- Email: [email protected]
Intracutaneous vaccination of rabbits with the cottontail rabbit papillomavirus (CRPV) L1 gene protects against virus challenge
0
Elsevier
PLI: SO264-41OX(96)00237-X
ELSEVIER
Vaccine, Vol. 15, No.-6/7, pp. 664-671, 1997 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-410x197 $17+0.00
Intracutaneous vaccination of rabbits with the cottontail rabbit papillomavirus (CRPV) Ll gene protects against virus challenge Pazhani
Sundaram*JJ,
Robert
E. Tigelaarf$$
and Janet L. Brandsma*§T
A DNA vaccine encoding the major capsid protein LI of cottontail rabbit papillomavirus (CRPV) was constructed and administered intracutaneously (i.c.) to rabbits as supercoiled plasmids bound to gold beads using a specialized delivery device (“gene gun “). LI DNA-vaccinated rabbits developed cellular proltferative responses to CRPV virus-like particles and developed high titered antibodies with neutralizing activity to CRPV Following experimental challenge with CRPV. all of the Ll DNA-vaccinated rabbits, vs none of the controls, were protectedfrom papilloma formation. These results demonstrate that i.e. vaccination of rabbits with the Lf papillomavirus capsid gene can induce antibodies that protect against subsequent papillomavirus infection. 0 1997 Elsevier Science Ltd. Keywords:
DNA
vaccination;
papillomavirus;
LI gene
The association of human papillomavirus (HPV) infection with cervical cancer in women and with skin cancers in patients with Epidermodysplasia verruciformis is well established’*2. Additionally, HPVs may be involved in head, neck, other anogenital and other skin cancers, bringing the estimated proportion of all cancers associated with HPV to 10%3. Precancerous HPV lesions may regress spontaneously, or they may persist for years. Therapies for the control of HPV disease are not always effective and do not usually eliminate infection. Therefore the search for a vaccine to prevent HPV infection, and hence HPV-associated cancer, has received much attention4. Vaccine development requires animal models, but species barriers prevent direct infection of animals with HPVS~,~. Using cottontail rabbit papillomavirus (CRPV) in rabbits, bovine papillomavirus (BPV) in cattle, and canine oral papillomavirus (COPV) in dogs, previous studies have shown that prophylactic immunity can be induced by vaccination with whole papilloma*Section of Comparative Medicine, Yale University School of Medicine. New Haven. CT 06520. USA. tDebartment of Dermatology, Yale University School of Medicine, New Haven, CT 06520, USA. ISection of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA. $Yale Skin Diseases Research Center, Yale University School of Medicine, New Haven, CT 06520, USA. fiDepartment of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT 06520, USA. IICurrent address: Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA. “To whom correspondence should be addressed. (Received 22 July 1996; revised 16 October 1996; accepted 16 October 1996)
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viruses, virus-like particles (VLPs), recombinant proteins or recombinant vaccinia virus6,m’3. Prophylactic immunity correlated with papillomavirus neutralizing antibodies to conformational epitopes of Ll and could not be induced with truncated or denatured Ll proteins14. A recent approach to vaccination that can induce potent and long-lived humoral and cell-mediated immunity is the direct inoculation of a host with a recombinant expression vector’ 5m1 7. This method eliminates the need to induce high level protein expression in vitro and to purify the protein product. DNAs can be delivered intramuscularly (i.m.) with a needle or intracutaneously (ic.) with a particle-mediated gene delivery device (gene gun). Both inoculation routes induce humoral and cellular immunity, but the i.c. route induces superior antibody responses183’9. Gene gun DNA vaccination of mice, monkeys, pigs, and ferrets has effectively induced immunity, presumably by inducing gene expression in Langerhans and other dendritic cells that process foreign proteins and, following migration to regional lym h nodes, present the antigens to the immune system’- ’ 21. The i .c . route induces gene expression in epidermal keratinocytes whose protein products can be ingested by adjacent dendritic cells for subsequent. processing and antigen presentation2’. We recently reported the optimization of gene gun-mediated DNA delivery conditions for gene expression in rabbit epidermis2’.2.. This report describes the application of i.c. DNA vaccination to rabbits and demonstrates that the CRPV Ll gene can induce strong and specific humoral and cellular immune responses to Ll and protect the rabbits against experimental challenge with CRPV virus.
