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Antibody-Mediated Neutralization of Human Papillomavirus Type 11 (HPV-l1) Infection in the Nude Mouse: Detection of HPV-ll mRNAs w. Bonnez, R. C. Rose, and R. C. Reichman
Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York
Early experiments with animal papillomaviruses demonstrated the existence of naturally occurring or experimentally raised neutralizing antibodies to cottontail rabbit (Shope) papillomavirus [I], bovine papillomavirus [2], and canine oral papillomavirus [3]. Recently, studies using the nude mouse xenograft model have reported antibody-mediated inhibition of human papillomavirus type II (HPV-II) infection [4, 5]. These experiments were suggestive of neutralization but used manifestations of infection (graft size and histopathologic features) that are indirect indicators ofHPVII genome expression. In addition, experiments required 3 months to complete. We investigated neutralization of HPV-I I particles by a specific antiserum, using the presence ofHPV-11 mRNAs as the primary marker of viral infection, in experiments lasting only 6 weeks. The system is based on reverse transcription (RT) and polymerase chain reaction (peR) amplification of HPV-I I mRNA followed by Southern blot detection using an HPV-II E4-specific riboprobe.
Methods P~oduction a/Viral particles. Female 6-week-old athymic nul nu mice on a I3ALB/c background (Taconic Farms, Germantown, NY) were used to support growth under the renal capsule
Received 2 July 1991; revised 14 October 1991. The study protocol was approved by the University of Rochester Committee on Animal Research. Grant support: AI-82509 (National Institutes of Health). Reprints or correspondence: Dr. William Bonnez, Infectious Disease Unit, Box 689, University of Rochester Medical Center, 642 Elmwood Ave., Rochester, NY 14642.
The Journal ofInfectious Diseases 1992;165:376-80 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6502-0026$01.00
of human neonate foreskin fragments infected with HPVII Hershey' according to the technique described by Kreider et al. [6, 7]. Discarded human neonatal foreskins were collected at circumcision in sterile vials containing MEM (Whittaker Bioproducts, Walkersville, MD) supplemented with penicillin (25,000 units/ml) and streptomycin (25 mg/ml). Foreskins were split parallel to the surface, and the side corresponding to the occluded foreskin was discarded. The remaining half was trimmed into 1- X I-mm squares before exposure to the virus and implantation. The infective viral suspension (lysate 83B) was prepared from a single HPV-I I-induced cyst using a pestle, mortar, sterile sand, and 30 ml of PBS. The suspension was clarified by centrifugation at 500 g for 6 min at 4°C. Virus preparation and antibody production. Purified viral particles were prepared by a series of high-speed and cesium chloride gradient centrifugations as previously described [8], and HPV particles were identified by electron microscopy. A New Zealand rabbit (Hazelton Research Products, Denver, PA) was immunized by the subcutaneous route for the first injection and by the intramuscular route for two subsequent injections I and 2 months later. Complete and incomplete Freund's adjuvants (GIBCD, Grand Island, NY) were used for the initial and subsequent immunizations, respectively. Serum was collected before immunization and 2 weeks after the second booster. The development of a specific antibody response was demonstrated by dot blot against the undenatured antigen and by Western blotting as previously described [9]. Neutralization and experimental design. In parallel experiments, 450 JLI of the infecting viral suspension was incubated for I h at 37°C with 50 JLI of either the preimmune rabbit serum (control) or the postimmune rabbit serum (test) (I: 10 final dilution). At the end of the incubation period, freshly prepared foreskin fragments were distributed evenly between the control and test vials and incubated at 37°C for I h. The neutralization experiment was done in duplicate on two different days with two different foreskins. Each day, both kidneys of 12 mice were grafted with one foreskin fragment each [7]. After random allocation, groups of 3 mice were alternately
Downloaded from http://jid.oxfordjournals.org/ at University of Manitoba on August 25, 2015
The nu/nu mouse xenograft is the only experimental system permitting the growth of human papillomaviruses (HPV). Previous studies demonstrating inhibition ofHPV-ll infection by antibodies against HPV-11 virions have used indirect markers of infection, such as graft size and histopathologic features. The presence of HPV-11 mRNAs was used as a direct marker of infection: Infectious HPV-11 was incubated with rabbit serum raised against purified HPV-11 virions or with the corresponding preimmune serum (controls) before use in the mouse xenograft model, and HPV-11 mRNAs were detected by a method using reverse transcription and amplification by polymerase chain reaction. Graft size, histopathologic features, and the presence of capsid antigen were also assessed. Six weeks after infection, 1 of 23 grafts in the test group contained HPV-ll mRNAs compared with 19 of 20 controls (P < .001). Therefore, antibody-mediated inhibition of infection by HPV-11 leads to blockade ofgenomic expression and is thus consistent with active prevention of viral penetration, that is, neutralization.
