Additive Protection against Congenital CMV Conferred by Combined gB/pp65 1 Vaccination using a Lymphocytic Choriomeningitis Virus (LCMV) Vector 2

3 1*Mark R. Schleiss, 2Ursula Berka, 2Elizabeth Watson, 2Mario Aistleithner, 2Bettina 4

Kiefmann, 3Bastien Mangeat, 1Elizabeth C. Swanson, 1Peter A. Gillis, 1Nelmary 5 Hernandez-Alvarado, 1Claudia Fernández-Alarcón, 1Jason C. Zabeli, 3Daniel D. 6

Pinschewer, 2Anders Lilja, 2Michael Schwendinger, 2Farshad Guirakhoo, 2Thomas P. 7 Monath, and 2*Klaus Orlinger 8

9 1Center for Infectious Diseases and Microbiology Translational Research 10

University of Minnesota Medical School 11 Department of Pediatrics 12

13 2Hookipa Biotech AG 14

Helmut-Qualtinger-Gasse 2 15 1030 Vienna 16

Austria 17 18

3 Department of Pathology and Immunology 19 University of Geneva 20 Geneva, Switzerland 21

22 1* CIDMTR, 2001 6th Street SE 23 Minneapolis, Minnesota 55455 24

Phone: 612-626-9913 25 Fax: 612-626-9924 26

Email: schleiss@umn.edu 27

CVI Accepted Manuscript Posted Online 26 October 2016Clin. Vaccine Immunol. doi:10.1128/CVI.00300-16Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 28 Subunit vaccines for prevention of congenital cytomegalovirus (CMV) infection based on 29 glycoprotein B (gB) and pp65 are in clinical trials, but it is unclear whether simultaneous 30 vaccination with both antigens enhances protection. We undertook evaluation of a novel 31 bivalent vaccine based on non-replicating lymphocytic choriomeningitis virus (rLCMV) 32 vectors expressing a cytoplasmic tail-deleted gB (gB(dCt)), and full-length pp65 from 33 human CMV in mice. Immunization with the gB(dCt) vector alone elicited a comparable 34 gB-binding antibody response, and a superior neutralizing response, to that elicited by 35 adjuvanted subunit gB. Immunization with the pp65 vector alone elicited robust T cell 36 responses. Comparable immunogenicity of the combined gB(dCt) and pp65 vectors with 37 the individual monovalent formulations was demonstrated. To demonstrate proof-of-38 principle for a bivalent rLCMV-based HCMV vaccine, the congenital guinea pig 39 cytomegalovirus (GPCMV) infection model was used to compare rLCMV vectors 40 encoding homologs of pp65 (GP83) and gB(dCt), alone and in combination, to Freund’s 41 adjuvanted recombinant gB. Both vectors elicited significant immune responses and no 42 loss of gB immunogenicity was noted with a bivalent formulation. Combined vaccination 43 with rLCMV-vectored GPCMV gB(dCt) and pp65 (GP83) conferred better protection 44 against maternal viremia than subunit or either monovalent rLCMV vaccine. The bivalent 45 vaccine also was significantly more effective in reducing pup mortality than the 46 monovalent vaccines. In summary, bivalent vaccines with rLCMV vectors expressing gB 47 and pp65 elicited potent humoral and cellular responses, and conferred protection in the 48 GPCMV model. Further clinical trials of LCMV-vectored HCMV vaccines are warranted. 49 241 words 50 51

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INTRODUCTION 52 53 Infection with human cytomegalovirus (HCMV) causes considerable burden in 54 immunocompromised solid organ and hematopoietic stem cell transplant patients, HIV-55 infected individuals, and newborns that acquire infection in utero (1, 2). By preventing 56 congenital HCMV infection, a preconception vaccine could provide a highly cost-effective 57 public health advance (3). Virus-neutralizing antibody targeting viral envelope 58 glycoproteins, as well as cellular immune responses (CD4+ and CD8+) targeting multiple 59 proteins play important roles in protection against acquisition and reactivation of infection 60 (4-8). Recombinant vaccines based on envelope glycoprotein B (gB), expressed in 61 mammalian cells and admixed with proprietary adjuvants such as the squalene based 62 oil-in-water adjuvant MF59, have demonstrated variable degrees of protection against 63 HCMV infection and/or disease in both immune competent women (9, 10) and immune 64 compromised solid organ transplant recipients (11). 65

Other expression strategies targeting HCMV proteins have been evaluated in phase I 66 and phase II studies in healthy volunteers and, in some cases, hematopoietic stem cell 67 transplant recipients (12-16). Two multivalent recombinant vaccine candidates have 68 been evaluated in the clinic. Propagation-defective alphavirus replicon particles 69 expressing gB and a pp65-IE1 fusion protein were immunogenic in a phase I study, but 70 not pursued further (15). A DNA vaccine expressing gB and pp65 has advanced through 71 phase II trials and has shown signs of efficacy. However, the data suggests only modest 72 immune responses to gB (12). Thus, the efficacy of a bivalent vaccine candidate that 73 elicits both strong humoral and cellular immune responses is unknown. 74

Because of the species-specificity of cytomegaloviruses, experimental HCMV 75 vaccines cannot be evaluated for efficacy against congenital infection in animal models. 76

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Guinea pig cytomegalovirus (GPCMV) recapitulates the pathogenesis observed for 77 HCMV infection in infants in many respects (17-19) and several GPCMV proteins, 78 including the gB and pp65 (GP83) homologs (18, 20), have been shown to provide some 79 protection against congenital GPCMV transmission. However, the combination of gB 80 and pp65 (GP83) homologs in the model demonstrated interference with the anti-gB 81 response when compared to gB antigen alone following administration MVA-vectored 82 vaccines (21). 83

In our studies we employed a replication-incompetent, single-round infectious vector 84 system based on the prototype Arenavirus, lymphocytic choriomeningitis virus (LCMV) 85 clone 13 (22, 23). This expression technology does not elicit vector-neutralizing antibody 86 responses, allowing for administration of homologous booster vaccinations. We describe 87 the construction and in vitro and in vivo characterization of this CMV vaccine candidate 88 expressing HCMV pp65 and gB. These constructs were tested in mice and rabbits and 89 induced significantly higher neutralizing antibody responses than adjuvanted gB protein. 90 Moreover, responses exceeded the titer of human convalescent sera used as 91 benchmark for comparison. Robust antigen-specific T-cell responses were also 92 generated against the vaccine antigen pp65. Finally, the efficacy of both the bivalent and 93 respective monovalent LCMV vectored-vaccines was assessed for protection against 94 congenital infection in the GPCMV model. 95

96 MATERIALS AND METHODS 97 98 Guinea pigs. Outbred Hartley guinea pigs, confirmed to be GPCMV-seronegative by 99 ELISA (18), were purchased from Elm Hill Laboratories (Chelmsford, MA) and housed 100 under university approved conditions. 101

