ceacba352a419d91a8e1039a43f8dc7a pmc_11359035.pdf 876868f1ca160e2470017192261b3850ccd55fcc pmc_11359035.pdf 32bc291b24052fe012735ceaec6d6173bf69f350e17e0c1b536c6f9b2a57c4de pmc_11359035.pdf Title: FlagT4G Vaccine Prevents Transplacental Transmission of Highly Virulent Classical Swine Fever Virus after Single Vaccination in Pregnant Sows Subject: The transplacental transmission of CSFV and the resulting persistent congenital infection in newborn piglets have been abundantly discussed in pregnant sows suffering from virus infection. Importantly, the availability of safe commercial vaccines with proven efficacy to prevent the generation of congenital and postnatal persistent infections in pregnant sows are critical tools for controlling the disease in CSF endemic areas. Here, we demonstrate the high efficacy of a single dose of the recombinant FlagT4G vaccine to provide solid protection in pregnant sows against transplacental transmission of a highly virulent CSFV. Pregnant sows vaccinated with FlagT4G at 44 days of gestation elicited a strong CSFV-specific antibody response, with neutralizing antibody levels above those required for protection against CSFV. Importantly, after the challenge with a highly virulent CSFV, all foetuses from FlagT4G-vaccinated sows lacked CSF macroscopic lesions and showed a complete absence of the challenge virus in their internal organs at day 79 of gestation. Therefore, pregnant sows safely vaccinated with FlagT4G without affecting reproductive efficacy are efficaciously protected, along with their foetuses, against the infection and disease caused by a CSFV virulent field strain. Keywords: classical swine fever virus; transplacental transmission; FlagT4G vaccine; protection; neutralizing antibodies; immune response; vaccine efficacy Author: Liani Coronado, Adriana Muñoz-Aguilera, Guillermo Cantero, Patricia Martínez, Mònica Alberch, Rosa Rosell, Douglas P. Gladue, Manuel V. Borca and Llilianne Ganges Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Wed Jul 24 11:20:29 2024 CEST ModDate: Wed Jul 24 11:33:38 2024 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3107027 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- IOVOOE+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 TCHIDI+URWPalladioL-Bold Type 1 Custom yes yes yes 16 0 CJQEJR+URWPalladioL-Roma Type 1 Custom yes yes yes 22 0 NVNKTU+URWPalladioL-Ital Type 1 Custom yes yes yes 27 0 OPZDHV+VnURWPalladioL Type 1 Custom yes yes yes 32 0 AAPCWM+CMSY10 Type 1 Builtin yes yes yes 62 0 ZXDKIP+EURM10 Type 1 Builtin yes yes yes 70 0 IOLAWT+PazoMath-Italic Type 1 Builtin yes yes yes 78 0 KBGMGA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 89 0 KBGMGB+PalatinoLinotype CID TrueType Identity-H yes yes yes 92 0 KBGMGC+PalatinoLinotype TrueType WinAnsi yes yes no 98 0 KBGMGE+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 101 0 KBGMOC+PalatinoLinotype TrueType MacRoman yes yes no 104 0 KBGOAM+PalatinoLinotype,Italic TrueType MacRoman yes yes no 107 0 KBGMGA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 114 0 KBGMGB+PalatinoLinotype CID TrueType Identity-H yes yes yes 117 0 KBGMGC+PalatinoLinotype TrueType WinAnsi yes yes no 123 0 KBGMGE+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 126 0 KBGMGA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 140 0 KBGMGB+PalatinoLinotype CID TrueType Identity-H yes yes yes 143 0 KBGMGC+PalatinoLinotype TrueType WinAnsi yes yes no 149 0 