Authors: Mélissanne de Wispelaere ,Meret Ricklin ,Philippe Souque,Marie-Pascale Frenkiel,Sylvie Paulous,Obdulio Garcìa-Nicolàs,Artur Summerfield,Pierre Charneau ,Philippe Desprès
Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in Southeast Asia. Vaccination of domestic pigs has been suggested as a “one health” strategy to reduce viral disease transmission to humans. The efficiency of two lentiviral TRIP/JEV vectors expressing the JEV envelope prM and E glycoproteins at eliciting protective humoral response was assessed in a mouse model and piglets.
A gene encoding the envelope proteins prM and E from a genotype 3 JEV strain was inserted into a lentiviral TRIP vector. Two lentiviral vectors TRIP/JEV were generated, each expressing the prM signal peptide followed by the prM protein and the E glycoprotein, the latter being expressed either in its native form or lacking its two C-terminal transmembrane domains. In vitro transduction of cells with the TRIP/JEV vector expressing the native prM and E resulted in the efficient secretion of virus-like particles of Japanese encephalitis virus. Immunization of BALB/c mice with TRIP/JEV vectors resulted in the production of IgGs against Japanese encephalitis virus, and the injection of a second dose one month after the prime injection greatly boosted antibody titers. The TRIP/JEV vectors elicited neutralizing antibodies against JEV strains belonging to genotypes 1, 3, and 5. Immunization of piglets with two doses of the lentiviral vector expressing JEV virus-like particles led to high titers of anti-JEV antibodies, that had efficient neutralizing activity regardless of the JEV genotype tested.
Immunization of pigs with the lentiviral vector expressing JEV virus-like particles is particularly efficient to prime antigen-specific humoral immunity and trigger neutralizing antibody responses against JEV genotypes 1, 3, and 5. The titers of neutralizing antibodies elicited by the TRIP/JEV vector are sufficient to confer protection in domestic pigs against different genotypes of JEV and this could be of a great utility in endemic regions where more than one genotype is circulating.
Mosquito-borne Japanese encephalitis virus is a member of the Flavivirus genus in the Flaviviridae family [1–4]. Flaviviruses contain a positive single-stranded RNA genome encoding a polyprotein that is processed into three structural proteins, the capsid (C), the precursor of membrane (prM) and the envelope (E), and seven non-structural proteins NS1 to NS5 . Viral assembly occurs in the lumen of the endoplasmic reticulum: the nucleocapsids associate with prM-E heterodimers to form an immature JEV virion. The latter transits through the secretory pathway, where it is matured through cleavage of prM into the membrane (M) protein by furin in the trans-Golgi . Additionally, JEV produces virus-like particles (VLPs), which are assembled solely from prM and E proteins, and undergo the same maturation process as genuine viral particles . These VLPs can be produced in the absence of any other viral component .
JEV is the etiologic agent of the most important viral encephalitis of medical interest in South Asia, with an incidence of 50,000 cases and about 10,000 deaths per year [1, 3, 6]. About 20 to 30% of the symptomatic human cases are fatal, while 30 to 50% of survivors can develop long-term neurologic sequelae. JEV is usually maintained in an enzootic cycle between Culex tritaeniorhynchus mosquitoes and amplifying vertebrate hosts, such as waterbirds and domestic pigs [1, 3, 7]. Horses and humans are thought to be dead-end hosts, since they do not develop a level of viremia sufficient to infect mosquitoes . In the past decades, there has been an expansion of the geographic distribution of JEV in Asia and a possible introduction of JEV into Europe has been documented recently [6, 8]. Phylogenetic studies based on the viral envelope protein sequences allow the division of JEV strains into genotypes G1 to G5 [1, 3, 9–15]. Initially, most of the circulating strains of JEV belonged to G3 and were at the origin of major epidemics in Southeast Asian countries. Recently a shift in prevalence from JEV G3 to G1 has been observed in several Asian countries, while some strains of JEV G5 have been occasionally isolated in China and South Korea [9–16].
We previously demonstrated that both integrative and non-integrative lentiviral vectors are promising vaccination vectors against arboviruses such as West Nile virus (WNV), a neurotropic Flavivirus that belongs to the JEV serocomplex [17, 18]. Immunization with a single minute dose of recombinant lentiviral TRIP vectors that express the soluble form of WNV E protein resulted to a robust protection against a lethal challenge with WNV in mice [17, 18]. The currently used lentiviral delivery vectors, mostly derived from human deficiency virus-1 (HIV-1), allow in vivo stable transduction of dendritic cells. This allows a sustained antigen presentation through the endogenous pathway, which in turn elicits robust both humoral and cellular adaptative immunity [19–21].