1ID 1992; 165 (February)
Concise Communications
scription in vitro ofa 351-bp E4 ORF fragment (HPV-ll nucleotides 3320-3671) cloned in the SmaI site of pBluescript KS- (Stratagene). For hybridization, 2 X 10 7 cpm of E4 probe was added to 10 ml of hybridization solution for each blot, followed by overnight incubation at 65°C. After washing, blots were air dried and exposed to Kodak XAR film overnight. Histology and immunocytochernistry. Selected samples fixed in formalin and embedded in paraffin were sectioned. For each tissue block, one section was stained with hemalum and eosin, and two unstained sections were recovered on slides coated with poly-L-lysine [14] for immunocytochemistry. Unstained slides were placed for 1 h in a 60°C oven and then deparaffinized by successive 3-min baths in the following solvents: xylene three times, 100% ethanol three times, 95% ethanol twice, and 70% ethanol once. The slides were then labeled by the biotin-streptavidin complex method as described elsewhere [14]. The rabbit primary antibody was directed against an Escherichia coli {3-galactosidase fusion protein derived from the Ll ORF ofHPV-6b, a construct named pEX-H6-LI-480 and containing the common papillomavirus antigen [9]. In addition to the postimmune primary antibody, a preimmune primary antibody was used on a second slide as negative control. A known positive HPV-II-infected human xenograft was used as a positive tissue control for the batch. The interpretation of the slides was done according to predefined criteria. For the hemalurn and eosin stain, the following morphologic criteria were recorded: papillomatosis, acanthosis, koilocytosis, parakeratosis, and hyperkeratosis. For the immunocytochemistry, the presence and intensity of the signal was recorded. Statistical methods. For comparisons, Fisher's exact test was used for categorical variables and the Mann-Whitney U test for quantitative variables. P values are two-tailed, with P =:;; .05 considered statistically significant.
Results In the control group, 24 kidneys were grafted, but 4 implants did not remain in place and were lost for analysis. Similarly, I graft of 24 was not recovered in the test group. Figure 1 shows the ethidium bromide-stained agarose gels containing cDNA samples after processing by the RT-PCR method. Bands consistent with the 736-bp HPY -II-specific amplification product were noted in 19 of 20 grafts in the control group compared with 0 of23 grafts in the test group. The predicted 1439-bp amplification product was also seen in some of the samples from the control group that had the 736-bp band. Southern blotting of the gel (not shown) confirmed that these amplification products were truly derived from HPV-ll RNAs and revealed that one sample from the test group contained HPV-II mRNAs. In summary, 19 of20 grafts in the control group compared with I of 23 in the test group contained HPV-Il mRNAs, a highly significant difference (P < .00 I). We also examined the size of the grafts in the two experimental groups. The median area of the graft was 4 mm 2 (range, 1-12) in the control group and I mm 2 (range, 1-2)
Downloaded from http://jid.oxfordjournals.org/ at University of Manitoba on August 25, 2015
grafted with the control or test foreskins. The order of assignment was reversed on the second day. Six weeks after grafting, the animals were sacrificed by cervical dislocation. Each kidney was placed in a clean, sterile Petri dish. Bidimensional areas (length X width) of the cysts were measured using the following rule: If the dimension was
Antibody-Mediated Neutralization of Human Papillomavirus Type 11 (HPV-l1) Infection in the Nude Mouse: Detection of HPV-ll mRNAs w. Bonnez, R. C. Rose, and R. C. Reichman
Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York
Early experiments with animal papillomaviruses demonstrated the existence of naturally occurring or experimentally raised neutralizing antibodies to cottontail rabbit (Shope) papillomavirus [I], bovine papillomavirus [2], and canine oral papillomavirus [3]. Recently, studies using the nude mouse xenograft model have reported antibody-mediated inhibition of human papillomavirus type II (HPV-II) infection [4, 5]. These experiments were suggestive of neutralization but used manifestations of infection (graft size and histopathologic features) that are indirect indicators ofHPVII genome expression. In addition, experiments required 3 months to complete. We investigated neutralization of HPV-I I particles by a specific antiserum, using the presence ofHPV-11 mRNAs as the primary marker of viral infection, in experiments lasting only 6 weeks. The system is based on reverse transcription (RT) and polymerase chain reaction (peR) amplification of HPV-I I mRNA followed by Southern blot detection using an HPV-II E4-specific riboprobe.