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Generation of LCMV-vectored HCMV and GPCMV vaccine constructs. rLCMV 102 vectors were generated and titrated as described previously (22, 23). Briefly, the coding 103 sequence (cDNA) of individual vaccine antigens was synthesized by Genscript (USA) 104 and inserted into a plasmid encoding a GP-deleted S segment of LCMV clone 13 under 105 the control of a murine pol I promoter. rLCMV-gB(dCt) encodes the ectodomain and 106 transmembrane domain of HCMV UL55 (gB; Genbank accession AY446894) derived 107 from strain Merlin (amino acids 1-772), followed by an additional arginine residue at 108 position 773 to aid in membrane anchoring. A corresponding vector construct was based 109 on guinea pig CMV gB derived from strain 22122 (Genbank accession: KC503762) and 110 designated rLCMV-GPgB(dCt), encoding the N-terminal 758 amino acids of the protein 111 (Fig. 1B). Analogously, vectors encoding the entire UL83/GP83 (pp65) protein of HCMV 112 strain AD169 or GPCMV strain 22122 were generated and designated rLCMV-pp65 or 113 rLCMV-GPpp65, respectively. Consensus sequencing confirmed transgene sequences 114 before recovering rLCMV vectors using a pol I/pol II rescue system (22). 115

Vector stocks were generated in suspension HEK293 production cells (293-GP) 116 genetically engineered to express the LCMV GP protein. Cells were seeded in shake 117 flasks at 3×105 cells/ml in 30 ml CDM4HEK293 medium (GE Healthcare) supplemented 118 with 4 mM stable Glutamine and 100 µg/ml Geneticin, and infected with rescued vector 119 at an MOI of 0.001. At day 3 post infection, supernatants were cleared from cells and 120 debris by low speed centrifugation (500×g, 5 min at 2-8°C), aliquoted and frozen at <-121 60°C. Vector stocks were titrated using an LCMV NP-specific focus forming unit (FFU) 122 assay as previously described (24). The generation and purification of recombinant 123 GPCMV gB expressed in baculovirus has been described previously (18). 124 Growth kinetics of vaccine vectors. 293-GP or non-complementing HEK293F cells 125 (Invitrogen) were inoculated with vector stocks generated as described above. Aliquots 126

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from individual cultures were drawn at 2, 24, 48, 72 and 96 hours post-infection. 127 Supernatant was cleared by low speed centrifugation, aliquoted and frozen at <-60°C. 128 Individual samples were titrated using by FFU. The wildtype LCMV virus (LCMV-wt) 129 used as a control in the growth curve analyses is a recombinant virus which expresses 130 an identical LCMV GP antigen sequence as the production cell line 293-GP. 131 Protein expression in vector-infected cells. HEK293 suspension cells constitutively 132 expressing LCMV GP (293-GP) were infected with vectors at an MOI of 0.001 and 133 incubated for 72 h. Whole cell lysates were resolved by 4-12% gradient SDS-PAGE 134 (NuPAGE 4-12% Bis-Tris Gel; NOVEX) and transferred to nitrocellulose membranes 135 (NOVEX) using iBlot (Invitrogen). Nonspecific binding sites were blocked by incubation 136 with 5% Blotting-grade Blocker (Bio-Rad Laboratories; Cat: 170-6404) in Tris-buffered 137 saline with 0.1% Tween 20 (1xTBST). Murine monoclonal anti-HCMV gB IgG antibody 138 (Sino Biological; diluted 1:750) that binds the C-terminal part of the gB ectodomain, 139 murine monoclonal anti-HCMV pp65 antibody (ABCAM; diluted 1:200) or a rabbit anti-140 LCMV sera (Prof. Doron Merkler, Faculty of Medicine, Department of Pathology and 141 Immunology; University of Geneva; diluted 1:2,000) were used for western blot 142 analyses. Secondary antibodies included HRP-conjugated donkey anti-mouse IgG 143 antibody (Jackson ImmunoResearch) or donkey anti-rabbit IgG antibody (Jackson 144 ImmunoResearch), respectively. ECL staining was visualized using an Amersham 145 chemiluminiscence reader (Amersham/GE Life Science). 146 Immunization of mice and rabbits. Groups of six-week old female C57BL/6 mice (N=5 147 or 10 animals), obtained from Charles River Laboratories (Châtillon-sur-Chalaronne, 148 France), were immunized at day 0 and boosted on day 21 or day 28 (in one experiment 149 boosted again on day 105) by intramuscular (i.m.) injection of 50 µl of vaccine 150 candidates in both hind legs (total volume 100 µl). rLCMV vectors were prepared in 151

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diluent (25 mM HEPES, 150 mM NaCl, 0.01% Pluronic F68, pH 7.4) with a final 152 concentration of 10% sorbitol and frozen at < -60°C until use. Control animals were 153 immunized with a 1:1 mixture of EM022 adjuvant (Infectious Disease Research Institute; 154 Seattle, WA) and 5 µg recombinant gB protein (Sino Biological) comprising the 155 extracellular domain (Met 1-Lys 700) linked with the cytoplasmic domain (Arg 777-Val 156 907) of the gB protein from human CMV strain Towne. Serum samples and/or spleens 157 were harvested at specified days. Mouse experiments were performed at Preclin 158 Biosystems AG (Epalinges, Switzerland). 159

Groups of five 13-15 week old female New Zealand white rabbits, sourced from 160 Charles River Laboratories (Châtillon-sur-Chalaronne, France), were immunized on day 161 0 and boosted on days 28 and 56 by i.m. injection of 250 µl of vaccine candidates at 4 162 sites (total volume 1000 µl). 163 CMV antibody analysis and mouse anti-HCMV ELISA. Antibody responses against 164 HCMV gB in mouse sera were measured by indirect ELISA using recombinant gB (Sino 165 Biological) as coating antigen on 96-well microplates (0.5 µg/ml, 100 µl per well, 166 overnight at 4°C) and a goat HRP-conjugated anti-mouse IgG polyclonal secondary 167 antibody (Jackson Immuno Research). Serial dilutions of a mouse monoclonal gB-168 specific IgG1 antibody (Sino Biologicals) served as reference standard expressed as 169 µg/ml mAb equivalent concentration. Titers were log-transformed prior to statistical 170 analysis. 171 Mouse IgG subclass-specific anti-HCMV ELISA. At the time of peak antibody 172 response (day 42), mouse IgG subclass-specific antibody responses (IgG1, IgG2b, 173 IgG2c, IgG3) against HCMV gB were measured using a protocol similar to the total IgG 174 mouse ELISA. Serial dilutions of the test sera were applied to the plate, and the endpoint 175 titer was determined as the reciprocal of the highest dilution that produced an 176