KBGMGE+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 152 0 KBGMGA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 175 0 KBGMGB+PalatinoLinotype CID TrueType Identity-H yes yes yes 178 0 KBGMGC+PalatinoLinotype TrueType WinAnsi yes yes no 184 0 KBGMGE+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 187 0 KBGMOC+PalatinoLinotype TrueType MacRoman yes yes no 190 0 KBGMGA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 199 0 KBGMGB+PalatinoLinotype CID TrueType Identity-H yes yes yes 202 0 KBGMGC+PalatinoLinotype TrueType WinAnsi yes yes no 208 0 KBGMGE+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 211 0 KBGMGA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 235 0 KBGMGB+PalatinoLinotype CID TrueType Identity-H yes yes yes 238 0 KBGMGC+PalatinoLinotype TrueType WinAnsi yes yes no 244 0 KBGMGE+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 247 0 KBGMOC+PalatinoLinotype TrueType MacRoman yes yes no 250 0 KBGMGA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 263 0 KBGMGB+PalatinoLinotype CID TrueType Identity-H yes yes yes 266 0 KBGMGC+PalatinoLinotype TrueType WinAnsi yes yes no 272 0 KBGMGE+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 275 0 KBGMOC+PalatinoLinotype TrueType MacRoman yes yes no 278 0 KBGMGA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 293 0 KBGMGB+PalatinoLinotype CID TrueType Identity-H yes yes yes 296 0 KBGMGC+PalatinoLinotype TrueType WinAnsi yes yes no 302 0 KBGMGD+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 305 0 KBGMGE+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 311 0 KBGMOC+PalatinoLinotype TrueType MacRoman yes yes no 314 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-09-19 02:49:23 CEST RepresentationInformation: pmc_11359035.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-09-18 02:30:46 CEST Size: 3107027 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 524 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Cells and Viruses Destination: subsection.2.1 Item: Title: Experimental Design Destination: subsection.2.2 Item: Title: Detection of Antibody Responses by ELISA and Virus Neutralization Tests Destination: subsection.2.3 Item: Title: CSFV RNA Detection Destination: subsection.2.4 Item: Title: Determination of IFN- Levels in Serum by ELISA Test Destination: subsection.2.5 Item: Title: CSFV Isolation in Tonsils Samples Destination: subsection.2.6 Item: Title: Results Destination: section.3 Children: Item: Title: FlagT4G Vaccine Protects Pregnant Sows from Clinical Signs after Challenge with a Highly Virulent CSFV Destination: subsection.3.1 Item: Title: FlagT4G Elicited Strong CSFV-Specific Antibody in Vaccinated Pregnant Sows Destination: subsection.3.2 Item: Title: The CSFV-Induced Interferon Alpha-Exacerbated Response Is Prevented by FlagT4G Vaccine Destination: subsection.3.3 Item: Title: FlagT4G Vaccine Avoids Systemic and Tissue Replication of the Highly Virulent Challenge Virus Destination: subsection.3.4 Item: Title: FlagT4G Vaccine Afforded Total Protection from CSFV Highly Virulent Challenge in the Foetuses from Vaccinated Sows Destination: subsection.3.5 Item: Title: FlagT4G Vaccine Colonizes the Foetuses from Vaccinated Sows with a High CSFV RNA Load at Systemic and Tissue Levels Destination: subsection.3.6 Item: Title: CSFV Antibody and IFN- Responses Were Not Found in the Foetuses from Vaccinated Sows Destination: subsection.3.7 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.6 