Methods P~oduction a/Viral particles. Female 6-week-old athymic nul nu mice on a I3ALB/c background (Taconic Farms, Germantown, NY) were used to support growth under the renal capsule
Received 2 July 1991; revised 14 October 1991. The study protocol was approved by the University of Rochester Committee on Animal Research. Grant support: AI-82509 (National Institutes of Health). Reprints or correspondence: Dr. William Bonnez, Infectious Disease Unit, Box 689, University of Rochester Medical Center, 642 Elmwood Ave., Rochester, NY 14642.
The Journal ofInfectious Diseases 1992;165:376-80 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6502-0026$01.00
of human neonate foreskin fragments infected with HPVII Hershey' according to the technique described by Kreider et al. [6, 7]. Discarded human neonatal foreskins were collected at circumcision in sterile vials containing MEM (Whittaker Bioproducts, Walkersville, MD) supplemented with penicillin (25,000 units/ml) and streptomycin (25 mg/ml). Foreskins were split parallel to the surface, and the side corresponding to the occluded foreskin was discarded. The remaining half was trimmed into 1- X I-mm squares before exposure to the virus and implantation. The infective viral suspension (lysate 83B) was prepared from a single HPV-I I-induced cyst using a pestle, mortar, sterile sand, and 30 ml of PBS. The suspension was clarified by centrifugation at 500 g for 6 min at 4°C. Virus preparation and antibody production. Purified viral particles were prepared by a series of high-speed and cesium chloride gradient centrifugations as previously described [8], and HPV particles were identified by electron microscopy. A New Zealand rabbit (Hazelton Research Products, Denver, PA) was immunized by the subcutaneous route for the first injection and by the intramuscular route for two subsequent injections I and 2 months later. Complete and incomplete Freund's adjuvants (GIBCD, Grand Island, NY) were used for the initial and subsequent immunizations, respectively. Serum was collected before immunization and 2 weeks after the second booster. The development of a specific antibody response was demonstrated by dot blot against the undenatured antigen and by Western blotting as previously described [9]. Neutralization and experimental design. In parallel experiments, 450 JLI of the infecting viral suspension was incubated for I h at 37°C with 50 JLI of either the preimmune rabbit serum (control) or the postimmune rabbit serum (test) (I: 10 final dilution). At the end of the incubation period, freshly prepared foreskin fragments were distributed evenly between the control and test vials and incubated at 37°C for I h. The neutralization experiment was done in duplicate on two different days with two different foreskins. Each day, both kidneys of 12 mice were grafted with one foreskin fragment each [7]. After random allocation, groups of 3 mice were alternately
Downloaded from http://jid.oxfordjournals.org/ at University of Manitoba on August 25, 2015
The nu/nu mouse xenograft is the only experimental system permitting the growth of human papillomaviruses (HPV). Previous studies demonstrating inhibition ofHPV-ll infection by antibodies against HPV-11 virions have used indirect markers of infection, such as graft size and histopathologic features. The presence of HPV-11 mRNAs was used as a direct marker of infection: Infectious HPV-11 was incubated with rabbit serum raised against purified HPV-11 virions or with the corresponding preimmune serum (controls) before use in the mouse xenograft model, and HPV-11 mRNAs were detected by a method using reverse transcription and amplification by polymerase chain reaction. Graft size, histopathologic features, and the presence of capsid antigen were also assessed. Six weeks after infection, 1 of 23 grafts in the test group contained HPV-ll mRNAs compared with 19 of 20 controls (P < .001). Therefore, antibody-mediated inhibition of infection by HPV-11 leads to blockade ofgenomic expression and is thus consistent with active prevention of viral penetration, that is, neutralization.