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absorbance of at least twice the absorbance of the negative control at that dilution. Goat 177 anti-mouse IgG-subclass-specific HRP-labelled conjugates (Jackson Immuno Research) 178 were used as secondary antibodies. A pool of naïve mouse sera served as a negative 179 control for determination of endpoint titer. Relative proportions of the four IgG 180 subclasses were calculated for each individual mouse using the endpoint titer results. 181 Rabbit IgG anti-HCMV gB ELISA. Antibody responses were determined analogously 182 to the mouse anti-HCMV ELISA, using a purchased rabbit anti-HCMV gB-specific 183 polyclonal antibody (Bioss) as reference standard and a goat anti-rabbit secondary 184 antibody (Jackson Immuno Research). Results were expressed as rabbit ELISA Units 185 (rEU) per ml. 186 Guinea pig anti-GPCMV gB ELISA. Titers were determined by ELISA using 187 recombinant His-tagged GPgB protein produced in CHO cells as target antigen (0.05 188 µg/mL, Evitria) in nickel-coated plates (Qiagen). Analogous to the mouse anti-HCMV 189 ELISA, the guinea pig ELISA titers were defined based on a pool of standard 190 hyperimmune guinea pig serum with an assigned endpoint titer as reference; results 191 were expressed as guinea pig ELISA units (gpEU) per ml. 192 GPCMV-specific neutralization assays. The GFP-tagged recombinant vJZ848 virus 193 was used for neutralization assays as previously reported (21). Neutralization assays 194 were performed on guinea pig fibroblast lung cells (ATCC CCL 158). Neutralizing titers 195 were defined as the dilution resulting in a reduction of ≥50% in the total number of GFP-196 positive foci. A hyperimmune serum from a guinea pig immunized with adjuvanted gB 197 and subsequently infected with GPCMV was used as a positive control. 198 HCMV-specific neutralization assays. Virus neutralization assays were performed at 199 Virginia Commonwealth University in the laboratory of Prof. M. McVoy (25-28) (Fig. 3A), 200

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and by the authors applying an analogous method (Fig. 3B). The virus neutralization 201 procedure is based on human ARPE-19 retinal pigment epithelial cells (ATCC CRL-202 2302) and a green fluorescent protein (GFP) expressing recombinant HCMV virus based 203 on strain Towne (TS15-rN (25). Three-fold serial dilutions of sera (50 μl) in medium were 204 prepared in duplicate in 96-well plates and mixed with 50 μl (300 pfu) TS15-rN in 205 medium containing 10% guinea pig serum (as a source of complement; Merck-Millipore) 206 per well. After incubation at 37 °C for 1 h, 50 μl aliquots were transferred to black-walled 207 clear/flat-bottomed half-area 96-well plates (Corning) that contained sub-confluent 208 ARPE-19 cells in 50 μl medium (MOI 0.02). The number of fluorescent cells was 209 measured 4 days post infection (Bioreader 6000 plate reader, BIO-SYS). A 4-parameter 210 sigmoidal curve was fitted through the data points with the neutralizing titer calculated 211 using GraphPad Prism, version 6.04 and expressed as the reciprocal of the serum 212 dilution that reduced the number of infected cells by 50% compared to medium control 213 (NT-50). 214 HCMV pp65 specific T-cell analytics. A flow-cytometry based intracellular cytokine 215 staining (ICS) assay was used to determine the frequencies of pp65-specific CD8 T-cells 216 expressing IFN-γ, TNF- α, or IL-2. Splenocytes from study animals (2×106 cells) were 217 incubated with a pool of seventeen H-2b restricted pp65 immunoreactive peptides (29) 218 at a concentration of 1 µg/ml per peptide or with medium alone (negative control) and 219 incubated for 1 h at 37°C 5% CO2. Next, 50 µl Brefeldin A (10 µg/ml) was added and 220 incubation was continued for 4 h. Cells were stained with anti-CD8-FITC, anti-CD3-221 PerCP, and anti-CD4-PacBlue (Biolegend) and stained intracellularly with anti-mouse-222 IFN-γ-APC, anti-TNF-α-PE-Cy7 and anti-IL2-PE (Biolegend) and analyzed by flow 223 cytometry (BD LSR II). The gating strategy was: first the gating of lymphocytes (FSC vs. 224 SSC), then the exclusion of doublets (FSC-A vs. FSC-H), followed by the gating of 225

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CD3+/CD4-/CD8+, CD8+ T cells specific for pp65 (defined as those expressing at least 226 one of the three cytokines IFN-γ, TNF-α, and IL2). Background signals obtained after 227 stimulation with medium were subtracted. Cytokine expression was evaluated on two 228 populations and compared by Kruskal-Wallis with Dunn’s multiple comparisons test. 229 HCMV T-cell assays were performed at Preclin Biosystems AG (Epalinges, Switzerland). 230 Vaccine and challenge study design. Five groups of 18 GPCMV-seronegative female 231 guinea pigs were immunized with 8x105 FFU of each vector. These groups consisted of: 232 monovalent rLCMV-GPgB(dCt); monovalent rLCMV-GPpp65; bivalent rLCMV-233 GPgB(dCt) plus rLCMV-GPpp65; recombinant baculovirus-expressed GPgB (truncated 234 at Pro692) admixed with incomplete Freund’s adjuvant (recGPgB + FA); and rLCMV-GFP 235 (negative control). The bivalent vaccine was combined in a single syringe and all 236 vaccines were administered i.m. 50 μg of recombinant baculovirus-expressed GPgB was 237 administered by subcutaneous (sc) administration. All animals were administered a 238 three-dose series at 30-day intervals. Neutralization titers were measured after the third 239 dose. Six animals from the monovalent rLCMV-GPpp65, the bivalent rLCMV-GPgB(dCt) 240 plus rLCMV-GPpp65 group, and the rLCMV-GFP group were sacrificed for ELISPOT 241 analyses 7 days following the second (n=3) or third (n=3) administration (described 242 below). Following vaccination, animals were mated, pregnancy established, and dams 243 challenged mid-gestation with salivary gland-adapted GPCMV (SG-GPCMV) at a dose 244 of 1×105 PFU by sc route (30). Pregnancy outcomes were then monitored. 245 ELISPOT assay. Mouse monoclonal anti-IFNγ antibodies (N-G3 and V-E4) were a gift 246 from Hubert Schäfer (21, 31). ELISPOT was carried out as previously described, with 247 minor modifications (32). Peptides spanned the coding sequence of GP83 in 9 amino 248 acid (aa) long fragments with 5 aa overlaps (140 total peptides). Spleens were harvested 249 from guinea pigs at 28-32 days following the third vaccination, and purified splenocytes 250