lntracutaneous vaccination with papillomavirus L 7 gene: I? Sundaram et al. MATERIALS
AND
METHODS
Construction of an expression vector for CRPV Ll protein Plasmid pCMV$ (Clonetech, Palo Alto, CA) expresses the @-galactosidase v-gal) gene from a cytomegalovirus (CMV) promoter/enhancer. It served as the parental vector after removal of the /?-galactosidase gene. Plasmid pCMV-Ll was generated by inserting a polymerase chain reaction (PCR) fragment at the Not I site of the pCMV parental plasmid. The PCR fragment was amplified from CRPV-pLAI1 template DNA using as sense primer: 5’-GT CAC GTC Age ggc cgc ATG GCA GTG TGG CTG TG-3’; and as antisense primer: S-TG ACA TCG Tgc ggc cgc TTA AGT ACG TCT CTT GCG-3’ (initiation and stop codons are italicized; Not I sites are lower case). It contained CRPV nucleotides 5828-7345 in the correct orientation, as determined by restriction endonuclease cleavage patterns. Plasmid pCMV-N contained the N gene of mouse hepatitis virus (MHV) under the control of a CMV promoter in vector pcDNA3 (Invitrogen, San Diego, CA). Supercoiled plasmid DNAs were purified using the Qiagen (Qiagen, Inc., Chatsworth, CA) method and used as supercoiled molecules. Transient transfection and imnnmofluorescent detection of Ll protein Six well plates of subconfluent CHO cells were transfected for 5 h at 37°C with occasional shaking in 1 ml of serum-free antibiotic-free medium containing 1 pug DNA and 5~1 lipofectamine (Life Technologies, Gaithersburg, MD). The transfection medium was then replaced with complete medium containing serum and antibiotics. The medium was changed after overnight incubation. Thirty-six hours after transfection, cells were fixed in cold acetone for 10 min, air dried, and incubated with a rabbit antiserum to CRPV Ll generated by protein immunization with a trpE-Ll fusion protein*. Cells were washed and incubated with a secondary, fluorescein-labeled goat anti-rabbit IgG. Administration of DNA vaccines DNA vaccines were prepared and administered to 2 kg New Zealand white rabbits, using an Accell@ gene delivery device (Auragen, Inc., Middleton, WI) with 350 p.s.i. of helium pressure, as described previously22. Rabbits were vaccinated on day 0, and booster vaccinations were given on days 21, 42, and 63. Rabbits were inoculated at each of 30 sites with 1 pg of DNA at each time point. The DNA dose was chosen arbitrarily. Two rabbits were vaccinated with pCMV-N, three with pCMV-/3, and five with pCMV-Ll. Two pCMV+’ immunized rabbits died after the first boost, due to causes unrelated to the experiment. Enzyme-linked immunosorbent assays (ELISAs) CRPV VLPs, prepared in a baculovirus expression system and composed of CRPV Ll and L2 proteins24, were generously_donated by John Schiller. ELISA plate wells were coated with the CRPV VLPs, or with P-galactosidase (Sigma Chemical Co., St. Louis, MO). Wells containing 100 ng of antigen were reacted with serial twofold dilutions of rabbit serum. Staphylococcal protein A coupled to horseradish peroxidase was used as
the enzyme conjugate. Serum dilutions giving an A,,, of 20.10 above the matched pre-immune sample were considered positive. Positive control antibodies were the rabbit antiserum to a TrpE-CRPV Ll fusion protein’, and a commercial mouse monoclonal antibody to /I-gal (Sigma Chemical Co., St. Louis, MO).
Western blots Western blots were prepared and processed as deprotein A was scribed previously22.25. Staphylococcal used to detect rabbit antibody, and goat anti-mouse IgG was used to detect mouse antibody.
In vitro proliferation of peripheral blood mononuclear cells (PBMC) In vitro proliferation assays were conducted 2 weeks after the final DNA booster vaccinations. Approximately 20 ml of blood per rabbit was collected from an ear vein into heparinized tubes, diluted 1:l with RPM1 1640 (GIBCO) buffered with 10 mM HEPES, layered over a half volume of Histopaque-1077 (1.077 g ml-‘, Sigma Diagnostics, St. Louis, MO) and centrifuged at 400g for 30 min at 25°C. PBMC at the interface were harvested, washed three times at 4”C, and then resuspended to 2 x lo6 viable PBMC ml- ’ in RPM1 1640 supplemented with 10% fetal calf serum, 25 mM HEPES buffer, 2 mM L-glutamine, 1 mM Na pyruvate, 0.1 mM nonessential amino acids, 5 x 10e5 M 2-mercaptoethanol, 100 U ml-’ penicillin and lOOpug ml-’ streptomycin (“complete” RPMI, or CRPMI). Proliferation assays were performed in 96-well round bottom microtiter plates. To each well was added a 100~1 aliquot of cells (2 x 105) and 100~1 of CRPMI containing either no antigen/mitogen (background response), 6 ,ug ml- ’ of the T cell mitogen concanavalin A (Sigma, St. Louis, MO) as a positive control for T cell proliferation, or graded concentrations (0.02 to 20 pg ml- I) of either /3-galactosidase (Sigma) or CRPV VLPS~~ (the concentrations refer to the estimated concentration of the Ll protein in the VLP preparations). Cultures were incubated at 37°C in humidified 5% CO, for 3 days to assay Con A responses and 5 days to assay responses to P-gal and Ll. Eighteen hours prior to harvest, wells were pulsed with 1 &i [“Hlthymidine (sp. act. 6.7 Ci mmol-‘; ICN, Costa Mesa, CA). Triplicate wells for each dose of antigen were harvested using a semi-automated cell harvester (Skatron Instruments, Vernon, VA); filter discs were placed in vials containing Ecoscint (National Diagnostics, Atlanta, GA), and counted in a liquid scintillation counter (LKB-Wallace, Turku, Finland). Because of substantial variability in the background responses (in the absence of antigen or mitogen) of individual rabbits, which ranged from 1830 f 890 to 12720 f 3980 (mean c.p.m. f S.E.M.), the data are expressed as the stimulation index (S.I.) f S.E.M., where: S.I.=
well containing antigen c p m. of experimental . mean c.p.m. of triplicate control wells (CRPMI only).
Differences between groups unpaired Student’s t-test.
were analyzed
by use of
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Experimental production of infectious CRPV virus A cottontai1 (Sylvilagus cuniculus) rabbit was anesthetized with ketamine (15 mg kg- ‘) and inoculated at each of 35 scarified sites with 30~1 of CRPV virus. The inoculum was prepared by adding to 1.2 ml of phosphate-buffered saline 150~1 of a crude extract of a naturally occurring wild cottontail rabbit papilloma, donated by Bettie Steinberg. Papillomas formed at all sites. Three months after inoculation, biopsies of two papillomas were obtained under ketamine anesthesia, minced in DMEM medium, and implanted subcutaneously into severe combifled immunodeficient (scid) mice for further growth. Subcutaneous papillomas developed at each inoculation site and were passaged to new scid mice 2 months after implantation. Papillomas were harvested from second passage mice 1 month later. The tissue was homogenized in a tissue grinder using ca 1 ml of PBS per 100 mg of tissue and then clarified by low-speed centrifugation. An equal volume of glycerol was added and the material was stored at - 80°C. The amount of virus required to induce papillomas in New Zealand white rabbits was estimated in a pilot experiment. Challenge with CRPV virus CRPV virus was diluted in PBS to l:lO, 1:40, and 1: 160. Six weeks after the last immunizations, three sites per rabbit were infected with each dilution, by applying 30~1 to scarified skin. Papilloma formation was monitored weekly in the second month after challenge and biweekly in the third month. Neutralization of CRPV infectivity Serial tenfold dilutions of preimmune and hyperimmune serum from a Ll DNA-vaccinated rabbit were prepared in PBS containing 1% bovine serum albumin. Fifteen microliters of diluted serum was mixed with 15 ~1 of a 1:60 dilution of CRPV virus, incubated on ice for 2-3 h, and applied by scarification to two sites on a naive rabbit. Two rabbits were inoculated with the preimmune mixture and two with the immune mixture. Each rabbit was also inoculated in duplicate with CRPV virus and no serum as a control.
