sosa, G., Brocchi, E., Schirrmeier, H., Wernike, K., Schelp, C., Beer, M., 2016. Analysis of the humoral immune response against the envelope glycoprotein Gc of Schmallenberg virus reveals a domain located at the amino terminus targeted by mAbs with neutralizing activity. J. Gen. Virol. 97, 571-580. doi: 10.1099/jgv.0.000377 [doi].
EP
Sailleau, C., Breard, E., Viarouge, C., Desprat, A., Doceul, V., Lara, E., Languille, J., Vitour, D., Attoui, H., Zientara, S., 2013. Acute Schmallenberg virus infections, France, 2012. Emerg. Infect. Dis. 19, 321-322.
AC C
Schuh, T., Schultz, J., Moelling, K., Pavlovic, J., 1999. DNA-based vaccine against La Crosse virus: protective immune response mediated by neutralizing antibodies and CD4+ T cells. Hum. Gene Ther. 10, 1649-1658. doi: 10.1089/10430349950017653 [doi]. Spik, K., Shurtleff, A., McElroy, A.K., Guttieri, M.C., Hooper, J.W., SchmalJohn, C., 2006. Immunogenicity of combination DNA vaccines for Rift Valley fever virus, tick-borne encephalitis virus, Hantaan virus, and Crimean Congo hemorrhagic fever virus. Vaccine 24, 4657-66. doi: S0264-410X(05)00842-X [pii] 10.1016/j.vaccine.2005.08.034. Varela, M., Pinto, R.M., Caporale, M., Piras, I.M., Taggart, A., Seehusen, F., Hahn, K., Janowicz, A., de Souza, W.M., Baumgartner, W., Shi, X., Palmarini, M., 2016. Mutations in the Schmallenberg Virus Gc Glycoprotein Facilitate Cellular Protein Synthesis Shutoff and Restore Pathogenicity of NSs Deletion Mutants in Mice. J. Virol. 90, 5440-5450. doi: 10.1128/JVI.00424-16 [doi]. 26
ACCEPTED MANUSCRIPT
Varela, M., Schnettler, E., Caporale, M., Murgia, C., Barry, G., McFarlane, M., McGregor, E., Piras, I.M., Shaw, A., Lamm, C., Janowicz, A., Beer, M., Glass, M., Herder, V., Hahn, K., Baumgartner, W., Kohl, A., Palmarini, M., 2013. Schmallenberg virus pathogenesis, tropism and interaction with the innate immune system of the host. PLoS Pathog. 9, e1003133. doi: 10.1371/journal.ppat.1003133 [doi].
RI PT
Wang, Q.M., Sun, S.H., Hu, Z.L., Zhou, F.J., Yin, M., Xiao, C.J., Zhang, J.C., 2004. Epitope DNA vaccines against tuberculosis: spacers and ubiquitin modulates cellular immune responses elicited by epitope DNA vaccine. Scand J Immunol 60, 219-25. doi: 10.1111/j.03009475.2004.01442.x SJI1442 [pii].
M AN U
SC
Wernike, K., Brocchi, E., Cordioli, P., Senechal, Y., Schelp, C., Wegelt, A., Aebischer, A., Roman-Sosa, G., Reimann, I., Beer, M., 2015a. A novel panel of monoclonal antibodies against Schmallenberg virus nucleoprotein and glycoprotein Gc allows specific orthobunyavirus detection and reveals antigenic differences. Vet. Res. 46, 27-015-0165-4. doi: 10.1186/s13567015-0165-4 [doi]. Wernike, K., Elbers, A., Beer, M., 2015b. Schmallenberg virus infection. Rev. Sci. Tech. 34, 363-373. Wernike, K., Hoffmann, B., Conraths, F.J., Beer, M., 2015c. Schmallenberg Virus Recurrence, Germany, 2014. Emerg. Infect. Dis. 21, 1202-1204. doi: 10.3201/eid2107.150180 [doi].
TE D
Wernike, K., Conraths, F., Zanella, G., Granzow, H., Gache, K., Schirrmeier, H., Valas, S., Staubach, C., Marianneau, P., Kraatz, F., Horeth-Bontgen, D., Reimann, I., Zientara, S., Beer, M., 2014. Schmallenberg virus-two years of experiences. Prev. Vet. Med. 116, 423-434. doi: 10.1016/j.prevetmed.2014.03.021 [doi].
EP
Wernike, K., Eschbaumer, M., Schirrmeier, H., Blohm, U., Breithaupt, A., Hoffmann, B., Beer, M., 2013a. Oral exposure, reinfection and cellular immunity to Schmallenberg virus in cattle. Vet. Microbiol. 165, 155-159. doi: 10.1016/j.vetmic.2013.01.040 [doi].
AC C
Wernike, K., Hoffmann, B., Breard, E., Botner, A., Ponsart, C., Zientara, S., Lohse, L., Pozzi, N., Viarouge, C., Sarradin, P., Leroux-Barc, C., Riou, M., Laloy, E., Breithaupt, A., Beer, M., 2013b. Schmallenberg virus experimental infection of sheep. Vet. Microbiol. 166, 461-466. doi: 10.1016/j.vetmic.2013.06.030 [doi]. Wernike, K., Nikolin, V.M., Hechinger, S., Hoffmann, B., Beer, M., 2013c. Inactivated Schmallenberg virus prototype vaccines. Vaccine 31, 3558-3563. doi: 10.1016/j.vaccine.2013.05.062 [doi]. Wernike, K., Breithaupt, A., Keller, M., Hoffmann, B., Beer, M., Eschbaumer, M., 2012. Schmallenberg virus infection of adult type I interferon receptor knock-out mice. PLoS One 7, e40380. doi: 10.1371/journal.pone.0040380 [doi].