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(1 x 105) were mixed with 50 μl stimulant (peptide pools at 20 µg/ml) or controls (no 251 stimulation DMSO control, or positive control concanavalin A at 20 µg/ml). Secondary 252 antibody and developing reagent were added as previously described (32) and spots 253 were counted with an AID Elispot Reader System using Elispot 6.0-iSpot (Autoimmune 254 Diagnostika GmbH, Straßberg, Germany). 255 Real-time qPCR Analysis. Maternal blood obtained on day 7 post-challenge with SG-256 GPCMV was analyzed for viral load by qPCR (30) with the results expressed as the 257 number of genome copies per mL of blood. Since the limit of detection was 258 approximately 200 copies/mL, a level of 100 copies/mL was assigned to negative 259 samples. Organs from stillborn pups or live-born pups sacrificed with 72 hours of 260 delivery (liver, lung and spleen) were homogenized and DNA extracted for qPCR to 261 evaluate for congenital GPCMV transmission as previously described (21). 262 Statistical analyses. GraphPad Prism (version 6.0) was used for statistical analyses. 263 Pup mortality and transmission were compared using Fisher’s exact test with two-sided 264 comparisons. Pup weights in pregnancy/challenge studies and pregnancy duration were 265 compared with Kruskal-Wallis followed by Dunn’s multiple comparison test. Guinea pig 266 serum neutralizing titers and rabbit serum neutralizing titers were compared using 267 ANOVA on log-transformed data. Mouse serum neutralizing antibody titers and ELISA 268 titers were compared using Student’s t-test on log-transformed data. CD8+ T cell 269 responses were compared with Student’s t-test. One-way ANOVA followed by Tukey’s 270 multiple comparisons was used for comparing each group to control for day 7 maternal 271 viremia assessment. Mann-Whitney analysis was employed to compare rLCMV-272 GPgB(dCt) versus the rLCMV-GPgB(dCt)/GPpp65 group. Pooled monovalent groups 273 were compared to the rLCMV-GPgB(dCt)/GPpp65 bivalent group by t-test. 274

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RESULTS 275 Design and generation of rLCMV vaccine vectors. A cDNA rescue system was used 276 to generate replication-incompetent rLCMV vaccine vectors (23) where the LCMV 277 surface glycoprotein was precisely replaced by HCMV or guinea pig CMV vaccine 278 antigens (Fig. 1A). The HCMV gB(dCt) construct encodes the full ectodomain of HCMV 279 gB, including the membrane anchor (33), followed by an extra arginine residue at 280 position 773 (Fig. 1B). Full-length gB is targeted to apical membranes of polarized cells 281 and an acidic cluster of amino acids located on the cytosolic part of the protein serves as 282 a signal for gB re-internalization (34, 35). The gB design of rLCMV-HgB(dCt) aimed to 283 transport gB to the cell surface, but to prevent protein re-internalization, which could 284 reduce immunogenicity. A second vector construct was generated to express the full-285 length sequence of HCMV UL83, encoding the tegument protein pp65. Nuclear 286 localization of pp65 protein after infection was verified using immunofluorescence 287 microscopy (data not shown). Analogous constructs were designed to express proteins 288 derived from guinea pig CMV and designated rLCMV-GPgB(dCt) (Fig. 1B) or rLCMV-289 GPpp65, respectively. 290 Characterization of rLCMV vaccine vectors. Owing to LCMV’s complete dependency 291 on GP for cell entry and thereby for virus propagation, the GP gene product was 292 supplemented during vaccine vector production (Fig. 1C) by a complementing cell line 293 stably expressing GP in trans, (293-GP). To examine the replication competence of 294 vaccine vectors in the production cell line, and conversely demonstrate the inability of 295 the vectors to spread in normal cells, 293-GP and HEK293 cells (293F) were infected 296 with rLCMV-gB(dCt), rLCMV-pp65 and wildtype LCMV virus (LCMV wt) as a control. 297 Both vectors grew efficiently in production cells, reaching titers comparable to wildtype 298 virus (Fig. 2A), whereas no infectious particles were formed in non-complementing 293F 299

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cells infected with the vector rLCMV-gB(dCt) (Fig. 2B). This finding verified that rLCMV 300 vector particles formed in normal cells were GP deficient and replication incompetent, 301 whereas GP trans-complementation in production cells provided for efficient particle 302 assembly of vectors. 303

To confirm expression of the vaccine antigens by rLCMV vectors, lysates of 293-GP 304 cells infected with rLCMV vectors expressing gB(dCt) or pp65 were analyzed by western 305 blot (Fig. 2C). rLCMV vector expressing green fluorescent protein (GFP) was used as 306 control. Native uncleaved gB migrates in the 150 kDa range. During export of the protein 307 to the cell surface, the gB precursor is cleaved by furin and a surface component with an 308 estimated molecular mass of 116 kDa is formed that is linked by disulfide bonds to a 309 transmembrane component with an estimated molecular mass of 55 kDa (36). Vector 310 expressed gB(dCt) appeared as single band in the 125 kDa range corresponding to the 311 uncleaved gB(dCt) precursor, whereas the C-terminal cleavage product was detected in 312 the 40 kDa and 25 kDa range. A predicted band in the ~65 kDa range was detected for 313 the pp65-expressing vector (37) but not in the GFP-expressing control (Fig. 2C). 314 Membranes were also probed with an antibody specific for LCMV NP protein, which 315 yielded bands of expected size and similar intensity for all vectors tested, confirming that 316 comparable amounts of LCMV vector expressed protein products were loaded on gels 317 (Fig. 2C). This analysis confirmed that the vaccine vectors expressed the gB(dCt) and 318 pp65 vaccine antigens in infected cells. 319 Immune response following rLCMV vector vaccination. Mice were immunized in a 320 three dose regimen (day 0, 21 and 105) using 105 FFU of rLCMV-gB(dCt), or 5 µg 321 recombinant gB protein adjuvanted with EM022, which provides a comparator for 322 adjuvant emulsions with composition comparable to MF59 (38). The induction of gB-323 specific antibody responses was analyzed by ELISA (Fig. 3A, left panel). Immunization 324

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stimulated potent gB-specific antibody responses. Responses peaked at day 42 with a 325 GMT of 235 µg/ml and 272 µg/ml serum of gB-specific IgG antibodies for rLCMV-326 gB(dCt) or adjuvanted gB, respectively. Antibody levels were stable during the 9 weeks 327 between the first and second booster. Antibody responses were boosted with a third 328 dose which increased gB-specific IgG levels from 94 µg/ml to 172 µg/ml serum for 329 LCMV-gB(dCt) and 128 µg/ml to 290 µg/ml serum for adjuvanted gB 330

Mouse sera obtained at day 42 were chosen for determination of HCMV neutralizing 331 antibody titers. These studies demonstrated a significantly higher neutralization capacity 332 of sera induced by rLCMV-gB(dCt) compared to adjuvanted recombinant gB protein and 333 a slightly higher neutralization capacity than the mean response of human convalescent 334 sera used as a benchmark for this analysis (Fig. 3A, right panel). 335

Neutralizing antibody titers directed against gB are significantly augmented by 336 adding complement in HCMV neutralization assays (39). This prompted us to analyze 337 the subclass of antibodies induced by vaccination, insofar as different subtypes of IgG 338 antibodies differ in their ability to activate complement. ELISA analysis of peak titer 339 mouse sera (d42) containing similar levels of total gB-specific IgG using IgG subclass-340 specific secondary antibodies indicated that animals vaccinated with adjuvanted 341 recombinant gB predominantly generated IgG1 antibodies (85%) which only poorly 342 activate complement, whereas the rLCMV vector induced mainly gB-specific IgG2b and 343 IgG2c antibodies (40% and 54%, respectively) which efficiently activate complement 344 (Table 1). 345

Vaccinated rabbits demonstrated potent and dose-dependent induction of gB-346 specific antibodies exhibiting HCMV neutralizing capacity in the medium and high dose 347 range (Fig. 3B). 348

