Attenuated and replication competent Vaccinia Virus strains

16 Replication competent poxvirus vectors with an attenuation phenotype and with high 17 immunogenic capacity of the foreign expressed antigen are being pursuit as novel 18 vaccine vectors against different pathogens. In this investigation we have examined the 19 replication and immunogenic characteristics of two vaccinia virus (VACV) mutants 20 M65 and M101. These mutants were generated after 65 and 101 serial passages of 21 persistently infected Friend erythroleukemia cells (FEL). In cultured cells of different 22 origins the mutants are replication competent and have growth kinetics similar or 23 slightly reduced in comparison with the parental Western Reserve (WR) virus strain. In 24 normal and immune suppressed infected mice the mutants showed different attenuation 25 levels, and pathogenicity in comparison with WR and MVA strains. Wide genome 26 analysis after deep sequencing revealed selected genomic deletions and mutations in a 27 number of viral open-reading frames (ORFs). Mice immunized in DNA prime/Mutant 28 boost regime with viral vectors expressing the LACK antigen of Leismania infantum 29 resulted in protection or delay in the onset of cutaneus leishmaniasis. Protection was 30 similar to that triggered by MVA-LACK. In immunized mice, both polyfunctional 31 CD4 and CD8 T cells with an effector memory phenotype were activated by the two 32 mutants, but DNA-LACK/M65-LACK protocol preferentially induced CD4 while 33 DNA-LACK/M101-LACK preferentially induced CD8 T cell responses. Altogether 34 our findings showed the adaptive changes of the WR genome during long-term virus35 host cell interaction and how replication competency of M65 and M101 mutants confers 36 distinct biological properties and immunogenicity in mice, as compared to the MVA 37 strain. These mutants could have applicability for understanding VACV biology and as 38 potential vaccine vectors against pathogens and tumors. 39 on Jauary 1, 2018 by gest http/jvi.asm .rg/ D ow nladed fom Introduction 40 Poxvirus vectors have emerged as prominent vehicles for delivering antigens of 41 pathogens from prevalent diseases. Different strains of vaccinia virus (VACV) 42 expressing antigens from different pathogen-causing diseases are used nowadays in 43 preclinical and clinical trials against HIV, malaria, tuberculosis, leishmaniasis and also 44 in cancer (1). The most promising vectors used in vaccination trials are the attenuated 45 canarypox, fowlpox, MVA and NYVAC strains (2-5). While those viruses do not 46 produce virus progeny in human cells, which assures safety, some evidence points out 47 that replication competent viruses with limited but amplified time of infection and 48 expression of heterologous antigen could provide more immunogenic vaccines (6). 49 Traditional smallpox vaccines have relied on replication competent and attenuated 50 vaccinia virus (VACV) vectors, but the side effects, particularly in immune 51 compromised individuals, preclude their use as recombinant viral vectors for current 52 vaccines. Recently, a replication competent NYVAC vector, from the Copenhagen 53 smallpox strain, with re-inserted host range genes and limited replication in tissues, was 54 shown to be a candidate vaccine vector against HIV (7). It is unclear how many rounds 55 of VACV vector replication are needed in vivo to activate effective immune responses 56 leading to long-term protection after pathogen challenge. Hence, we decided to 57 investigate VACV vectors that can replicate in vivo for several rounds for their capacity 58 as recombinants to induce immune responses with protection against a pathogen. These 59 vectors have been isolated during a persistent VACV infection (WR strain) in the Friend 60 erythroleukemia cell line (FEL) (8). It has been previously reported that persistent 61 infection with the IHD-W strain of VACV can