Info: Title: FlagT4G Vaccine Prevents Transplacental Transmission of Highly Virulent Classical Swine Fever Virus after Single Vaccination in Pregnant Sows Author: Liani Coronado, Adriana Muñoz-Aguilera, Guillermo Cantero, Patricia Martínez, Mònica Alberch, Rosa Rosell, Douglas P. Gladue, Manuel V. Borca and Llilianne Ganges Subject: The transplacental transmission of CSFV and the resulting persistent congenital infection in newborn piglets have been abundantly discussed in pregnant sows suffering from virus infection. Importantly, the availability of safe commercial vaccines with proven efficacy to prevent the generation of congenital and postnatal persistent infections in pregnant sows are critical tools for controlling the disease in CSF endemic areas. Here, we demonstrate the high efficacy of a single dose of the recombinant FlagT4G vaccine to provide solid protection in pregnant sows against transplacental transmission of a highly virulent CSFV. Pregnant sows vaccinated with FlagT4G at 44 days of gestation elicited a strong CSFV-specific antibody response, with neutralizing antibody levels above those required for protection against CSFV. Importantly, after the challenge with a highly virulent CSFV, all foetuses from FlagT4G-vaccinated sows lacked CSF macroscopic lesions and showed a complete absence of the challenge virus in their internal organs at day 79 of gestation. Therefore, pregnant sows safely vaccinated with FlagT4G without affecting reproductive efficacy are efficaciously protected, along with their foetuses, against the infection and disease caused by a CSFV virulent field strain. Keywords: classical swine fever virus; transplacental transmission; FlagT4G vaccine; protection; neutralizing antibodies; immune response; vaccine efficacy Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Wed Jul 24 11:20:29 CEST 2024 ModDate: Wed Jul 24 11:33:38 CEST 2024 ID: 0xa119be8077ac6c9a7b2ccdc4b548a5d1, 0xa119be8077ac6c9a7b2ccdc4b548a5d1 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: OPZDHV+VnURWPalladioL FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: OPZDHV+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: TCHIDI+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 241 FontDescriptor: FontName: TCHIDI+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: CJQEJR+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: CJQEJR+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: ZXDKIP+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: ZXDKIP+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true ToUnicode: true Font: BaseFont: IOVOOE+VnURWPalladioL-Bold FontSubset: true FirstChar: 189 LastChar: 225 FontDescriptor: FontName: IOVOOE+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: NVNKTU+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 150 FontDescriptor: FontName: NVNKTU+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: AAPCWM+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: AAPCWM+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true ToUnicode: true Font: BaseFont: IOLAWT+PazoMath-Italic FontSubset: true FirstChar: 97 LastChar: 97 FontDescriptor: FontName: IOLAWT+PazoMath-Italic Flags: Symbolic FontBBox: -70, -277, 902, 733 FontFile: true ToUnicode: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 104, 281, 152, 290 Annotation: Subtype: Link Rect: 34, 269, 143, 278 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 125, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 149, 87 Annotation: Subtype: Link Rect: 34, 756, 150, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 125, 642, 137, 655 Annotation: Subtype: Link Rect: 282, 642, 293, 655 Annotation: Subtype: Link Rect: 309, 629, 320, 643 Annotation: Subtype: Link Rect: 539, 629, 550, 643 Annotation: Subtype: Link Rect: 453, 160, 460, 169 ActionDest: 327 Annotation: Subtype: Link Rect: 250, 135, 256, 143 ActionDest: 327 Annotation: Subtype: Link Rect: 257, 134, 264, 143 ActionDest: 327 Annotation: Subtype: Link Rect: 549, 122, 556, 131 ActionDest: 327 Annotation: Subtype: Link Rect: 539, 72, 546, 81 ActionDest: 327 Annotation: Subtype: Link Rect: 549, 72, 556, 81 ActionDest: 327 Annotation: Subtype: Link Rect: 112, 17, 266, 28 Annotation: Subtype: Link Rect: 406, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 390, 87, 409 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 370, 731, 377, 740 ActionDest: 327 Annotation: Subtype: Link Rect: 378, 731, 385, 740 ActionDest: 327 Annotation: Subtype: Link Rect: 530, 718, 537, 727 ActionDest: 327 Annotation: Subtype: Link Rect: 392, 656, 399, 665 ActionDest: 327 Annotation: Subtype: Link Rect: 261, 605, 268, 614 ActionDest: 327 Annotation: Subtype: Link Rect: 484, 593, 491, 602 ActionDest: 327 Annotation: Subtype: Link Rect: 492, 593, 504, 602 ActionDest: 327 Annotation: Subtype: Link Rect: 498, 530, 510, 539 ActionDest: 327 Annotation: Subtype: Link Rect: 510, 530, 522, 539 ActionDest: 327 Annotation: Subtype: Link Rect: 266, 492, 278, 501 ActionDest: 327 Annotation: Subtype: Link Rect: 346, 455, 358, 464 ActionDest: 327 Annotation: Subtype: Link Rect: 544, 442, 556, 451 ActionDest: 327 Annotation: Subtype: Link Rect: 279, 392, 291, 401 ActionDest: 327 Annotation: Subtype: Link Rect: 431, 379, 443, 388 ActionDest: 327 Annotation: Subtype: Link Rect: 353, 329, 364, 338 ActionDest: 327 Annotation: Subtype: Link Rect: 221, 304, 233, 313 ActionDest: 327 Annotation: Subtype: Link Rect: 233, 304, 245, 313 ActionDest: 327 Annotation: Subtype: Link Rect: 507, 304, 519, 313 ActionDest: 327 Annotation: Subtype: Link Rect: 423, 279, 430, 288 ActionDest: 327 Annotation: Subtype: Link Rect: 430, 279, 437, 288 ActionDest: 327 Annotation: Subtype: Link Rect: 438, 279, 449, 288 ActionDest: 327 Annotation: Subtype: Link Rect: 409, 241, 416, 250 ActionDest: 327 Annotation: Subtype: Link Rect: 416, 241, 428, 250 ActionDest: 327 Annotation: Subtype: Link Rect: 429, 241, 441, 250 ActionDest: 327 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 341, 743, 353, 752 ActionDest: 327 Annotation: Subtype: Link Rect: 330, 718, 342, 727 ActionDest: 327 Annotation: Subtype: Link Rect: 481, 718, 493, 727 ActionDest: 327 Annotation: Subtype: Link Rect: 389, 567, 401, 576 ActionDest: 327 Annotation: Subtype: Link Rect: 544, 517, 555, 526 ActionDest: 327 Annotation: Subtype: Link Rect: 349, 241, 361, 250 ActionDest: 330 Annotation: Subtype: Link Rect: 315, 203, 327, 212 ActionDest: 330 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 200, 731, 212, 740 ActionDest: 330 Annotation: Subtype: Link Rect: 346, 718, 358, 727 ActionDest: 330 Annotation: Subtype: Link Rect: 329, 656, 341, 665 ActionDest: 330 Annotation: Subtype: Link Rect: 330, 630, 342, 639 ActionDest: 330 Annotation: Subtype: Link Rect: 433, 605, 445, 614 ActionDest: 330 Annotation: Subtype: Link Rect: 218, 555, 230, 564 ActionDest: 330 Annotation: Subtype: Link Rect: 231, 555, 243, 564 ActionDest: 330 Annotation: Subtype: Link Rect: 535, 441, 547, 451 ActionDest: 330 Annotation: Subtype: Link Rect: 397, 379, 409, 388 ActionDest: 327 Annotation: Subtype: Link Rect: 431, 277, 438, 289 ActionDest: 83 Annotation: Subtype: Link Rect: 269, 226, 276, 238 ActionDest: 83 Annotation: Subtype: Link Rect: 533, 214, 540, 226 ActionDest: 83 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 368, 456, 375, 468 ActionDest: 163 Annotation: Subtype: Link Rect: 321, 406, 329, 418 ActionDest: 83 Annotation: Subtype: Link Rect: 549, 369, 556, 381 ActionDest: 83 Annotation: Subtype: Link Rect: 250, 331, 257, 343 ActionDest: 163 Annotation: Subtype: Link Rect: 474, 306, 481, 318 ActionDest: 83 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 485, 372, 492, 384 ActionDest: 163 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 387, 690, 394, 702 ActionDest: 225 Annotation: Subtype: Link Rect: 492, 665, 499, 677 ActionDest: 225 Annotation: Subtype: Link Rect: 199, 577, 206, 589 ActionDest: 225 Annotation: Subtype: Link Rect: 254, 539, 261, 551 ActionDest: 225 Annotation: Subtype: Link Rect: 406, 501, 413, 513 ActionDest: 225 Annotation: Subtype: Link Rect: 198, 464, 205, 476 ActionDest: 225 Annotation: Subtype: Link Rect: 198, 315, 205, 327 ActionDest: 257 Annotation: Subtype: Link Rect: 486, 202, 493, 214 ActionDest: 257 Page: Sequence: 8 Page: Sequence: 9 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 198, 677, 205, 689 ActionDest: 257 Annotation: Subtype: Link Rect: 367, 514, 374, 526 ActionDest: 283 Annotation: Subtype: Link Rect: 524, 488, 531, 500 ActionDest: 283 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 208, 690, 215, 699 ActionDest: 327 Annotation: Subtype: Link Rect: 215, 690, 222, 699 ActionDest: 327 Annotation: Subtype: Link Rect: 223, 690, 230, 699 ActionDest: 327 Annotation: Subtype: Link Rect: 230, 690, 242, 699 ActionDest: 327 Annotation: Subtype: Link Rect: 295, 640, 302, 649 ActionDest: 327 Annotation: Subtype: Link Rect: 420, 565, 427, 574 ActionDest: 327 Annotation: Subtype: Link Rect: 427, 565, 439, 574 ActionDest: 327 Annotation: Subtype: Link Rect: 279, 527, 291, 536 ActionDest: 330 Annotation: Subtype: Link Rect: 207, 464, 214, 473 ActionDest: 327 Annotation: Subtype: Link Rect: 214, 464, 226, 473 ActionDest: 327 Annotation: Subtype: Link Rect: 237, 376, 249, 385 ActionDest: 330 Annotation: Subtype: Link Rect: 544, 364, 556, 373 ActionDest: 330 Annotation: Subtype: Link Rect: 208, 314, 215, 323 ActionDest: 327 Annotation: Subtype: Link Rect: 215, 314, 227, 323 ActionDest: 327 Annotation: Subtype: Link Rect: 489, 264, 501, 272 ActionDest: 330 Annotation: Subtype: Link Rect: 504, 175, 511, 185 ActionDest: 327 Annotation: Subtype: Link Rect: 222, 138, 234, 147 ActionDest: 330 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 265, 718, 277, 727 ActionDest: 330 Annotation: Subtype: Link Rect: 372, 655, 379, 665 ActionDest: 327 Annotation: Subtype: Link Rect: 467, 605, 479, 614 ActionDest: 330 Annotation: Subtype: Link Rect: 482, 605, 494, 614 ActionDest: 330 Annotation: Subtype: Link Rect: 436, 593, 448, 602 ActionDest: 330 Annotation: Subtype: Link Rect: 342, 555, 354, 564 ActionDest: 330 Annotation: Subtype: Link Rect: 354, 555, 366, 564 ActionDest: 330 Annotation: Subtype: Link Rect: 210, 530, 222, 539 ActionDest: 330 Annotation: Subtype: Link Rect: 223, 530, 234, 539 ActionDest: 330 Annotation: Subtype: Link Rect: 235, 530, 247, 539 ActionDest: 330 Annotation: Subtype: Link Rect: 292, 492, 304, 501 ActionDest: 330 Annotation: Subtype: Link Rect: 304, 492, 316, 501 ActionDest: 330 Annotation: Subtype: Link Rect: 319, 492, 331, 501 ActionDest: 330 Annotation: Subtype: Link Rect: 322, 455, 334, 464 ActionDest: 330 Annotation: Subtype: Link Rect: 411, 392, 423, 401 ActionDest: 327 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 250, 663, 287, 674 Annotation: Subtype: Link Rect: 293, 663, 329, 674 Annotation: Subtype: Link Rect: 474, 652, 510, 663 Annotation: Subtype: Link Rect: 104, 630, 142, 640 Annotation: Subtype: Link Rect: 148, 630, 184, 640 Annotation: Subtype: Link Rect: 223, 606, 261, 617 Annotation: Subtype: Link Rect: 109, 572, 146, 582 Annotation: Subtype: Link Rect: 126, 549, 164, 559 Annotation: Subtype: Link Rect: 216, 525, 254, 536 Annotation: Subtype: Link Rect: 236, 491, 273, 502 Annotation: Subtype: Link Rect: 280, 491, 316, 502 Annotation: Subtype: Link Rect: 405, 479, 442, 490 Annotation: Subtype: Link Rect: 170, 445, 207, 456 Annotation: Subtype: Link Rect: 186, 387, 224, 398 Annotation: Subtype: Link Rect: 230, 387, 266, 398 Annotation: Subtype: Link Rect: 90, 342, 128, 352 Annotation: Subtype: Link Rect: 519, 306, 557, 317 Annotation: Subtype: Link Rect: 59, 296, 95, 306 Annotation: Subtype: Link Rect: 122, 261, 159, 271 Annotation: Subtype: Link Rect: 166, 261, 202, 271 Annotation: Subtype: Link Rect: 224, 226, 262, 237 Annotation: Subtype: Link Rect: 268, 226, 304, 237 Annotation: Subtype: Link Rect: 95, 192, 132, 202 Annotation: Subtype: Link Rect: 138, 192, 174, 202 Annotation: Subtype: Link Rect: 217, 157, 254, 168 Annotation: Subtype: Link Rect: 261, 157, 297, 168 Annotation: Subtype: Link Rect: 174, 135, 210, 145 Annotation: Subtype: Link Rect: 383, 111, 421, 122 Annotation: Subtype: Link Rect: 361, 88, 399, 99 Annotation: Subtype: Link Rect: 512, 76, 550, 87 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 232, 730, 269, 741 Annotation: Subtype: Link Rect: 131, 707, 168, 718 Annotation: Subtype: Link Rect: 324, 684, 362, 694 Annotation: Subtype: Link Rect: 317, 649, 355, 660 Annotation: Subtype: Link Rect: 463, 614, 501, 625 Annotation: Subtype: Link Rect: 59, 592, 96, 602 Annotation: Subtype: Link Rect: 298, 545, 336, 556 Annotation: Subtype: Link Rect: 86, 512, 124, 522 Annotation: Subtype: Link Rect: 201, 489, 239, 499 Annotation: Subtype: Link Rect: 245, 489, 281, 499 Annotation: Subtype: Link Rect: 120, 466, 157, 476 Annotation: Subtype: Link Rect: 164, 466, 200, 476 Annotation: Subtype: Link Rect: 494, 442, 531, 453 Annotation: Subtype: Link Rect: 131, 408, 168, 418 Annotation: Subtype: Link Rect: 140, 385, 177, 395 Annotation: Subtype: Link Rect: 184, 385, 220, 395 Annotation: Subtype: Link Rect: 131, 362, 168, 372 Annotation: Subtype: Link Rect: 191, 327, 228, 338 Annotation: Subtype: Link Rect: 234, 327, 270, 338 Annotation: Subtype: Link Rect: 270, 304, 308, 315 Annotation: Subtype: Link Rect: 338, 281, 376, 292 Annotation: Subtype: Link Rect: 90, 247, 128, 257 Annotation: Subtype: Link Rect: 192, 213, 230, 223 Annotation: Subtype: Link Rect: 243, 189, 281, 200 Annotation: Subtype: Link Rect: 378, 166, 415, 177 Checksum: 40601c0e Type: CRC32 Checksum: ceacba352a419d91a8e1039a43f8dc7a Type: MD5 Checksum: 876868f1ca160e2470017192261b3850ccd55fcc Type: SHA-1 Checksum: 32bc291b24052fe012735ceaec6d6173bf69f350e17e0c1b536c6f9b2a57c4de Type: SHA-256