RESULTS Construction of a CRPV Ll expression vector Plasmid pCMV-Ll was constructed to transcribe the full length Ll gene of CRPV from the CMV immediate early promoter. Its ability to express Ll protein was verified in transiently transfected CHO cells by immunofluorescent staining with a CRPV Ll antibody’. pCMV-Ll transfected cells expressed Ll protein in the nucleus 36 h after transfection (Figure 1). Cells transfected with the parental pCMV vector and stained with the Ll antibody were negative. Replicate wells stained with a non-immune rabbit serum also were negative. DNA vaccination and induction of humoral immunity to conformational epitopes Rabbits were vaccinated by gene gun inoculation of skin with DNA plasmids bound to gold beads. The experimental vaccine pCMV-Ll expressed CRPV Ll
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Figure 1 Ll expression induced by pCMV-Ll transfection of CHO cells. The top panel shows immunofluorescent staining of cells transfected with pCMV-Ll. The bottom panel shows cells transfected in parallel with the parental pCMV vector and stained
120,000 100,ooo
80,000 60,000 40,000 20,000 0 Serum:
Ls A
t
Pre Bl
B2 B3
B
Pre Bl
B2 B3
Vaccine:
pCMV-CRLl
pCMV-P
Antigen:
VLP
Pgd
Figure 2 Antibody development in DNA-vaccinated rabbits. Sera obtained prior to vaccination (Pre), and after the first (Bl), second (B2) and third (83) boosts were tested. Control antibody to p-gal reacted at 1:6,400 in the p-gal ELISA, and control antiserum to Ll reacted at 1:120000 in the CRPV VLP ELISA. Arithmetic mean titers and their standard deviations are shown
protein. Plasmid pCMV$ expressed P-galactosidase Cpgal) and served as a positive control for induction of humoral and cellular immune responses. pCMV-/? and pCMV-N served as negative controls for protection from CRPV infection. Five of five rabbits vaccinated with pCMV-Ll and three of three rabbits vaccinated with pCMV$ developed specific humoral immune responses to CRPV VLPs or P-gal, respectively. Antibody titers were high after the first boost and increased after the second boost to a mean of 1:96000 in rabbits vaccinated with pCMV$ and 1: 104000 in rabbits vaccinated with pCMV-Ll (Figure 2). Increases after the third boost were minimal. Western blot analyses confirmed the ELISA findings and provided further documentation of the specificity of the humoral immune responses (Figure 3). Rabbits vaccinated with pCMV-P
lntracutaneous
#l
#2
#3
#4
#5
c
Rabbit Figure 3 Specificity of antibody responses in DNA-vaccinated rabbits. Western blots contained 1 pg of CRPV VLPs (A) or P-galactosidase (B) and were probed with preimmune (P) or immune sera (I). (A) Probes are antiserum from rabbits immunized with pCMV-Ll (Nos l-5), control antiserum to CRPV Ll (Ll), or antiserum from a rabbit immunized with pCMV$ (B). Molecular weight markers are 106, 80, 49.5, 32.5, 27.5, and 18.5 kDa. (B) Probes are antiserum from rabbits immunized with pCMV-8 (Nos 6-8) control antibody to /I-gal (8). or antiserum from a rabbit immunized with pCMV-Ll (Ll). Molecular weight markers are 205, 116.5. 80, 49.5, and 32.5 kDa
developed antibodies that recognized P-gal but not CRPV VLPs. Similarly, rabbits vaccinated with pCMV-Ll developed antibodies to the Ll protein in the CRPV VLPs but not to P-gal. The nature of the Ll epitopes recognized by antisera from the Ll DNA-vaccinated rabbits was characterized by ELISAs using VLPs that had been denatured by boiling for 10 min as well as VLPs in the native conformation. In each case, the titer to denatured epitopes was about an order of magnitude lower than the titer to conformational epitopes (Figure 4). In the same assay, stronger reactivity to denatured than native VLPs was observed by a control rabbit antiserum to disrupted CRPV VLPs. We therefore conclude that gene gun vaccination with the Ll gene induced humoral immunity to conformational epitopes predominantly. In vitro proliferative responses Data summarizing the day 5 proliferative responses of PBMC from the eight vaccinated rabbits in this study are presented in Table 1. Each rabbit’s cells demonstrated a vigorous response on day 3 to the T cell mitogen Con A (data not shown). PBMC from each of the five rabbits vaccinated with pCMV-Ll exhibited significantly greater responses to Ll than to P-gal, and while their mean proliferative response to B-gal was not significantly higher than their background response to media alone (mean S.I.=l.2&0.4, mean c.p.m.= 11030& 2770), their mean response to Ll was highly significant (mean S.I.=3.3 f 0.6, mean c.p.m.=32890& 2180, PcO.005). In contrast, PBMC from control rabbit No. 6 vaccinated with pCMV+gal proliferated significantly in response to P-gal but not to Ll (S.I. 5.6& 1 vs 0.9&012), while control rabbits Nos 7 and 8 vaccinated with pCMV-N exhibited minimal responses either to Ll (S.I. 1.2-1.9) or to /?-gal (S.I. 1.5-1.6).
vaccination with papillomavirus
L 1 gene: I? Sundaram et al.