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To analyze the immunogenicity of the rLCMV vector expressing the T-cell antigen 349 pp65, mice were vaccinated i.m. utilizing 105 or 103 FFU on days 0 and 28 of the 350 experiment. Vaccination with 105 FFU induced robust pp65-specific CD8+ responses, 351 with a mean magnitude of 1.8% pp65-specific CD8+ cells after prime and 3.0% after a 352 booster vaccination (Fig. 3C). pp65-specific T-cells were also detected in the low-dose 353 group, reaching mean levels of 0.86% pp65-specific CD8+ cells after prime or 1.0% after 354 boost, respectively. 355 Interference in bivalent vector composition. Observations suggesting interference 356 between gB and pp65 antigens in previous studies (21, 40) prompted us to analyze if 357 any interference was observed when rLCMV-gB(dCt) and rLCMV-pp65 vaccines were 358 co-administered compared to monovalent immunization. Immunogenicity of monovalent 359 (rLCMV-gB(dCt) or rLCMV-pp65) and bivalent (rLCMV-gB(dCt) plus rLCMV-pp65 mixed 360 in 1:1 ratio) was analyzed after i.m. vaccination of C57BL/6 mice using a dose of 105 361 FFU per vector on days 0 and 28. gB-specific IgG ELISA analysis was conducted using 362 sera sourced on day 49 and indicated no significant difference in magnitude of gB-363 specific antibody induction in the monovalent versus bivalent vaccination (Fig. 3D, left 364 panel). Analogously, determination of IFN-γ, TNF-α, or IL-2 producing pp65-specific 365 CD8+ responses indicated no significant difference between the monovalent versus 366 bivalent vaccines (Fig. 3D, right panel), confirming no interference when vaccine was 367 administered as a bivalent combination vaccine. 368 Immunogenicity and protective efficacy of rLCMV vectors expressing gB(dCt) and 369 pp65 from guinea pig CMV. Antibody responses measured by ELISA in guinea pig 370 dams vaccinated with constructs expressing GPCMV antigens homologous to the 371 HCMV-specific antigens demonstrated excellent immunogenicity with strong and uniform 372 (narrow 95% CI) antibody responses to GPgB(dCt) in both the monovalent and bivalent 373

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vaccines, in agreement with results from mouse studies (Fig. 4A). The rLCMV-374 GPgB(dCt) vaccine elicited robust neutralizing antibody titers alone and when 375 administered in combination with rLCMV-GPpp65, indicating no interference between 376 antigens (22; Fig. 4B, in both cases p<0.0001 compared to irrelevant vector control). 377 Indeed, animals receiving the bivalent combination of rLCMV-GPgB(dCt) and rLCMV-378 GPpp65 had a significantly (p=0.0006) stronger neutralizing response than animals 379 vaccinated with rLCMV-GPgB(dCt). Consistent with the ELISA data, sera from animals 380 immunized with rLCMV-GPgB(dCt) were significantly more neutralizing than sera from 381 animals immunized with adjuvanted GPgB protein (p<0.0001). 382

Splenocytes from animals immunized with rLCMV-GPpp65 showed stronger 383 reactivity in ELISPOT assays than cells from control animals receiving a vector 384 expressing an irrelevant protein (rLCMV-GFP) (Fig. 4C). There was not a significant 385 difference in reactivity between splenocytes from animals immunized with monovalent 386 rLCMV-GPpp65 and animals immunized with the bivalent vaccine. Peptide mapping of 387 GP83 epitopes following rLCMV-GPpp65 vaccination was comparable to that observed 388 during natural GPCMV infection (data not shown). Responses to two previously 389 identified immunodominant peptides (LGIVHFFDN and CQEQVFVKS) were observed 390 following vaccination (32). 391 Reduction of viral load following rLCMV-vectored vaccination. Viral loads in all 392 vaccinated groups were significantly lower at day 7 compared to rLCMV-GFP control. 393 The viral load after rLCMV-GPgB(dCt) was 1.6±0.3×106 copies/mL; compared to 394 1.7±0.6×106 copies/mL in the rLCMV-GPpp65 group; 5.8±3.1×105 copies/mL in the 395 bivalent rLCMV-GPgB(dCt) plus rLCMV-GPpp65 group; 1.1±0.6×106 copies/mL in the 396 GPgB/Freund’s adjuvant group; and 4.5±1.0×106 copies/mL in rLCMV559 GFP controls 397

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(Fig. 5A). Administration of the bivalent rLCMV-gB(dCt)/GPpp65 demonstrated a 398 significant reduction of viral load when compared with rLCMV gB(dCt) vaccine 399 administered alone (p=0.0386, Fig. 5A). The analysis of reduction in viral load after the 400 bivalent vaccine when compared with rLCMV GPpp65 vaccine alone did not 401 demonstrate statistical significance (p=0.07). However, the viral load of 1.65±0.3×106 402 copies/mL when the single-antigen vaccine groups (rLCMV-GPgB(dCt) and rLCMV-403 GPpp65) were combined was significantly higher than that of the bivalent rLCMV-404 GPgB(dCt)/GPpp65 combination group (p = 0.03, t-test, Fig. 5B). 405 Protection against mortality and disease following rLCMV-vectored vaccination. 406 Preconception vaccination resulted in a significant decrease in pup mortality after 407 challenge (Table 2). The mortality rate was 13% in the rLCMV-GPgB(dCt) group, 39% in 408 rLCMV-GPpp65, 8% in bivalent rLCMV-GPgB(dCt)/GPpp65, 20% in recGPgB/Freund’s 409 adjuvant, and 93% in the rLCMV-GFP control group. Furthermore, the mortality rate in 410 pups born to dams vaccinated with single antigen rLCMV-GPpp65 was significantly 411 higher than those born to the rLCMV-GPgB(dCt) vaccinated group (p = 0.0059), and to 412 those born to bivalent rLCMV-GPgB(dCt)/GPpp65 vaccinated group (p = 0.0024, 413 Fisher’s exact test). The were no significant differences in mortality between the rLCMV-414 GPgB(dCt) (13%) and rLCMV-GPgB(dCt)/GPpp65 (8%) groups. Pup weights were also 415 significantly higher in the vaccinated versus rLCMV-GFP control groups (Fig. 6). 416

Duration of pregnancy following SG-GPCMV challenge in the control group was 417 13.4±2.6 days, which was significantly lower (p<0.01, Kruskal-Wallis) than all of the 418 vaccine groups (23.9±2.4 in rLCMV-GPgB(dCt) group; 25.3±4 in rLCMV-GPpp65 group; 419 33.9±6.4 in the bivalent rLCMV-GPgB(dCt)/GPpp65 group; and 35.1±4.4 in the recGPgB 420 + FA group). In the rLCMV-GPgB(dCt) vaccine group, 41/55 pups (75%), compared to 421 22/33 (67%) in the rLCMV-GPpp65 group, had congenital GPCMV infection, as 422