Humoral immunity plays a pivotal role in protection against JEV infection [22–25] and consequently, the elicitation of a protective antibody response is critical in the development of safe JEV vaccines . In the present study, we evaluated the ability of two lentiviral vectors TRIP/JEV expressing JEV G3 prM and E to induce a protective humoral immune response against JEV infection in a mouse model and in pigs. Both TRIP/JEV.prME vectors were efficient at producing broad anti-JEV neutralizing antibodies in a mouse model. Immunization of piglets with a TRIP/JE vector expressing JEV VLPs elicited high titers of specific neutralizing antibodies that could give a sufficient protection against different JEV genotypes.
Mosquito Aedes albopictus C6/36 cells were maintained at 28°C in Leibovitz medium (L15) supplemented with 10% heat-inactivated fetal bovine serum (FBS). Vero cells and were maintained at 37°C in Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% FBS. BHK-21, SK-N-SH, and HEK-293T cells were maintained at 37°C in DMEM supplemented with 10% FBS. Highly purified mouse anti-pan Flavivirus E monoclonal antibody (mAb) 4G2 was produced by RD Biotech (Besançon, France). Mouse mAb anti-JEV NS5 was kindly provided by Y. Matsuura . Antibodies against Calnexin and SNAP-tag were purchased from Enzo Life Sciences and New England Biolabs, respectively. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG antibodies were obtained from Bio-Rad Laboratories. HRP-conjugated goat anti-pig antibody was obtained from Bethyl Laboratories. Alexa Fluor 488-conjugated goat anti-mouse IgG antibody was obtained from Jackson ImmunoResearch.
The JEV G1 strain G1 CNS769_Laos_2009  was kindly provided by R. Charrel . The JEV G3 strain Nakayama was obtained from the National Collection of Pathogenic Viruses (NCPV, Salisbury, UK) and passaged twice on Vero cells. The molecular clone of JEV G3 strain RP-9 was kindly provided by Y-L. Lin  and modified to produce pBR322(CMV)-JEV-RP9, as described previously . To produce infectious virus, the molecular clone was transfected into HEK-293T cells using Lipofectamine 2000 (Life Technologies). At three days post-transfection, viral supernatants were collected and used to infect C6/36 cells in order to grow final virus stocks for experiments. The chimera JEV S-g5/NS-g3 (G5/G3) which express the structural proteins C, prM, and E from the JEV G5 strain XZ0934 fused to the nonstructural proteins of JEV G3 RP-9 has been already described . The chimeric JEV S-g1/NS-g3 (G1/G3) virus containing the structural protein region from the JEV G1 CNS769_Laos_2009 fused to the RP-9 nonstructural protein region was produced as follows. A silent mutation that created a unique restriction site (AflII) at position 2208–2213 (residues 705 and 706 of the viral polyprotein) was introduced directly in pBR322(CMV)-JEV-RP9 through PCR mutagenesis using primers 5’-actgggaaaggctttcacgaccactcttaagggtgctcagagac-3’ and 5’-gtctctgagcacccttaagagtggtcgtgaaagcctttcccagt-3’ (the Afl II site is underlined). The resulting pBR322(CMV)-JEV-RP9(Afl II) plasmid was used as template to generate the chimeric JEV. The fragment corresponding to nucleotides 114 to 2213 and flanked by the unique sites ApaI and AflII was substituted with the homologous fragments of JEV G1 strain (nucleotides 115–2214). Both chimeric viruses were produced through transfection of the cDNA infectious clone, as described for JEV G3 strain RP-9.
For the construction of recombinant lentiviral vectors expressing JEV proteins, modifications that optimize the expression of prM and E genes in mammalian cells were done on the original sequence of JEV strain RP-9 of G3 using a synthetic gene (Genecust, Lux.). The mammalian codon-optimized sequence coding for prM signal peptide followed by prM and E glycoproteins was cloned into the BamH I and Xho I restriction sites of the pTRIPΔU3CMV plasmid, to generate pTRIPΔU3CMV/JEV.prME. The optimized sequence was further modified by mutagenesis PCR to generate pTRIPΔU3CMV/JEV.prMEΔTM that contains the genes encoding prM and E lacking its two transmembrane domains (EΔTM).