Protection against challenge with CRPV virus following DNA vaccination Effective vaccination should protect naive animals from infection. The ability of vaccinated rabbits to resist CRPV-induced papilloma formation was evaluated by virus challenge at three sites each with l:lO, 1:40, and 1: 160 dilutions of CRPV. Papillomas developed at 27 of 27 infected sites in three rabbits vaccinated with pCMV-/I or pCMV-N (Table 2). In contrast, 44 of the 45 sites (98%) in the pCMV-Ll-vaccinated rabbits remained free of disease for 3 months after challenge (the duration of the experiment) (Table 2). Furthermore, the single papilloma that formed was at all times smaller than any in the control rabbits (Figure 5). Fifteen weeks after infection it was 10 times smaller than control papillomas: 0.64 cm3 vs 9.6 f 5.1 cm3 (mean f SD.), indeed it was smaller than every one of the control lesions. These results demonstrate clearly that i.c. DNA vaccination with pCMV-Ll elicited strong prophylactic immunity. Neutralizing antibody to CRPV Previous studies have shown that prophylactic immunity to papillomavirus infection correlates with the presence of neutralizing antibody. We used an in vivo assay to show that CRPV virus was neutralized by immune serum at dilutions of I 10e3 but not by preimmune serum at the same dilutions (Table 3). This difference was highly significant (one-tailed Student’s r-test, two-sample unequal variance, P
DISCUSSION This study demonstrates that Ll antigen-specific humoral immunity and highly prophylactic immunity against CRPV infection can be achieved by i.c. vaccination of rabbits with the CRPV Ll capsid gene. Rabbits vaccinated with pCMV-Ll developed high titered antisera that reacted specifically with Ll antigens (Figure 2 and Figure 3), recognized predominantly conformational Ll epitopes (Figure 4), and neutralized CRPV (Table 3). Furthermore, Ll DNA-vaccinated rabbits were protected from papilloma formation at 44 of 45 sites (97.8%) challenged with CRPV virus, whereas control rabbits developed papillomas at all infected sites (Table 2). Moreover, the single papilloma in a Ll DNA-vaccinated rabbit was more than ten times smaller than the mean size of control papillomas (Figure 5). Interestingly, this rabbit had the lowest titer to native VLPs, suggesting that protection requires a titer of 2 1:60000, as measured by ELISA to native VLPs. While our work was in progress, Donnelly et al. published a Ll DNA vaccination study in the CRPVrabbit model using an i.m. route of vaccination26. The most important difference between our study design and
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L 1 gene: t? Sundaram et al.
vaccination with papillomavirus
Rabbit:
# 1
#4
#6
#5 I
301 25 % = w .M VI
20 15 -
!J =: ‘& n”
10 5 -
o5 8 11
5 8 11
I
j 8 11
5 8 11
5 8 11
5 8 11
5 8 11
5 8 11
Weeks after challenge Figure 4 Reactivity to conformational epitopes of CRPV VLPs among antisera from pCMV-Ll vaccinated rabbits. Dark bars represent the mean titer of reactivity against native VLPs; light bars, against denatured VLPs. Rabbits Nos l-5 were vaccinated with pCMV-Ll. Rabbit “C” was immunized with denatured VLPs. The sera were diluted to 1:50000
Table 1 Day 5 proliferative responses vaccinated rabbits to Ll (VLP) and b-gal Stimulation
of PBMC
from
index (SI)&.E.M.
to:
Vaccine
Rabbit
Ll (VLP)
P-gal
pCMV-CRLl
No. No. No. No. No.
pCMV-/I pCMV-N
No. 6 No. 7 No. 8
3.7*0.3 5.1rt0.2 3.2kO.2 3.2*0.2 1.4*0.4 Mean=3.3*0.6 0.9*0.2 1.9iO.6 1.2io.4
1.5to.2 1.8kO.2 1.2*0.2 0.9*0.2 0.5*0.0 Mean=l.2*0.4 5.6&l 1.5*0.3 1.6*0.4
Table 2 Frequency with CRPV
1 2 3 4 5
of papilloma
formation
DNA-
following
challenge
Papilloma formationa Vaccine
Rabbit
Sites inoculated at: 1:lO 1:40 1:160
pCMV-CRLl
No. No. No. No. No.
o/3 o/3 o/3 o/3 o/3
o/3 o/3 o/3 o/3 o/3
o/3 o/3 o/3 113 o/3
Controlsb
No. 6 No. 7 No. 8
313 b 313 313
313 313 313
313 313 313
1 2 3 4 5
All sites
Protection W)
o/9 o/9 o/9 l/9 o/9 1145 919 919 919 27127
100 100 100 88.9 100 97.8 : :
sNo. of papilloma-forming siteslNo. of challenge sites; bRabbit No. 6 was vaccinated with pCMV-/I; rabbits Nos 7 and 8 with pCMV-N
theirs is the route of DNA delivery, although other differences-the sequences of the Ll constructs and the sources of baculovirus-derived VLPs and CRPV virus stocks-may also have influenced the results. Bearing in mind that the i.c. and i.m. routes were not directly compared, the following observations can nevertheless be made. In both studies, Ll-vaccinated rabbits developed neutralizing antibodies and were completely protected from papilloma formation at 97.8% (Table 2)
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Vaccine 1997 Volume 15 Number 6/7
or 96.7%26 of CRPV challenge sites. However, since papillomas formed at c 100% (28/30=940/o) of their control sites, our challenge appears to have been more stringent. Moreover, our CRPV virus stock (used here at dilutions of I 1: 160) is sufficient to induce papillomas at 100% of challenge sites infected with a 1:270 dilution (data not shown). In addition, the specific antibody titers induced by Ll DNA-vaccination appeared to be higher in our study: ca 1: 100000 (Figure 2) vs I 1:ll 000Z6, as measured by ELISA using 0.1 pug VLP per well here or 0.5pg VLP per well in Ref. 26. Since i.c. routes of DNA vaccination, when compared directly to i.m. routes, induce superior humoral immune responses’ *.19, our results should help guide the development of a Ll-based HPV DNA vaccine. Transient assays of pCMV$ expression in rabbit skin22.23 strongly suggest that, following vaccination, the CRPV Ll gene product was expressed in epidermal keratinocytes and epidermal Langerhans cells or other i.c. dendritic cells. Ll protein expressed by the vaccine gene localized to the nucleus of transfected cells and, in vivo, may have spontaneously assembled into empty CRPV capsids, as suggested by experimental studies in insect and mammalian ce11s24.27,28.Capsids may have assembled in highly differentiated keratinocytes, as in natural infection, or in other cells. Release of Llcontaining empty CRPV capsids would be expected to result in their phagocytosis or endocytosis by dendritic cells, followed by endosomal/lysosomal processing into peptides for presentation, in the context of MHC class II molecules, to the CD4+ subset of rabbit T cells that provide help for B cells to produce antigen specific IgG antibodies that recognize conformational (threedimensional) epitopes29.30. Indeed, PBMCs from Ll DNA-vaccinated rabbits demonstrated antigen specific proliferative responses in vitro to CRPV Ll-containing VLPs; similar responses were not observed in control animals vaccinated with other DNA preparations (Table 1). These responses were virtually certainly those of T lymphocytes, as documented previously in cells from rabbits bearing virus-induced papillomas or carcinostudies using antibody-mediated mas31. Additional depletion of the CD4 subset of rabbit T cells32 will be
lntracutaneous
#2
vaccination with papillomavirus
#4
#3
#5
#6
L 1 gene: I? Sundaram et al.