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evidenced by positive PCR detection in pup lung, liver, or spleen. Congenital infection 423 was observed in 22/40 (55%) of the recGPgB + FA group, and in 20/40 (50%) of pups in 424 the control group. In the bivalent rLCMV-GPgB(dCt)/GPpp65 vaccine group, congenital 425 GPCMV infection was observed in 18/38 pups (53%). One pup from the control group 426 and six pups from the rLCMV-GPpp65 were unavailable for PCR testing. When the 427 bivalent rLCMV-GPgB(dCt)/GPpp65 group was compared to both monovalent LCMV 428 vector groups combined, the rate of congenital transmission was noted to be significantly 429 reduced (53% compared to 72%; p<0.05, Fisher’s exact test). 430 431 DISCUSSION 432 Vaccines against HCMV-associated disease targeting both congenital infection (41) and 433 HCMV-associated disease in the solid organ and hematopoietic stem cell transplant 434 patients are needed (42, 43). All vectored and subunit vaccines evaluated in clinical 435 trials to date have included the immunodominant gB glycoprotein, and several have 436 included the dominant T cell target, pp65 (ppUL83), with or without the major immediate 437 early protein-1 (IE1) gene product (4-7). However, it is not known whether inclusion of 438 T-cell targets improves protective efficacy compared to gB administered alone. 439 Moreover, one study using a recombinant MVA system suggested, in a guinea pig 440 model, that inclusion of the pp65 homolog in a two-component vaccine decreased the 441 immunogenicity of gB, compared to MVA-vectored gB administered alone (21). In the 442 present study, we demonstrated that immunization with both HCMV and GPCMV gB and 443 pp65 rLCMV vectors did not show interference; moreover, there was a significant 444 additive benefit of both gB and pp65, both in reducing the magnitude of maternal viremia 445 following viral challenge and in improving pregnancy and pup outcomes. 446

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The vaccines described in this report are based on a recombinant clone 13 strain of 447 LCMV that exhibits a natural tropism for dendritic cells, and elicits both robust CD8+ T 448 cell and antibody responses (23). Vectors were generated by replacing the essential GP 449 gene with the HCMV or GPCMV antigens of interest. Previous work with rLCMV 450 vectored vaccines elicited responses that were equivalent or superior to those elicited by 451 recombinant adenovirus 5 or recombinant vaccinia virus, both in magnitude of CTL 452 response and cytokine profiles. These rLCMV vaccines were also more protective in 453 several models, including a Listeria monocytogenes challenge model (23). Importantly, 454 in contrast to recombinant adenovirus 5, rLCMV has been previously shown not to elicit 455 any vector-specific antibody immunity, allowing for the potential of re-administration for 456 booster vaccination (23). This would be of particular relevance for a maternal HCMV 457 vaccine, where periodic booster immunizations of women of child-bearing age may be 458 required to confer protection against re-infection and subsequent congenital 459 transmission (44) throughout their childbearing years. 460

rLCMV vectors expressing the HCMV antigens gB(dCt) and pp65 elicited robust, 461 antigen-specific humoral and cellular immune responses in mice and rabbits in a dose-462 dependent manner and the gB-binding antibody response was comparable in magnitude 463 and durability to adjuvanted subunit gB protein. However, the HCMV-neutralizing titers 464 of mice immunized with rLCMV-gB(dCt) exceeded responses of mice immunized with 465 adjuvanted gB. 466

In contrast to a previous report by Cardin (24), the current study demonstrated that 467 gB-containing LCMV vaccines were superior to GPpp65 LCMV vaccines in protecting 468 against pup mortality and in reducing pup viral load. Cardin et al. had observed 23% pup 469 mortality in the GPgB vaccinated group and 26% in the pp65 vaccinated group, 470 compared to 49% mortality in control pups (24). In the present study, we observed a 471

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93% pup mortality in the control group, which was reduced to 39% in the GPpp65 472 vaccinated and 13% in the GPgB vaccinated groups. One reason for these differences 473 might be the improved immunogenicity of the GPgB vaccine, since the GPCMV HK1-474 GPgB(dTM) LCMV vector construct used in the Cardin study encoded a 475 transmembrane-deleted protein, comprising the entire extracellular domain of GPCMV 476 gB (through Arg685) and the entire intracellular domain (aa 762-901). In contrast, the dCt 477 construct in the currently reported study was a C-terminally truncated gB consisting of 478 the N-terminal 758 amino acids of gB (encoding the predicted full ectodomain of gB) and 479 the membrane anchor that transports gB to the cell surface and prevents protein re-480 internalization via deletion of cytosolic internalization signals (33-35). Although we 481 propose that the enhanced immunogenicity of gB in the dCt construct was responsible 482 for the improved pregnancy outcomes observed when compared to the monovalent 483 rLCMV-GPpp65 vaccine, the ELISA and neutralization assays were performed using 484 different methodologies, making direct comparisons of the serologic results between the 485 two studies problematic. 486

Notably, immunization with bivalent rLCMV-GPgB(dCt) plus rLCMV-GPpp65 resulted 487 in a statistically significant reduction of maternal DNAemia, and reduced pup mortality, 488 when compared to monovalent vaccination with either vaccine alone. Although there 489 have been no side-by-side comparisons of single-antigen HCMV gB vaccines with multi-490 antigen gB and pp65 vaccines in clinical trials using acquisition of HCMV infection as a 491 study endpoint, it is noteworthy that monovalent adjuvanted recombinant gB vaccine has 492 demonstrated only modest efficacy, in the range of 40-50%, in clinical trials of 493 adolescents and young women (9, 10). Since a major goal of vaccination is to confer 494 protection against HCMV transmission in infants born to young women of childbearing 495

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age, the additive benefit of combining both gB and pp65 in the guinea pig model may be 496 relevant to future bivalent HCMV vaccines to be evaluated in clinical trials. 497

Our study is in marked contrast to a prior study of MVA-vectored gB and pp65 498 vaccines, where it was observed that the addition of pp65 to a gB subunit vaccine not 499 only interfered with antibody responses, but also correlated with diminished protection 500 against congenital GPCMV infection, compared to gB vaccine given alone (21). 501 Interestingly in our study, the neutralizing response was significantly higher in animals 502 administered the bivalent vector vaccine compared to animals received only rLCMV-503 GBgB(dCt). This may have been due to the higher total vector dose in the bivalent 504 vaccine group that contributed to the stronger overall immune response, although these 505 differences were not observed in the ELISA assay. 506

In conclusion, these data demonstrate that both vectors, LCMV-gB(dCt) and LCMV-507 pp65, are highly immunogenic for both HCMV and GPCMV antigens. Immunization with 508 the HCMV vaccine constructs induces robust CMV gB-specific neutralizing antibody 509 responses and robust pp65-specific IFN-y producing CD8+ responses in mice. Similarly, 510 an LCMV-vectored vaccine based on the GPCMV gB homolog induces high-titer ELISA 511 and neutralizing antibody responses, with superior immunogenicity when compared to 512 gB with Freund’s adjuvant, in guinea pigs. The GPCMV rLCMV-GPpp65 vaccine 513 construct was demonstrated to induce T cell responses, as measured by an IFNγ 514 ELISPOT assay, in guinea pigs. Finally, this study demonstrated an additive benefit of 515 combining both a neutralizing antibody target (gB) and a T-cell target (GP83, pp65 516 homolog) in a bivalent vaccine strategy targeting prevention of maternal and fetal 517 disease in the GPCMV model, and supports further testing of rLCMV-based HCMV 518 vaccines in clinical trials. 519