be established in FEL cells and that the 62 virus produced was indistinguishable from the parental virus (9). In the case of the WR 63 strain, during persistent infection of FEL cells mutants with large deletions of about 8 64 MDa at the left terminus of the viral genome (10), alterations in some of the structural 65 proteins with roles in the morphogenetic pathway, a small plaque size phenotype 66 compared with WR parental virus and replication capacity in some mammalian cell 67 lines (11) were produced. Recombinants based on these mutants at early passages in 68 FEL cells and expressing parasite antigens for malaria and leishmania have been shown 69 in prime/boost regimens in mice to elicit protection after challenge with parasites (12, 70 13). 71 72 on Jauary 1, 2018 by gest http/jvi.asm .rg/ D ow nladed fom To continue the further exploration of mutants from the persistent FEL infection and to 73 define their adaptive changes during long-term virus persistence and immunological 74 properties as vaccine vectors, in this investigation we selected two mutants from 75 infected FEL cells after 65 (referred as M65) and 101 (M101) passages, and have 76 characterized their biological, genetic and immune properties. Pathogenesis of both 77 mutants was monitored in normal and immune deficient mice by weight loss after 78 systemic inoculation (14); the duration of gene expression and replication capacity was 79 followed in mice infected with the mutants expressing the sensitive firefly luciferase 80 gene as marker, and by virus yields, as described previously (6, 15). Genomic status 81 (10) was defined by a wide genome analysis after deep DNA sequencing of M65 and 82 M101. Moreover, we analyzed if the expression of a parasite antigen in a prime/boost 83 regimen with M65 and M101 recombinant viruses could induce protection against 84 experimental challenge. We focused on leishmaniasis as this is one of the most 85 neglected tropical diseases, prevalent in 88 countries and presenting an estimated annual 86 incidence of 2 million and about 12 million cases worldwide (16). Among all 87 leishmania antigens used, we selected one of the most promising genes: Leishmania 88 homologue for receptors of activated C kinase or LACK (17). The model of infection 89 chosen was the permissive BALB/c mouse model inoculated in the footpad, as this 90 strain of mice is highly susceptible and develop cutaneous lesions after the inoculation 91 of L.major and L.amazonensis. Immunogenicity was evaluated by intracellular cytokine 92 staining (ICS). 93 Overall, this study demonstrates how the VACV genomes get adapted after 65 and 101 94 passages during a persistent virus infection, and how the different biological 95 characteristics acquired for M65 and M101 impact on the immune responses of the host. 96 Moreover, comparative studies with the prototype vaccine vector MVA reveals 97 differences in replication capacity and immunological behavior of the mutants. These 98 novel characteristics of M65 and M101 could be useful in the design of new poxvirus99 based vaccine strains against prevalent diseases. 100 101 102 on Jauary 1, 2018 by gest http/jvi.asm .rg/ D ow nladed fom Materials and methods 103 Ethics Statement 104 The Ethical Committee of Animal Experimentation (CEEA-CNB) of Centro Nacional 105 de Biotecnología (CNB-CSIC) approved the animal studies in accordance with national 106 and international guidelines and with the Royal Decree (RD 1201/2005). Permit 107 number: 011045. 108 Cells, plasmids and viruses 109 Cells were maintained in a humidified air 5% CO2 atmosphere at 37oC. Primary chicken 110 embryo fibroblasts (CEF) were obtained from pathogen-free (SPF) 11-day-old eggs 111 (Intervet, Salamanca, Spain) and were grown in Dulbecco’s modified Eagle’s medium 112 (DMEM) supplemented with 10% FCS. African green monkey kidney cells (BSC40) 113 and human cells (HELA) were grown in Dulbecco’s modified Eagle’s medium 114 (DMEM) supplemented with 10% newborn calf serum (NCS). 