_j
_-o 7 1115
7 1115
#8
#7
7 1115
Weeks
7 1115
7 1115
7 1115
after challenge
Figure 5 Papilloma size and growth rate in DNA-vaccinated rabbits. Each panel represents one rabbit. Rabbits Nos 1-5 were vaccinated with pCMV-Ll , rabbit No. 6 with pCMV-8, and rabbits Nos 7 and 8 with pCMV-N. Measurements obtained 7, 11, and 15 weeks after CRPV challenge are shown. Open circles represent the size of individual papillomas (nine per rabbit), and bars represent their mean size
Table 3 Neutralization DNA-vaccinated rabbit
of CRPV
infectivity
by serum from a Ll
Papilloma formation Serum
Dilution
lb
2
Both
Papilloma sizes mean+S.D.
Preimmune
10 o lo-’ 1o-* 10-S 1o-4 1o-5
212 = 212 212 212 212 212 212
212 212 212 l/2 Z 212
414 414 414 314 414 414 414
N.D. N.D. N.D. N.D. N.D. N.D. N.D.
3
4
Both
o/2 o/2 o/2 l/2 Z
l/2 o/2 l/2 212 212 212 212
114 o/4 II4 314 414 414 414
None
Immune
None
10 o 1o-’ 1o-* 1 o-3 1 o-4 1 o-5
2l2
2.1 1.7 0.2*0.1 N.D. N.D. 43.oZt9.0
=Mean size (cm3)+S.D. Measurements were obtained 7 weeks after infection; %abbit No.; ‘No. of papillomas/No. of infected sites
necessary to determine whether the proliferative response is contained exclusively within the T helper subset of T cells. DNA vaccination of skin can induce strong and long-lived cytotoxic T cell responses15-19 and may have done so in this study. Even proteins that contain nuclear localization signals33 can be presented as peptides in the context of MHC class I molecules on professional antigen presenting cells (APCs), such as Langerhans cells, to activate naive CD8+ T cells that then can lyse target cells expressing the appropriate peptide:MHC class I complexes (reviewed in Ref. 34). If the Ll DNA vaccine induced specific cytotoxicity and if Ll gene functions are required for papilloma formation, the vaccine might be able to prevent the induction or maintenance of papillomas. Genetic experiments suggest that Ll gene functions are required for papilloma formation in rabbits, since CRPV mutant genomes with a deletion in Ll do not induce papillomas35 (and our unpublished data). Ll expression in CPRV papillomas is low but appropriately localized in differentiated kera-
tinocytes36 (and our unpublished data) and sufficient for most rabbits to mount Ll-specific antibody37 and T cell 24 months after infection. No studies to responses3’ date have addressed the question of whether cytotoxic T cells do indeed recognize foreign antigens (such as ones derived from Ll) on differentiated keratinocytes in vivo (in other words, whether T cell-mediated immune surveillance extends into the spinous layers of differentiating keratinocytes in papillomas). On the other hand, the presence of lymphocytes within the epidermis immediately adjacent to differentiating keratinocytes is a regularly observed histopathologic phenomenon in a variety of inflammatory skin lesions, including spontaneously that there is no a regressing papillomas 38,39, indicating priori reason to assume that cytotoxic cells cannot gain access to this site. T cell responses were not evaluated in the study by Donnelly et CII.~~, and they did not discuss the regression of a papilloma that occurred in one of their Ll DNA-vaccinated rabbits. However, since papillomas in their control rabbits did not regress, we suggest cytotoxicity induced by the Ll vaccine as an explanation. Even if papillomas can form without Ll, progeny virus in a permissive host cannot be replicated without the major capsid protein. Ll cytotoxicity might therefore be useful for killing Ll-expressing permissive cells, inhibiting the production of infectious viral progeny, and hence interrupting viral transmission. The value of a therapeutic vaccine to prevent transmission of a sexually transmitted disease as widespread as papillomavirus cannot be overestimated. This possibility could be tested by monitoring virus production in papillomas of the permissive cottontail rabbit host after vaccination with pCMV-Ll. Having established that three inoculations of 3Opg pCMV-Ll each are effective, further studies are warranted to determine the minimal effective DNA dose. Most likely, lower doses will be sufficient, since gene gun delivery of as little as 16 ng of DNA can induce effective immune responses in mice4’. Vaccination with a viral gene eliminates the risk of infection while retaining the advantages of in vivo expression. The promoter can be engineered to control the level of antigen expression, the target cell or its state of differentiation. The vaccine gene can be engineered to inactivate potentially pathogenic
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domains. In addition, DNA vaccines, as compared to protein-based vaccines, have the advantage that they are more easily purified. Furthermore, different constructs can be combined in a single DNA vaccine4’, and any formulation issues about different types of vaccines would be eliminated. Assuming that antigenic competition will not be an impediment, DNA vaccination could offer the potential for simultaneous immunization against multiple papillomavirus strains.