Lentiviral particles were produced by transient calcium co-transfection of HEK-293T cells as described previously , but with the following modifications: at 24h hours post-transfection, the cell culture medium was replaced by serum-free DMEM. Supernatants were collected at 48 hours post-transfection, clarified by several rounds of low-speed centrifugation, and stored at -20°C. The recombinant lentiviral vectors were pseudotyped with VSV-G envelope protein of serotype Indiana (IND) or New Jersey (NJ) . In the resulting vectors TRIP/JEV.prME and TRIP/JEV.prMEΔTM the CMV immediate early promoter (CMVie) drives the constitutive expression of recombinant JEV proteins. The TRIP/JEV vector stocks were titrated by real-time PCR on cell lysates from transduced HEK-293T cells and expressed as transduction unit (TU) per ml [17,18]. Titers of non-concentrated TRIP/JEV.prME vector bearing IND or NJ VSV.G envelope protein were 6.8 log10 TU.mL-1 and 6.2 log10 TU.mL-1, respectively.
Titers of lentiviral TRIP/JEV.prMEΔTM vector bearing IND or NJ VSV.G envelope protein were 7.1 log10 TU.mL-1 and 6.2 log10 TU.mL-1, respectively. Lentiviral vector stocks were adjusted by dilution in sterile PBS and were inoculated in mice or pigs without further concentration.
Human HEK-293T cells were transduced with recombinant lentiviral vectors. The supernatants were collected at 2 days post-transduction and clarified by centrifugation at 3,000g for 5 min at 4°C. The clarified supernatant was loaded over a 15% sucrose cushion in TNE buffer (10 mM Tris-HCl [pH 7.5], 2.5 mM EDTA, 50 mM NaCl), and centrifuged at 100,000g for 2.5 h at 4°C. The supernatants were discarded, and the purified virus-like particles (VLPs) were suspended in 50 μl of TNE buffer.
For titration of JEV infectivity, focus-forming assays (FFA) were performed on BHK-21 cells as previously described . Virus titers were given in focus forming units per ml (FFU.mL-1).
Large flasks of Vero cell monolayers were inoculated with JEV at low multiplicity of infection. The supernatant fluids of JEV-infected (JEV antigen) or mock-infected (normal cell antigen or NCA) cells were harvested and clarified. The supernatants were precipitated with 7% w/v PEG 6,000 (Fluka), centrifuged, and the viral pellet was suspended in cold PBS supplemented with 0.1% ß-propiolactone in 0.1 M Sorensen buffer (pH 9.0) for JEV inactivation. The working dilution of inactivated JEV antigen (1:200) was estimated based on an « in-house » indirect ELISA using well-characterized human positive JEV serum samples and already validated JEV antigen.
The DES expression system (Life Technologies) was required for the production of recombinant viral antigens in Drosophila S2 cells. A synthetic gene coding for prM followed by EΔTM from JEV G3 strain SA-14 was cloned into the shuttle vector pMT/BiP/SNAP, a derived pMT/BiP/V5-HisA plasmid (Life Technologies) in which the SNAP-tag sequence (Covalys BioSciences AG) had been inserted in frame with the insect BiP signal peptide. The resulting plasmid pMT/BiP/JEV.prMEΔTM-SNAP encodes prM followed by EΔTM in fusion with the N-terminus of SNAP-tag. The recombinant domain III from the E protein (EDIII) of JEV G1 strain JaNAr0102/Japan/2002/Mosquito, JEV G3 strain GP05, and JEV G5 strain 10–1827 were fused in frame to the C-terminus of SNAP-tag into the plasmid pMT/BiP/SNAP. The resulting plasmids pMT/BiP/JEV.prMEΔTM-SNAP and pMT/BiP/SNAP-JEV.EDIII were transfected into S2 cells to establish stable cell lines S2/JEV.prMEΔTM-SNAP and S2/SNAP-JEV.EDIII for G1, G3, and G5 as previously described . The production and the purification of recombinant viral antigens from stable S2 cell lines were performed as previously described .
Western blot assay was essentially performed as previously described . For immunofluorescence (IF) assay, cells were fixed with 3.2% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS. JEV E protein was detected with the mAb 4G2, followed by incubation with AlexaFluor488-conjugated secondary antibody. The cover slips were mounted with ProLong Gold Antifade Reagent with DAPI (Life Technologies). The slides were examined using a fluorescent microscope (Axioplan 2 Imaging, Zeiss).