1ID 1992; 165 (February)
Concise Communications
scription in vitro ofa 351-bp E4 ORF fragment (HPV-ll nucleotides 3320-3671) cloned in the SmaI site of pBluescript KS- (Stratagene). For hybridization, 2 X 10 7 cpm of E4 probe was added to 10 ml of hybridization solution for each blot, followed by overnight incubation at 65°C. After washing, blots were air dried and exposed to Kodak XAR film overnight. Histology and immunocytochernistry. Selected samples fixed in formalin and embedded in paraffin were sectioned. For each tissue block, one section was stained with hemalum and eosin, and two unstained sections were recovered on slides coated with poly-L-lysine [14] for immunocytochemistry. Unstained slides were placed for 1 h in a 60°C oven and then deparaffinized by successive 3-min baths in the following solvents: xylene three times, 100% ethanol three times, 95% ethanol twice, and 70% ethanol once. The slides were then labeled by the biotin-streptavidin complex method as described elsewhere [14]. The rabbit primary antibody was directed against an Escherichia coli {3-galactosidase fusion protein derived from the Ll ORF ofHPV-6b, a construct named pEX-H6-LI-480 and containing the common papillomavirus antigen [9]. In addition to the postimmune primary antibody, a preimmune primary antibody was used on a second slide as negative control. A known positive HPV-II-infected human xenograft was used as a positive tissue control for the batch. The interpretation of the slides was done according to predefined criteria. For the hemalurn and eosin stain, the following morphologic criteria were recorded: papillomatosis, acanthosis, koilocytosis, parakeratosis, and hyperkeratosis. For the immunocytochemistry, the presence and intensity of the signal was recorded. Statistical methods. For comparisons, Fisher's exact test was used for categorical variables and the Mann-Whitney U test for quantitative variables. P values are two-tailed, with P =:;; .05 considered statistically significant.
Results In the control group, 24 kidneys were grafted, but 4 implants did not remain in place and were lost for analysis. Similarly, I graft of 24 was not recovered in the test group. Figure 1 shows the ethidium bromide-stained agarose gels containing cDNA samples after processing by the RT-PCR method. Bands consistent with the 736-bp HPY -II-specific amplification product were noted in 19 of 20 grafts in the control group compared with 0 of23 grafts in the test group. The predicted 1439-bp amplification product was also seen in some of the samples from the control group that had the 736-bp band. Southern blotting of the gel (not shown) confirmed that these amplification products were truly derived from HPV-ll RNAs and revealed that one sample from the test group contained HPV-II mRNAs. In summary, 19 of20 grafts in the control group compared with I of 23 in the test group contained HPV-Il mRNAs, a highly significant difference (P < .00 I). We also examined the size of the grafts in the two experimental groups. The median area of the graft was 4 mm 2 (range, 1-12) in the control group and I mm 2 (range, 1-2)
Downloaded from http://jid.oxfordjournals.org/ at University of Manitoba on August 25, 2015
grafted with the control or test foreskins. The order of assignment was reversed on the second day. Six weeks after grafting, the animals were sacrificed by cervical dislocation. Each kidney was placed in a clean, sterile Petri dish. Bidimensional areas (length X width) of the cysts were measured using the following rule: If the dimension was