16 17
18
19
ACKNOWLEDGEMENTS We are indebted to John Schiller for the CRPV VLPs, Felix Wettstein for antibody to CRPV Ll, Bettie M. Steinberg for CRPV virus, and Auragen Inc., Middleton, WI, for providing the Accell@ gene delivery device, operational protocols, gold beads and helpful discussions. Andrew Makarovskiy, Yanwen Yang, and Namitha Kumar contributed technical assistance. We thank Daniel C. DiMaio and Robert 0. Jacoby for helpful comments on the manuscript. This work was supported by Grants ROl-CA57970, POl-16038, T32CA09159, AI27404 and AR41942.
20
21
22
23
24
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zur Hausen, H. Molecular pathogenesis of cancer of the cervix and its causation by specific human papillomavirus types. Cum Top. Microbial. Immunol. 1994, 186, 131-l 56 Orth, G. Epidermodysplasia verruciformis. In: The Papovaviridae, vol. 2, The Papillomaviruses (Eds Salzman, N.P. and Howley, P.M.). Plenum, New York, 1987, pp. 199-243 zur Hausen, H. and devilliers, E-M. Human papillomaviruses. A. Rev. Microbial. 1994, 48, 427-447 Schiller, J.T. and Okun, M.M. Papillomavirus vaccines: current status and future prospects. Adv. Dermatol. 1996, 11, 355-380 Brandsma, J.L. Animal models of human papillomavirusassociated oncogenesis. Intervirology 1994, 37, 189-200 Brandsma, J.L. Animal models for HPV vaccine development. Pap. Rep. 1994,5,105-111 Shope, R.E. Immunization of rabbits to infectious papillomatos/s. J. Exp. Med. 1937, 65, 219-231 Lin, Y.-L., Borenstein, L.A., Selvakumar, R., Ahmed, Ft. and Wettstein, F.O. Effective vaccination against papilloma development by immunization with Ll or L2 structural protein of cottontail rabbit papillomavirus. Virology 1992, 167, 612-619 Christensen, N.D., Hopfl, R., DiAngelo, S.L. et al. Assembled baculovirus-expressed human papillomavirus type 11 Ll capsid protein virus-like particles are recognized by neutralizing monoclonal antibodies and induce high titers of neutralizing antibodies. J. Gen. Viral. 1994, 75, 1171-1176 Breitburd, F., Kirnbauer, R., Hubbert, N.L. et a/. Immunization with virus-like particles from cottontail rabbit papillomavirus (CRPV) can protect against experimental CRPV infection. J. Virol. 1995, 69, 3959-3963 Bell, J.A., Sundberg, J.P., Ghim, S.J., Newsome, J., Jenson, A.B. and Schlegel, R.A. Formalin-inactivated vaccine protects against mucosal papillomavirus infection: a canine model. Pathobiology 1994, 62, 194-l 98 Jansen, K.U., Rosolowsky, M., Schultz, L.D. et a/. Vaccination with yeast-expressed cottontail rabbit papillomavirus (CRPV) virus-like particles protects rabbits from CRPV-induced papilloma formation. Vaccine 1995, 13, 1509-1514 Christensen, N.D., Reed, C.A., Cladel, N.M., Han, R. and Kreider, J.W. Immunization with viruslike particles induces longterm protection of rabbits against challenge with cottontail rabbit papillomavirus. J. Viral. 1996, 70, 960-965 Lin, Y.L., Borenstein, L.A., Ahmed, R. and Wettstein, F.O. Cottontail rabbit papillomavirus-Li -protein-based vaccines: protection is achieved only with a full-length, nondenatured product. J. Viral. 1993, 67, 41544162 Pardoll, D.M. and Beckerleg, A.M. Exposing the immunology of naked DNA vaccines. immunity 1995, 3, 165-169
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McDonnell, W.M. and Askari, F.K. DNA vaccines. N. Engl. J. Med. 1996, 334, 42-45 Ciernik, I.F., Berzofsy, J.A. and Carbone, D.P. Induction of cytotoxic T lymphocytes and antitumor immunity with DNA vaccines expressing single T cell epitopes. J. fmmunot. 1996, 156, 2369-2375 Raz, E., Carson, D.A., Parker, S.E. et a/. lntradermal gene immunization: the possible role of DNA uptake in the induction of cellular immunity to viruses. Proc. NaNAcad. Sci. USA 1994, 91, 9519-9523 Pertmer, T.M., Eisenbraun, M.D., Prayaga, S.K., Fuller, D.H. and Haynes, J.R. Gene gun-based nucleic acid immunization: ellicitation of humoral and cytotoxic T lymphocyte responses following epidermal delivery of nanogram quantitites of DNA. Vaccine 1995,13, 1427-l 430 Fynan, E.F., Webster, R.G., Fuller, D.H., Haynes, J.R., Santoro, J.C. and Robinson, H.L. DNA vaccines-a novel approach to immunization. Int. J. Immunopharmacol. 1995, 17, 79-83 Sprecher, E. and Becker, Y. Role of Langerhans cells and other dendritic cells in viral diseases. Arch. Viral. 1993, 132, l-28 Sundaram, P., Xiao, W. and Brandsma, J.L. Particle-mediated delivery of recombinant expression vectors to rabbit skin induces high-titered polyclonal antisera (and circumvents purification of a protein immunogen). Nucl. Acids Res. 1996, 24, 1375-l 377 Xiao, W. and Brandsma, J.L. High efficiency long term clinical expression of cottontail rabbit papillomavirus (CRPV) DNA in rabbit skin following particle-mediated DNA transfer. Nucl. Acids Res. 1996, 24,2620-2622 Kirnbauer, R., Taub, J., Greenstone, H. et al. Efficient selfassembly of human papillomavirus type 16 Ll and Ll-L2 into virus-like particles. J. Viral. 1993, 67, 6929-6936 Sundaram, P. and Brandsma, J.L. Rapid, efficient, large-scale purification of unfused, non-denatured E7 protein of cottontail rabbit papillomavirus. J. Viral. Meth. 1996, 57, 61-70 Donnelly, J.J., Martinez, D., Jansen, K.U., Ellis, R.W., Montgomery, D.L. and Liu, M.A. Protection against papillomavirus with a polynucleotide vaccine. J. Infect. Dis. 1996, 173, 314-320 Zhou, J., Sun, X.Y., Stenzel, D.J. and Frazer, I.H. Expression of vaccinia recombnant HPV16 Ll and L2 ORF proteins in epithelial cells is sufficient for assembly of HPV virion-like particles. Virology 1991, 165, 251-257 Kirnbauer, R., Booy, F., Cheng, N., Lowy, D.R. and Schiller, J.T. Papillomavirus Ll major capsid protein self-assembles into virus-like particles that are hiqhlv immunoqenic. Proc. Nat/ Acad. Sci.