27
ACCEPTED MANUSCRIPT
Yilmaz, H., Hoffmann, B., Turan, N., Cizmecigil, U.Y., Richt, J.A., Van der Poel, W.H., 2014. Detection and partial sequencing of Schmallenberg virus in cattle and sheep in Turkey. Vector Borne Zoonotic Dis. 14, 223-225. doi: 10.1089/vbz.2013.1451 [doi].
AC C
EP
TE D
M AN U
SC
RI PT
Zivcec, M., Safronetz, D., Scott, D., Robertson, S., Ebihara, H., Feldmann, H., 2013. Lethal Crimean-Congo hemorrhagic fever virus infection in interferon alpha/beta receptor knockout mice is associated with high viral loads, proinflammatory responses, and coagulopathy. J. Infect. Dis. 207, 1909-1921. doi: 10.1093/infdis/jit061 [doi].
28
ACCEPTED MANUSCRIPT
FIGURE LEGENDS Fig. 1- Schematic of the constructs used for DNA vaccination, and verification of in vitro
RI PT
expression. (A) DNA vaccine constructs based on the S and M-segments of SBV were cloned into either pOPINE or pOPING expression vectors (see Materials and Methods). Full-length nucleoprotein (N) was generated based on the SBV S-segment, while GN-ecto, GC-ecto1 and GC-
SC
ecto2 (indicated by the double-headed arrows) were generated based on the SBV M-segment; the inset represents the tridimensional homology model generated by Phyre2 as cartoon and colored
M AN U
dark blue to red from N-terminus to C-terminus. (B) Verification of in vitro expression of the DNA vaccine constructs. The ubiquitinated and non-ubiquitinated N constructs (Ub-N and N, respectively) was transformed in BL21 E.coli, and expression was detected by 12% SDS-PAGE and western blotting using anti-histidine antibodies (left panel). All of the constructs were transfected in HEK293T cells (see Materials and Methods), and expression was detected by 12%
TE D
SDS-PAGE and western blotting using anti-histidine antibodies. The putative protein bands are indicated by arrowheads. The theoretical molecular weights are indicated at the bottom of each
EP
lane.
Fig. 2- ELISA of sera taken from vaccinated Sv/129 IFNAR-/- mice. (A) Following the third
AC C
DNA vaccination, sera were collected from all mice, and serial dilutions starting at 1/100 were used to detect partially purified SBV. Each point indicates the mean absorbance value of each group at 450 nm. Vaccination groups with p-values of 0.0131 (Ub-N; 1/200 diluition), 0.0484 (Ub-N; 1/100 dilution) and 0.0488 (N; 1/100 diluition) are indicated by one asterisk; the variation of measurements within each group is shown in panel B. The one-sided error bars at each point represent the standard error of the mean. (B) Dot plot of ELISA absorbance values at
29
ACCEPTED MANUSCRIPT
1/100 and 1/200 dilution of individual mice within each vaccinated group; the red horizontal bars indicate the mean.
RI PT
Fig. 3- Change in the average weight of vaccinated Sv/129 IFNAR-/- following challenge with virulent SBV. All six groups were monitored for 15 days following inoculation with virulent SBV. (A) The mean weight of each vaccinated group, measured at timepoints 0 to 15 dpi is
SC
indicated at the indicated data point. (B) Dot plot of each mouse in all vaccinated groups. The individual weights of each mouse is plotted from 0 to 10 dpi, with the mean average of the first 7
M AN U
days indicated in red. Vaccination groups with a p-value of less than 0.001 are indicated by the three asterisks. The one-sided bars indicated at each point represent the standard error. Fig. 4- Viremia of vaccinated Sv/129 IFNAR-/- following challenge with virulent SBV. Viremia at 3 and 6 dpi was measured by extracting RNA from 50 uL of blood from each mouse, and the
TE D
presence of SBV was quantified using real-time RT-PCR (see Materials and Methods). Results are presented as copy number/mL of blood, with the red-brick dash in each scatter plot denoting
EP
the mean.
Fig. 5- Cellular proliferation of CD8+ T-cells-. Splenocytes extracted from vaccinated mice were
AC C
incubated with and without the presence of SBV antigen, and incubated for 3 days with CPE (see Materials and Methods). CD8+ T cells were analyzed through flow cytometry. The cellular proliferation index was calculated as the degree of CD8+T cell proliferation observed in the presence of SBV, relative to proliferation observed without antigen. The red-brick dash in each scatter plot denotes the mean. Vaccination groups with a p-value of less than 0.001 (using a student t-test) are indicated by the three asterisks.
30
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
2
Highlights •
IFNAR-/- mice.
3 4
cDNAs of Schmallenberg virus were evaluated for their ability to act as DNA vaccines in
•
RI PT
1
Two constructs encoding for the nucleoprotein N and for a portion of glycoprotein GC confer protection.
5
•
For both constructs, immunity in IFNAR-/- appeared to be mediated by CD8+ T-cells.
7
•
These results suggest that SBV has at least two distinct immunological targets.
AC C
EP
TE D
M AN U
SC
6
1