3T3 murine cells and 115 baby hamster kidney cells (BHK-21) were grown in Dulbecco’s modified Eagle’s 116 medium (DMEM) supplemented with 10% calf serum (CS). The origin of FEL cells (a 117 subclone of line 745A) and conditions for the establishment of virus persistence with 118 the WR strain of VACV has been previously described (52, 53). Briefly, on the second119 third day of infection with 1 PFU/cell of purified WR strain, about 90% of the cells 120 have died, surviving cells removed and resuspended in fresh medium. After recovery 121 (2-3 weeks), the cells were passage every 4-5 days. 122 The mammalian expression plasmid vector pCI-neo-LACK was previously described in 123 (18). The empty plasmid pCI-neo (Promega) was used as control (DNAφ). Both 124 constructs were purified using the Quiagen plasmid purification kit (Quiagen). 125 The viruses used in this study included VACV WR parental strain, WR recombinant for 126 the luciferase gene of Photinus Pyralis (WR-Luc) (15), and attenuated MVA strain 127 recombinant for the luciferase gene (MVA-Luc) (19). 128 M65 and M101 were grown in BHK21 cells and purified by sucrose gradient 129 centrifugation for DNA extraction and in BSC40 for the rest of the studies. M65130 LACK, M101-LACK, M65-Luc and M101-Luc were grown in BSC40 cells. 131 Purification and titration of viruses was performed as described before (20, 21). 132 Plasmids and viruses were diluted for inoculation in endotoxin-free phosphate buffered 133 saline (PBS). 134 Construction of vaccinia virus recombinants expressing firefly luciferase gene. 135 on Jauary 1, 2018 by gest http/jvi.asm .rg/ D ow nladed fom Vaccinia insertion plasmid pSC11-luc derives from pSC11 insertion plasmid for TK 136 locus (J2R gene) and contains the gene encoding the firefly luciferase of Photinus 137 pyralis under the vaccinia early/late promoter p7.5. This plasmid also contains the 138 Escherichia coli β-galactosidase (Lac Z) gene under the control of the p11 late viral 139 promoter (15). M65-Luc and M101-Luc were obtained by transfecting with pSC11-luc 140 plasmid BSC-40 cells infected with M65 strain or M101 and harvested 48-72 hours 141 post-infection (h.p.i.) and β-galactosidase producing plaques were selected by the 142 addition of 5-bromo-4-chloro-3-indolil-β-D-galactopyranoside (X-Gal) to the agar. For 143 the generation of MVA-LACK, the plasmid pHLZ-LACK was used and recombinants 144 selected after plaque assay by the addition of 5-bromo-4-chloro-3-indolil-β-D145 glucuronic (X-Gluc) acid to the agar. The β-glucuronic acid-producing plaques were 146 picked, cloned by plaque isolation, and amplified following standard procedures (22). 147 The purity of M65-Luc, M101-Luc and MVA-LACK was assessed by PCR. 148 Construction of vaccinia virus recombinants expressing Leishmania infantum 149 LACK antigen (M65-LACK and M101-LACK). 150 Vaccinia insertion plasmid pHLZ-LACK contains the gene encoding the L. infantum 151 LACK protein cloned into the SmaI site of the pHLZ VACV insertion plasmid under 152 the control of the synthetic early/late pE/L viral promoter and the hemagglutinin (HA) 153 flanking sites (18). This plasmid contains the Escherichia coli β-glucuronidase gene 154 under the control of the p7.5 early/late viral promoter. M65-LACK and M101-LACK 155 were obtained by transfecting with pHLZ-LACK plasmid BSC-40 cells infected with 156 M65 or M101 viruses, harvested 48-72 hours post-infection (h.p.i.) and virus plaques 157 selected after the addition of X-Gluc acid to the agar. The β-glucuronidase-producing 158 plaques were picked, cloned by plaque isolation, and amplified following standard 159 procedures (22). The purity of M65-LACK and M101-LACK was assessed by PCR. 160 Parasite strains and animals 161 L. major (WHOM/IR/-173) was a kind gift from Dr Nicholas Glaichenhaus (CNSR, 162 Valbonne, France). Promastigotes were cultured at 27 °C in Schneider's medium (Gibco 163 BRL, UK), supplemented with 20% fetal calf serum (FCS, Gibco BRL, UK) and 164 antibiotics. The virulence of the strain was preserved by periodic passage through 165 BALB/c mice. Frozen stocks grown in culture until the stationary phase were used for 166 the experiments. In order to purify only infective metacyclic promastigotes, stationary 167 phase cultures were resuspended in PBS at a final concentration of 1-5x10 168 on Jauary 1, 2018 by gest http/jvi.asm .rg/ D ow nladed fom promastigotes/ml and treated with 10 mg/ml of Peanut Agglutinin. After centrifugation 169 at 1000 rpm, metacyclic promastigotes were collected from the supernatant, washed 170 twice in PBS and resuspended in the same buffer for inoculation. 171 L. amazonensis (LTB0016 strain) was a kind gift from Professor Diane McMahon-Pratt 172 (Yale School of Medicine, New Haven, USA). Promastigotes were cultured at 27 °C in 173 Schneider's medium (Gibco BRL, UK), supplemented with 20% fetal calf serum (FCS, 174 Gibco BRL, UK) and antibiotics. The virulence of the strain was preserved by periodic 175 passage through BALB/c mice. Fresh aspirates were synchronized in culture until 176 stationary phase, passaged twice and grown for 5 days before inoculation in animals. 177 All mice used in this study were female BALB/c mice ranging from 6 to 8 weeks of age 178 purchased from Harlan (IN, USA) and housed in the Animal Facility of the Centro 179 Nacional de Biotecnología-CSIC (Madrid, Spain) under pathogen-free conditions. 180 DNA Extraction from VACV-infected cells 181 5x10 pfu were resuspended in 1ml of buffer containing 50 mM Tris-Hcl pH 8, 50 mM 182 NaCl, 50 mM MgCl2. Suspension was incubated with 20 units of DNAse/RNAse free 183 (Roche Applied Science, USA) 30 min at 37 oC. After treatment with DNAse, Sarkosyl 184 was added at a final concentration of 0.5%. Samples were later treated 1 hour at 37 oC 185 with Proteinase K (Quiagen) at a final concentration of 200 μg/ml. 1 volume of 186 Equilibrated Phenol was added, sample was mixed by inversion and centrifuged 13000 187 rpm for 5 min. Supernatant was collected and added 2 volumes of Chorophorm/Isoamyl 188 alcohol (1:1). Samples were centrifuged 12000 rpm 5 min. NaCl was added to 2 189 volumes of 100% ethanol to a final concentration of 0.2 M and samples were left 190 overnight at -20 oC. Samples were then centrifuged 10000 rpm 5 min. Pellets were 191 washed twice in 75% Ethanol, air-dried and resuspended in 200 μl of H20. DNA 192 concentration was measured in a NanoDrop (Thermo Scientific, Wilmington, USA). 193 Wide Genome Analysis and Deep Sequencing 194 Sequences generation: 195 Library preparation was according to the recommendations by Illumina. Briefly, 5 196 micrograms of genomic DNA were fragmented using a Nebulizer to a peak size of 250 197 bases. Following end-repair and A-tailing, internally indexed adapters were ligated to 198 the DNA. Size selection after adapter ligation was performed on a 2% agarose gel, and a 199 narrow size fraction corresponding to an insert size of 250 bases was recovered using 200 GeneCatcher tips. The DNA was recovered from the gel using the QIAquick gel 201 extraction method, and the DNA was amplified by 12 cycles of PCR using standard 202 on Jauary 1, 2018 by gest http/jvi.asm .rg/ D ow nladed fom Illumina PE 1.1 and 2.0 primers. The quality of the libraries was confirmed with the 203 Agilent 2100 Bioanalyzer. The quantified libraries were loaded as a pool onto one lane 204 of an Illumina flow cell and sequenced on the Illumina Genome Analyzer IIx using a 205 36-cycle recipe. 206 Sequence alignment: 207 Short reads were aligned to viral genomes with BWA (23) with default parameters for 208 single-end data. Alignment files in SAM format were transformed to BAM format, 209 sorted and indexed with SAMtools (24). 