‘USA 1992, 69, 1218ki2184 Germain. R.N. and Marauiles. D.H. The biochemistrv and cell biology of antigen process and presentation. A. Rev.‘lmmunol. 1993, 11, 403-450 Miller, J. and Sant, A.J. MHC class II-restricted antigen presentation cells. Sem. Immunol. 1995, 7, 343-346 Selvakumar, R., Borenstein, L.A., Ahmed, R. and Wettstein, F.O. T-cell response to cottontail rabbit papillomavirus structural proteins in infected rabbits. J. Viral. 1994, 66, 4043-4048 Kotani, M., Yamamura, Y., Tamatani, T., Kitamura, F. and Miyasaka, M. Generation and characterization of monoclonal antibodies against rabbit CD4, CD5, and CDlla antigens. J. Immunol. Meth. 1993, 157, 241-252 Ulmer, J.B., Donnelly, J.J., Parker, S.E. et al. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 1993, 259, 1745-l 749 Rock, K.L. and Unanue, E.R. Antigen recognition, an editorial overview. Curr. Opinion Immunol. 1995, 7, 65-68 Nasseri, M., Meyers, C. and Wettstein, F.O. Genetic analysis of CRPV pathogenesis: the Ll open reading frame is dispensable for cellular transformation but is required for papilloma formation. Virology 1989, 165, 251-257 Zeltner, R., Borenstein, L.A., Wettstein, F.O. and Iftner, T. Chanqes in RNA expression pattern during the malignant progression of cottontail rabbit papillomavirus induced tumors in rabbits. J. Viral. 1994. 66, 3620-3630 Lin, Y.-L., Borenstein, L.A.,. Selvakumar, R., Ahmed, R. and Wettstein, F.O. Progression from papilloma to carcinoma is accompanied by changes in antibody response to papillomavirus proteins. J. Viral. 1993, 67, 382-389 Murphy G.F. Dermatopathology: A Practical Guide to Common Disorders; chapter 3, Spongiotic Dermatitis. W.B. Saunders Co. Philadelphia, 1995, pp. 49-71
lntracutaneous 39
40
Berman, A. and Winkelmann, R.K. Involuting common warts; clinical and histopathologic findings. J. Am. Acad. Dermatol. 1980, 3,356-362 Haynes, JR, Fuller, D.H., Eisenbraun, M.D., Ford, M.J. and Pertmer, T.M. Accell particle-mediated DNA immunization elicits
vaccination with papillomavirus
41
L 7 gene: I? Sundaram et al.
humoral, cytotoxic, and protective immune responses. AlDS Res. Hum. Retroviruses 1994, 102, S43-S45 Barry, M.A., Lai, W.C. and Johnston, S.A. Protection against mycoplasma infection using expression-library immunization. Nature 1995, 377, 632-635
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Elsevier
PLI: SO264-41OX(96)00237-X
ELSEVIER
Vaccine, Vol. 15, No.-6/7, pp. 664-671, 1997 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-410x197 $17+0.00
Intracutaneous vaccination of rabbits with the cottontail rabbit papillomavirus (CRPV) Ll gene protects against virus challenge Pazhani
Sundaram*JJ,
Robert
E. Tigelaarf$$
and Janet L. Brandsma*§T
A DNA vaccine encoding the major capsid protein LI of cottontail rabbit papillomavirus (CRPV) was constructed and administered intracutaneously (i.c.) to rabbits as supercoiled plasmids bound to gold beads using a specialized delivery device (“gene gun “). LI DNA-vaccinated rabbits developed cellular proltferative responses to CRPV virus-like particles and developed high titered antibodies with neutralizing activity to CRPV Following experimental challenge with CRPV. all of the Ll DNA-vaccinated rabbits, vs none of the controls, were protectedfrom papilloma formation. These results demonstrate that i.e. vaccination of rabbits with the Lf papillomavirus capsid gene can induce antibodies that protect against subsequent papillomavirus infection. 0 1997 Elsevier Science Ltd. Keywords:
DNA
vaccination;
papillomavirus;
LI gene
The association of human papillomavirus (HPV) infection with cervical cancer in women and with skin cancers in patients with Epidermodysplasia verruciformis is well established’*2. Additionally, HPVs may be involved in head, neck, other anogenital and other skin cancers, bringing the estimated proportion of all cancers associated with HPV to 10%3. Precancerous HPV lesions may regress spontaneously, or they may persist for years. Therapies for the control of HPV disease are not always effective and do not usually eliminate infection. Therefore the search for a vaccine to prevent HPV infection, and hence HPV-associated cancer, has received much attention4. Vaccine development requires animal models, but species barriers prevent direct infection of animals with HPVS~,~. Using cottontail rabbit papillomavirus (CRPV) in rabbits, bovine papillomavirus (BPV) in cattle, and canine oral papillomavirus (COPV) in dogs, previous studies have shown that prophylactic immunity can be induced by vaccination with whole papilloma*Section of Comparative Medicine, Yale University School of Medicine. New Haven. CT 06520. USA. tDebartment of Dermatology, Yale University School of Medicine, New Haven, CT 06520, USA. ISection of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA. $Yale Skin Diseases Research Center, Yale University School of Medicine, New Haven, CT 06520, USA. fiDepartment of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT 06520, USA. IICurrent address: Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA. “To whom correspondence should be addressed. (Received 22 July 1996; revised 16 October 1996; accepted 16 October 1996)