Groups of 6-week-old female BALB/c mice (n = 6) were intraperitoneally (i.p.) inoculated with recombinant lentiviral vectors in 0.1 ml DPBS supplemented with 0.2% endotoxin-free serum albumin. Animals were bled by puncturing at the retro-orbital sinus level. A very low individual variability exists within each group of mice inoculated with recombinant lentiviral vectors justifying the use of pooled sera in subsequent experiments . A control group of 3-week-old female BALB/c mice (n = 6) was inoculated with 3 log10 FFU of JEV G3 (strain RP-9) and immune sera was collected and pooled at 21 days post-inoculation.
For passive seroprotection experiments, pooled immune sera were transferred i.p. into groups of 3-week-old C57BL/6 female mice (n = 6 or 12) one day before a challenge with 10 FFU of JEV G3 (strain RP-9) inoculated by the i.p. route. The challenged mice were monitored for signs of morbidity and mortality. Euthanasia was applied on animals showing the symptoms of viral encephalitis.
Groups of 7-week-old specific pathogen free Swiss Land Race piglets from in-house breeding were housed in groups, and an adaptation time to the new environment of one week was given before starting the experiment.
For immunization, the TRIP/JEV.prME lentiviral vector was diluted to a final volume of 0.5 ml with PBS (Life Technologies). Immunization with the TRIP/GFP lentiviral vector was used as a negative control . From a group of 5 piglets, four were vaccinated intramuscularly with various doses of the TRIP/JEV.prME vector and one was injected with the equivalent dose of control lentiviral vector TRIP/GFP. Immunized animals were bled before the first vaccination and then weekly until the end of the experiment. Four weeks after the first vaccination, all animals got a booster vaccination with the same dose of recombinant lentiviral vectors as at the first time point. For ethical reasons no lethal challenge was performed as protection in pigs. As a control, 3 animals were inoculated by the oronasal route with 7 log10TCID50 of live JEV Nakayama G3. All pigs developed temporary fever and viremia and recovered completely after 4–6 days. The animal sera were examined weekly for anti-JEV antibody.
Indirect ELISA measured the production of anti-JEV IgGs in immunized mice and piglets. A series of 96-well ELISA plates (Nunc) was coated with 0.1 ml of inactivated native JEV antigen or highly purified recombinant JEV antigens diluted in PBS at the concentration of 1 μg.mL-1 at 4°C overnight. NCA and SNAP served as negative control antigens. Indirect ELISA were performed as previously described (29). The Immune Status Ratio (ISR) of each group of immunized mice is obtained by dividing the average of JEV antigen OD450 values by the average control antigen OD450 values. The end-point titers of anti-JEV antibodies in mouse sera were calculated as the reciprocal of the last dilution of serum having ISR value > 3.0. Pig sera were tested as described for the mice, using HRP-conjugated goat anti-pig antibody as a secondary antibody. Pig sera obtained prior immunization were used as a negative control.
Neutralizing ability of mouse and pig serum antibodies against JEV was determined by focus (FRNT) or plaque (PRNT) reduction neutralization tests on Vero cells, respectively. Mouse serum samples from each group were pooled. Pig sera were tested individually in triplicates starting at a 1:5 serum dilution. Pooled mouse or individual pig serum were two-fold serial diluted in DMEM supplemented with 2% FBS, with a starting dilution of 1:10, and incubated for 2 h at 37°C with an equal volume of viral suspension containing 100 FFU of JEV. The end-point titer was calculated as the reciprocal of the highest serum dilution tested that reduced the number of FFU (FRNT50) or PFU (PRNT50) by 50%.
A Log-rank (Mantel-Cox) test was used to compare survival data. Antibody levels between groups of immunized pigs were compared by Mann Whitney U test and the level of significance was set at 5%. GraphPad Prism (GrapPad Software Inc. La Jolla, CA, USA) was used for all statistical analysis.
All mice were housed under pathogen-free conditions at the Institut Pasteur animal facility. The protocols and subsequent experiments were ethically approved by the CETEA (Ethic Committee for Control of Experiments on Animals: http://cache.media.enseignementsup-recherche.gouv.fr/file/Encadrement_des_pratiques_de_recherche/58/1/Charte_nationale_portant_sur_l_ethique_de_l_experimentation_animale-version_anglaise_243581.pdf) at the Institut Pasteur (C2A N°89/CETEA) with the reference n°2013–0071 and declared to the French Ministère de l’Enseignement Supérieur et de la Recherche (reference n° 000762.1) in accordance with the articles R.214-24 et R.214-125 du Code Rural et de la Pêche Maritime and R.214-120 du décret n°2013–118 du 1er février 2013 in France. Experiments were conducted following the guidelines of the Office Laboratory of Animal Care at the Institut Pasteur. The protocols and subsequent experiments on pigs were ethically approved by the cantonal ethical committee of Bern (number BE 118–13) and the FSVO (Federal Food Safety and Veterinary Office, website: http://www.blv.admin.ch/index.html?lang=en) in Switzerland. Pig experiments were conducted following the guidelines of Swiss Animal Welfare Regulations (Veterinary Service of LANAT).