210 Variant detection: 211 For detecting statistically sound SNPs and short INDELs, GenomeAnalysisToolKit 212 (GATK) (25) was used (–stand_call_conf = 30.0 and –stand_emit_conf = 10.0). 213 IndelRealigner option was applied for refining alignments of reads based on 214 misalignments due to the presence of indels. 215 Variant annotation: 216 In-house developed PHP scripts were used to classify the effect of SNPs as: Silent: 217 substitutions that do not change any amino acid; Change: substitutions that change an 218 amino acid; Upstr: substitutions that occurs in the 200bp upstream region of a gene; 219 Downstr: substitutions that occurs in the 200bp downstream region of a gene. 220 (NOTE: Copenhagen annotations are not available) 221 Immunohistochemical studies 222 Immunohistochemical staining were performed on 5 μm 4% paraformaldehyde-fixed 223 and paraffin-embedded lung sections using the Dako Autostainer (DAKO Corp., 224 Carpinteria, CA) with rabbit anti-WR primary antibody. After incubation with the 225 secondary antibodies, positive cells were visualized using 3,3-diaminobenzidine 226 tetrahydrochloride plus as a chromogen. All sections were counterstained with 227 hematoxylin. Images were taken using a Zeiss Axiophot microscope (Carl Zeiss 228 Microimaging GmbH, Gottingen, Germany), converted into standard TIFF format and 229 analyzed with the Nikon Digital Sight software. 230 Growth Curves 231 To determine virus yields produced intracellularly and released from cells in the 232 different cell lines, confluent CEF, HeLa or 3T3 cells grown in 24-well plates were 233 infected in duplicates with a MOI of 0.01 or 5 pfu/cell. At 6, 24 and 48 hpi, cells were 234 recovered from the well, freeze-thawed three times, sonicated and virus titration was 235 on Jauary 1, 2018 by gest http/jvi.asm .rg/ D ow nladed fom performed in BSC40 cells as previously described (21). Extracellular virus yields were 236 determined titrating fresh supernatant of cells infected for 24 and 48 hpi. 237 Measurement of Luciferase activity in mouse tissue 238 Gene expression of recombinant viruses was monitored by the luciferase assay to 239 quantify heterologous gene expression in tissues from mice inoculated intraperitoneally 240 (i.p) with WR-Luc, M65-Luc, M101-Luc or MVA-Luc. Tissues were collected at 6, 24, 241 48 or 72 hpi. At indicated times post-inoculation, animals were sacrificed, and spleen, 242 liver, draining lymph nodes and ovaries were dissected and stored at -80oC. Peritoneal 243 cells were harvested by mouse peritoneal cavity washed with 10 ml of sterile PBS, 244 centrifuged for 5 min at 1500 rpm and stored at -80oC. Tissues from individual mice 245 were homogenized in luciferase extraction buffer (200 μl/spleen and 200 μl/ovary, 246 lymph node or peritoneal extracts) (Promega Corp., Madison, Wis.) using an 247 Eppendorf-fitted Ultraturrax T8 homogenizer (Janke&Kunkel, Staufen, Germany). 248 Luciferase activity was measured in the presence of ATP and luciferin according to the 249 manufacturer’s instructions using a Lumat LB 9501 luminometer (Berthold, Nashua, 250 N.H.), and was expressed as Reference Luciferase Units (RLU) per milligram of 251 protein. Protein in tissue extracts was measured with BCA Protein Assay Kit (Pierce, 252 Thermo Scientific, Rockford, USA). Tissues from individual mice were collected and 253 homogenized in complete DMEM media to test for the production of infectious virus by 254 plaque assay in BSC-40 cells. The virus titer was expressed as Plaque Forming Units 255 (PFU) per milligram of protein. 256 Immunization and parasite challenge 257 BALB/c mice, 6–8 weeks of age, were primed intradermally (i.d) in the abdomen with 258 100 g of DNA-LACK, or empty DNA-φ in 100 l volume per mouse. At day 14 mice 259 were boosted i.p with 2x10 PFU/mouse of MVA-LACK, M65-LACK, M101-LACK or 260 non-recombinant vaccinia virus and PBS as control. At 3 weeks or 8 weeks days after 261 boosting, six animals per group were challenged subcutaneously (s.c) in the right hind 262 footpad with 5x10 metacyclic PNA-purified L. major, or 2×10 stationary phase 263 L.amazonensis promastigotes resuspended in 10 μl using BD Micro-Fine (BD 264 Biosciences) 0.5 ml 30G needles. 