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ACKNOWLEDGEMENTS 521 This work was supported by grants R03HD082273, T32HD068229 and R56AI114013 522 and R01s HD044864 and HD079918 from the NIH, and by the Austrian Research 523 Promotion Agency (Die Österreichische Forschungsförderungsgesellschaft, FFG). The 524 authors thank Dr. Mike McVoy, Medical College of Virginia/Virginia Commonwealth 525 University, for performing neutralization assays and providing reagents. 526 527 Manuscript Word Count: 5276 528 529 530 531 532 533 534 535 536 537 538 539 540

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27. Saccoccio FM, Gallagher MK, Adler SP, McVoy MA. 2011. Neutralizing activity of 631 saliva against cytomegalovirus. Vaccine 33:7328-36. 632 28. Saccoccio FM, Sauer AL, Cui X, Armstrong AE, Habib el-SE, Johnson DC, 633 Ryckman BJ, Klingelhutz AJ, Adler SP, McVoy MA. 2011. Peptides from 634 cytomegalovirus UL130 and UL131 proteins induce high titer antibodies that block viral 635 entry into mucosal epithelial cells. Vaccine 29:2705-11. 636 29. Shedlock DJ, Talbott KT, Wu SJ, Wilson CM, Muthumani K, Boyer JD, Sardesai 637 NY, Awasthi S, Weiner DB. 2012. Vaccination with synthetic constructs expressing 638 cytomegalovirus immunogens is highly T cell immunogenic in mice. Hum Vaccin 639 Immunother 8:1668-81. 640 30. Schleiss MR, Bierle CJ, Swanson EC, McVoy MA, Wang JB, Al-Mahdi Z, 641 Geballe AP. 2015. Vaccination with a live attenuated cytomegalovirus devoid of a 642 protein kinase R inhibitory gene results in reduced maternal viremia and improved 643 pregnancy outcome in a guinea pig congenital infection model. J Virol 89:9727-38. 644 31. Schäfer H, Kliem G, Kropp B, Burger R. 2007. Monoclonal antibodies to guinea pig 645 interferon-gamma: tools for cytokine detection and neutralization. J Immunol Methods 646 328:106-17. 647 32. Gillis PA, Hernandez-Alvarado N, Gnanandarajah JS, Wussow F, Diamond DJ, 648 Schleiss MR. 2014. Development of a novel, guinea pig-specific IFN-γ ELISPOT assay 649 and characterization of guinea pig cytomegalovirus GP83-specific cellular immune 650 responses following immunization with a modified vaccinia virus Ankara (MVA)-vectored 651 GP83 vaccine. Vaccine 32:3963-70. 652

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33. Tugizov S, Maidji E, Xiao J, Zheng Z, Pereira L. 1998. Human cytomegalovirus 653 glycoprotein B contains autonomous determinants for vectorial targeting to apical 654 membranes of polarized epithelial cells. J Virol 72:7374-86. 655 34. Radsak K, Eickmann M, Mockenhaupt T, Bogner E, Kern H, Eis-Hübinger A, 656 Reschke M. 1996. Retrieval of human cytomegalovirus glycoprotein B from the infected 657 cell surface for virus envelopment. Arch Virol 141:557-72. 658 35. Tugizov S, Maidji E, Xiao J, Pereira L. 1999. An acidic cluster in the cytosolic 659 domain of human cytomegalovirus glycoprotein B is a signal for endocytosis from the 660 plasma membrane. J Virol 73:8677-88. 661 36. Spaete RR, Thayer RM, Probert WS, Masiarz FR, Chamberlain SH, Rasmussen 662 L, Merigan TC, Pachl C. 1988. Human cytomegalovirus strain Towne glycoprotein B is 663 processed by proteolytic cleavage. Virology 167:207-25. 664 37. Nowak B, Gmeiner A, Sarnow P, Levine AJ, Fleckenstein B. 1984. Physical 665 mapping of human cytomegalovirus genes: identification of DNA sequences coding for a 666 virion phosphoprotein of 71 kDa and a viral 65-kDa polypeptide. Virology 134:91-102. 667 38. Fox CB, Haensler J. 2013. An update on safety and immunogenicity of vaccines 668 containing emulsion-based adjuvants. Expert Rev Vaccines 12:747-58. 669 39. Britt WJ, Vugler L, Stephens EB. 1988. Induction of complement-dependent and -670 independent neutralizing antibodies by recombinant-derived human cytomegalovirus 671 gp55-116 (gB). J Virol 62:3309-18. 672 40. Selinsky C, Luke C, Wloch M, Geall A, Hermanson G, Kaslow D, Evans T. 2005. 673 A DNA-based vaccine for the prevention of human cytomegalovirus-associated 674 diseases. Hum Vaccin 1:16-23. 675

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41. Swanson EC, Schleiss MR. 2013. Congenital cytomegalovirus infection: new 676 prospects for prevention and therapy. Pediatr Clin North Am 60:335-49. 677 42. Beam E, Dioverti V, Razonable RR. 2014. Emerging cytomegalovirus management 678 strategies after solid organ transplantation: challenges and opportunities. Curr Infect Dis 679 Rep 16:419. 680 43. Boeckh M, Gilbert PB. 2016. Search continues for a CMV vaccine for transplant 681 recipients. Lancet Haematol 3:e58-9. 682 44. Ross SA, Arora N, Novak Z, Fowler KB, Britt WJ, Boppana SB. 2010. 683 Cytomegalovirus reinfections in healthy seroimmune women. J Infect Dis 201:386-9. 684 685 686 687 688 689 690 691 692 693 694 695 696

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697 698 % IgG1 % IgG2b % IgG2c % IgG3 Rec. gB + Adj 85 11 3.8 0.1

rLCMV-gB(dCt) 4.0 40 54 1.5

699 Mean percentages calculated from subclass titers for individual animals 700 e.g.: % IgG1 = 100 x IgG1 / (IgG1 + IgG2b + IgG2c + IgG3) 701 702

Table 1: IgG subclass analysis 703 704 705 706 707 708 709 710 711 712 713 714 715

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716 717 718

Vaccine Group Total Litters

Live Pups

Dead Pups

Mortality

rLCMV-GPgB(dCt)

17 48 7 13%*

rLCMV-GPpp65 10 24 15 39%*

rLCMV-GPgB(dCt) plus rLCMV-GPpp65

(Combined)

12 35 3 8%*∫

recGPgB + FA 13 32 8 20%*

rLCMV-GFP 11 3 38 93%

719 720

* p<0.0001 v. rLCMV-GFP 721 ∫ p<0.01 v. rLCMV-GPgB(dCt) 722 723 Table 2: Mortality comparisons 724