We have reported earlier that a single minute dose of a non-replicative lentiviral vector expressing the soluble form of WNV E glycoprotein induced a robust protective humoral response in a mouse model of WNV encephalitis [17, 18]. To assess the potential of lentiviral vectors expressing JEV proteins at eliciting protective humoral response against JEV infection, a mammalian codon-optimized gene encoding prM and E from the JEV strain RP9 of G3 was inserted into the lentivirus TRIP vector (Fig 1). We generated two lentiviral vectors, expressing the prM signal peptide followed by the prM protein and the E glycoprotein, the latter being expressed either in the native form (TRIP/JEV.prME) or lacking its two C-terminal transmembrane domains (TRIP/JEV.prMEΔTM). In these constructs, prM contributes to the folding, stability, and efficient secretion of the glycoprotein E. Lentiviral vectors which expressed JEV proteins were pseudotyped with VSV-G protein of the IND serotype. Non-replicative TRIP/JEV.prME and TRIP/JEV.prMEΔTM particles were produced from HEK-293T cells, achieving titers of about 7 log10 TU.mL-1.
The antigenicity of recombinant JEV proteins was assessed by transducing HEK-293T cells with the TRIP/JEV.prME or TRIP/JEV.prMEΔTM vectors (Fig 2A). An empty vector served as a control. At 48 h post-transduction, the intracellular form of the E protein was detected using the anti-E MAb 4G2 by IF assay. A similar staining pattern for E was observed in TRIP/JEV-transduced cells expressing prME or prMEΔTM. Immunoblot assays using mouse anti-JEV antisera detected recombinant prM and E in lysates from HEK-293T cells transduced with TRIP/JEV vectors (Fig 2B). We observed an efficient release of E protein to the supernatants of HEK-293T cells transduced with either TRIP/JEV vectors (Fig 2C, top). The presence of prM was only detected in supernatants from cells transduced with the TRIP/JEV.prME vector (Fig 2C, top). Because JEV prM and E have the capacity to self-assemble into VLPs, we assessed whether VLPs were secreted from HEK-293T cells transduced with TRIP/JEV vectors. The VLPs were detected by immunoblot assay using anti-E mAb 4G2 and anti-JEV immune serum (Fig 2C, bottom). Extracellular JEV VLPs containing prM and E accumulated in the supernatant of HEK-293T cells transduced with TRIP/JEV.prME vector but not with TRIP/JEV.prMEΔTM vector. Thus, transduction of cells with TRIP/JEV.prME vector leads to efficient secretion of recombinant JEV VLPs.
To evaluate humoral responses induced by the lentiviral TRIP/JEV vectors, adult BALB/c mice were inoculated with increasing doses of TRIP/JEV.prME or TRIP/JEV.prMEΔTM (3 to 5 log10 TU per animal) by i.p. route. At 21 days post-immunization, sera were collected from each group of mice and pooled. Indirect ELISA was performed to detect anti-JEV IgGs using inactivated JEV particles as capture antigens (Table 1). NCA served as a control antigen. There was little to no antibody responses against JEV at TRIP/JEV vector doses lower than 5 log10 TU per animal. The dose of 5 log10 TU induced a significant production of anti-JEV specific antibodies with a mean titer reaching 1,600 for TRIP/JEV.prME and 400 for TRIP/JEV.prMEΔTM (Table 1). At the highest dose (6 log10 TU) inoculated to mice, the mean titer of TRIP/JEV.prME antibody reached 10,000. The latter dose was not further used due to the too large volume of non-concentrated TRIP/JEV vector inoculated to mice by i.p. route. We therefore decided to select the unique dose of 5 log10TU in subsequent mouse immunizations. To determine the time course of anti-JEV production, BALB/c mice that received 5 log10TU of TRIP/JEV.prME or TRIP/JEV.prMEΔTM were bled at 7, 14 and 21 days post-immunization (Table 1). Anti-JEV antibodies were detectable at day 14 of immunization and reached significant titers at day 21.
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