265 Intracellular Cytokine Staining assay (ICS) 266 The phenotypes of responding T cells were analyzed by ICS and fluorescence-activated 267 cell sorting analysis as described elsewhere (26). After an overnight rest, 5x10 268 splenocytes (depleted of red blood cells) were stimulated with 2 μg/ml of LACK157-173 269 on Jauary 1, 2018 by gest http/jvi.asm .rg/ D ow nladed fom peptide or 5x10 A20 cells nucleofected with pCIneo-LACK plasmid (using a 4D 270 nucleofector, LONZA, Germany) during 6 hours in RPMI 1640 medium supplemented 271 with 10% FCS and containing 1 μl/well Golgiplug (BD Biosciences) to inhibit cytokine 272 secretion. After stimulation, cells were washed, stained for the surface markers, fixed, 273 permeabilized using the BD Cytofix/CytopermTM Kit (Becton Dickinson) and stained 274 intracellularly using the appropriate fluorochromes. For memory analyses, the following 275 antibodies were used: CD4-Alexa 700, CD8-V500, CD62L-FITC, CD44-SPRD, 276 CD127-PECy5.5, IFN -PECy-7, TNF -PE and IL-2-APC. All antibodies were from 277 BD Biosciences. Cells were acquired using an LSRII flow cytometer (Becton 278 Dickinson) equipped with a high throughput system. The number of events ranged 279 between 10 and 10. Dead cells were excluded using the violet LIVE/DEAD stain kit 280 (Invitrogen). Lymphocytes were gated on a forward scatter area versus side scatter area 281 pseudo-color dot plot. CD4 and CD8 events were gated versus CD44 and CD62L or 282 CD62L and CD127 to analyze the memory phenotype. IFN , TNF and IL-2 were 283 gated in the different memory populations and then combined together using the 284 boolean operator. Sample analysis was performed using FlowJo version 8.5.3 (Tree 285 Star, Ashland, OR). 286 Measurement of Vaccinia-specific antibodies by ELISA 287 11 days and 8 weeks after immunization, serum was collected from each group of 288 animals and the presence of specific anti-vaccinia antibodies were analyzed by ELISA. 289 In brief, 96-well Maxisorp plates (Nunc, Denmark) were coated overnight at 4 °C with 290 extracts of BSC-40 cells infected with VACV Western Reserve (WR). Plates were 291 washed with PBS-0.05% Tween-20 (PBS-T) and blocked with 5% Milk in PBS 292 (blocking buffer) overnight at 4 °C. Serum samples were diluted 50-fold in PBS, 0.1% 293 tween, 1%milk, added in 50 l per well and incubated for 1 h at 37 °C. Plates were 294 washed five times. Peroxidase-conjugated goat anti-mouse total IgG (Sigma), was 295 added and incubated for 1 h at 37 °C. Plates were then reacted with peroxidase substrate 296 TMB (Sigma) and absorbance was read at 450 nm on a Labsystem Multiskan plus plate 297 reader (Tecan Maguellan, Sunrises). 298 299 Statistical analysis 300 Statistical significance (p < 0.05 (*), p < 0.005 (**) or p < 0.001 (***)) of differences 301 between immunization groups of mice was determined by Student's t-test (2 tail, type 302 3). 303 on Jauary 1, 2018 by gest http/jvi.asm .rg/ D ow nladed fom For the statistical analysis of ICS data we used a novel approach that corrects 304 measurements for the medium response (RPMI) and at the same time allows the 305 calculation of confidence intervals and p-values of hypothesis tests (27, 28). 306 The data analysis program, Simplified Presentation of Incredibly Complex Evaluations 307 (SPICE, version 4.1.5, Mario Roederer, Vaccine Research Center, NIAID, NIH), was 308 used to analyze and generate graphical representations of T cell responses detected by 309 polychromatic flow cytometry. All values used for analyzing proportionate 310 representation of responses are background-subtracted. 311