725 726 727 728

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729 FIGURE LEGENDS 730 731 Figure 1: Schematic drawing of rLCMV vector design and production. (A) LCMV vector 732 particle and encapsidated genomic segments; GP: surface Glycoprotein; NP: 733 nucleoprotein; Z: RING finger protein; L: Polymerase; and genetic organization of the 734 two encapsidated genomic segments; S: Short; L: Long. (B) Representation of the full-735 length HCMV gB open reading frame HgB(FL), the truncated isoform HgB(dCt) and the 736 corresponding design for guinea pig CMV gB (GPgB(dCt)) expressed in vaccine vectors. 737 Numbers represent amino acid positions and shaded boxes represent the proposed 738 transmembrane region of the protein. Position 773 in HgB(dCt) is an extraneous 739 arginine residue. (C) Schematic drawing of rLCMV vaccine vector rescue, stock 740 production and single round infectious character of rLCMV vectored vaccination. 741 Figure 2: rLCMV vector characterization. (A) Growth kinetics of rLCMV vectors and 742 LCMV wildtype virus in the production cell line 293-GP. Titers represent means of 2 743 independent experiments, with standard deviations (error bars). (B) rLCMV-gB(dCt) was 744 selected as representative vector to demonstrate deficiency in formation of infectious 745 progeny in cells that do not provide LCMV GP protein in trans (293F). All cell types were 746 infected with 0.001 FFU/cell. Samples for individual time points were analyzed by means 747 of a focus forming unit assay (FFU) based on LCMV GP complementing 293T cells. 748 Dotted lines indicate detection limits of the FFU assay. (C) gB(dCt) and pp65 vaccine 749 antigen expression was analyzed by Western blot. HCMV gB and HCMV pp65-specific 750 monoclonal antibodies were used to detect vaccine antigen expression and a polyclonal 751 anti-LCMV serum reactive with LCMV NP was used for the infection/loading control. 752

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Figure 3. Immunogenicity analyses of rLCMV-gB and pp65 vectors. Error bars represent 753 95% confidence intervals for serological assays, and standard deviation for T-cell 754 analyses. (A) Immunogenicity in C57BL/6 mice: 1 x 105 FFU per dose of rLCMV or 5µg 755 per dose of adjuvanted recombinant gB were administered i.m. on days 0 and 21; Left: 756 Antibody induction and persistence measured by ELISA.; Right: Levels of neutralizing 757 antibodies in sera collected on day 42 after two administrations of rLCMV-gB(dCT) or 758 adjuvanted recombinant gB, in comparison to 5 human sera from CMV infected subjects 759 which cover a typical range of titers for naturally induced humoral immunity. 760 (B) Immunogenicity in New Zealand White rabbits: 1×107 FFU, 1×105 FFU, or 1×103 761 FFU of rLCMV-gB(dCt) was administered i.m. on days 0, 28 and 56; Left: Antibody 762 induction and persistence measured by ELISA; Right: Levels of neutralizing antibodies 763 in sera collected on day 69 after three administrations of rLCMV-gB(dCt) (C) T-cell 764 responses induced by pp65 vector in C57BL/6 mice; a dose of 1×105 or 1×103 FFU 765 was administered i.m. on days 0 and 28; T-cell analysis of splenocytes was performed at 766 day 10 (d10 post prime) and day 38 (day 10 post boost) by ICS. Frequencies of pp65-767 specific CD8 T-cells expressing at least one of the cytokines IFN-γ, TNF-α or IL2 are 768 shown. (D) Immunogenicity of monovalent and bivalent rLCMV formulations in C57BL/6; 769 1x105 FFU of rLCMV vector were administered i.m. on days 0 and d28; gB-specific 770 antibody induction and pp65-specific CD8 T-cell responses were measured at day 49 by 771 ELISA and ICS. 772 Figure 4. Immunogenicity analyses of rLCMV GPCMV vectors in guinea pigs. Error bars 773 represent 95% confidence intervals. 8x105 FFU per dose of each rLCMV vector were 774 administered i.m. or 50 µg per dose of adjuvanted recombinant GPgB was administered 775 s.c. on days 0, 30 and 60. Animals were challenged with 1×105 PFU GPCMV after day 776 155. (A) Antibody induction measured by ELISA. (B) Neutralizing titers in sera at day 777

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103. The titers in individual sera are shown with geometric mean and 95% CI indicated. 778 Sera without neutralizing effect at the lowest dilution were assigned a titer of half the 779 LOD. Hyperimmune serum was derived from an animal immunized with adjuvanted 780 recombinant gB protein (50 μg in IFA) and subsequently infected with GPCMV. 781 Statistical comparisons were made by ANOVA analysis on log-transformed values. 782 783 Figure 5. Reduction of maternal viral load following rLCMV-vectored vaccination. (A) 784 Mean (±SEM) maternal viral loads were analyzed by qPCR in vaccine and control 785 groups at 7 days following SG-GPCMV challenge. All vaccine groups had significantly 786 lower viral loads at day 7 compared to the rLCMV-GFP control (*p<0.05). In addition, 787 maternal viral load at day 7 was significantly reduced by the combination of rLCMV-788 vectored gB(dCt) and GPpp65 compared with rLCMV gB(dCt) vaccine administered 789 alone (**p=0.0386). (B) Immunization with both gB and pp65 (GP83) confers 790 significantly improved protection compared to either antigen administered alone. 791 Combined analysis of single-antigen vaccine groups rLCMV-GPgB(dCt) and rLCMV-792 GPpp65 identified mean day 7 magnitude of DNAemia of 1.65±0.3×106 copies/mL, 793 compared to 5.8±3×105 copies/mL in the bivalent rLCMV-GPgB(dCt)/GPpp65 group (p = 794 0.03, t-test). 795 796 Figure 6. Improved pup weights and reduced pup mortality conferred by pre-conception 797 vaccination. Pup weights were significantly improved in all vaccine groups compared to 798 the rLCMV-GFP control group (p < 0.0001, Kruskal-Wallis and Dunn’s multiple 799 comparison tests). Open circles, live-born pups; closed circles, still-born pups. 800

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Figure 1

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Figure 2

A

B

C

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Figure 3

A

B

C

D

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Figure 4

A

B

C

rLC

MV

-GFP

rLC

MV

-G

Pp

p65

rLC

MV

-G

PfB

(dC

t) +

rL

CM

V-

GP

pp65

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Figure 5

A

B

Gro

up 1 [r

LCM

V-

GPgB

(dCt)] +

Gro

up 2

[rLC

MV-G

Ppp65

]

Gro

up 3 [r

LCM

V-

GPgB

(dCt)

+ rL

CM

V-

GPpp65

]

0

2×106

4×106

6×106

CM

V g

en

om

e c

op

ies/m

L

* p = 0.03

rLCMV-G

PgB(dCt)

rLCMV-G

Ppp65

rLCMV-G

PgB(dCt)

+ rLCMV-G

Ppp65

gB + F

reund's

rLCMV-G

FP101

102

103

104

105

106

107

108

CM

V g

en

om

e c

op

ies/m

L

*

** p = 0.0386

nsns ns

nsns

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Figure 6

****p < 0.0001, Kruskal-Wallis and Dunn’s multiple comparison tests

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