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viruses, virus-like particles (VLPs), recombinant proteins or recombinant vaccinia virus6,m’3. Prophylactic immunity correlated with papillomavirus neutralizing antibodies to conformational epitopes of Ll and could not be induced with truncated or denatured Ll proteins14. A recent approach to vaccination that can induce potent and long-lived humoral and cell-mediated immunity is the direct inoculation of a host with a recombinant expression vector’ 5m1 7. This method eliminates the need to induce high level protein expression in vitro and to purify the protein product. DNAs can be delivered intramuscularly (i.m.) with a needle or intracutaneously (ic.) with a particle-mediated gene delivery device (gene gun). Both inoculation routes induce humoral and cellular immunity, but the i.c. route induces superior antibody responses183’9. Gene gun DNA vaccination of mice, monkeys, pigs, and ferrets has effectively induced immunity, presumably by inducing gene expression in Langerhans and other dendritic cells that process foreign proteins and, following migration to regional lym h nodes, present the antigens to the immune system’- ’ 21. The i .c . route induces gene expression in epidermal keratinocytes whose protein products can be ingested by adjacent dendritic cells for subsequent. processing and antigen presentation2’. We recently reported the optimization of gene gun-mediated DNA delivery conditions for gene expression in rabbit epidermis2’.2.. This report describes the application of i.c. DNA vaccination to rabbits and demonstrates that the CRPV Ll gene can induce strong and specific humoral and cellular immune responses to Ll and protect the rabbits against experimental challenge with CRPV virus.
lntracutaneous vaccination with papillomavirus L 7 gene: I? Sundaram et al. MATERIALS
AND
METHODS
Construction of an expression vector for CRPV Ll protein Plasmid pCMV$ (Clonetech, Palo Alto, CA) expresses the @-galactosidase v-gal) gene from a cytomegalovirus (CMV) promoter/enhancer. It served as the parental vector after removal of the /?-galactosidase gene. Plasmid pCMV-Ll was generated by inserting a polymerase chain reaction (PCR) fragment at the Not I site of the pCMV parental plasmid. The PCR fragment was amplified from CRPV-pLAI1 template DNA using as sense primer: 5’-GT CAC GTC Age ggc cgc ATG GCA GTG TGG CTG TG-3’; and as antisense primer: S-TG ACA TCG Tgc ggc cgc TTA AGT ACG TCT CTT GCG-3’ (initiation and stop codons are italicized; Not I sites are lower case). It contained CRPV nucleotides 5828-7345 in the correct orientation, as determined by restriction endonuclease cleavage patterns. Plasmid pCMV-N contained the N gene of mouse hepatitis virus (MHV) under the control of a CMV promoter in vector pcDNA3 (Invitrogen, San Diego, CA). Supercoiled plasmid DNAs were purified using the Qiagen (Qiagen, Inc., Chatsworth, CA) method and used as supercoiled molecules. Transient transfection and imnnmofluorescent detection of Ll protein Six well plates of subconfluent CHO cells were transfected for 5 h at 37°C with occasional shaking in 1 ml of serum-free antibiotic-free medium containing 1 pug DNA and 5~1 lipofectamine (Life Technologies, Gaithersburg, MD). The transfection medium was then replaced with complete medium containing serum and antibiotics. The medium was changed after overnight incubation. Thirty-six hours after transfection, cells were fixed in cold acetone for 10 min, air dried, and incubated with a rabbit antiserum to CRPV Ll generated by protein immunization with a trpE-Ll fusion protein*. Cells were washed and incubated with a secondary, fluorescein-labeled goat anti-rabbit IgG. Administration of DNA vaccines DNA vaccines were prepared and administered to 2 kg New Zealand white rabbits, using an Accell@ gene delivery device (Auragen, Inc., Middleton, WI) with 350 p.s.i. of helium pressure, as described previously22. Rabbits were vaccinated on day 0, and booster vaccinations were given on days 21, 42, and 63. Rabbits were inoculated at each of 30 sites with 1 pg of DNA at each time point. The DNA dose was chosen arbitrarily. Two rabbits were vaccinated with pCMV-N, three with pCMV-/3, and five with pCMV-Ll. Two pCMV+’ immunized rabbits died after the first boost, due to causes unrelated to the experiment. Enzyme-linked immunosorbent assays (ELISAs) CRPV VLPs, prepared in a baculovirus expression system and composed of CRPV Ll and L2 proteins24, were generously_donated by John Schiller. ELISA plate wells were coated with the CRPV VLPs, or with P-galactosidase (Sigma Chemical Co., St. Louis, MO). Wells containing 100 ng of antigen were reacted with serial twofold dilutions of rabbit serum. Staphylococcal protein A coupled to horseradish peroxidase was used as
the enzyme conjugate. Serum dilutions giving an A,,, of 20.10 above the matched pre-immune sample were considered positive. Positive control antibodies were the rabbit antiserum to a TrpE-CRPV Ll fusion protein’, and a commercial mouse monoclonal antibody to /I-gal (Sigma Chemical Co., St. Louis, MO).
Western blots Western blots were prepared and processed as deprotein A was scribed previously22.25. Staphylococcal used to detect rabbit antibody, and goat anti-mouse IgG was used to detect mouse antibody.
In vitro proliferation of peripheral blood mononuclear cells (PBMC) In vitro proliferation assays were conducted 2 weeks after the final DNA booster vaccinations. Approximately 20 ml of blood per rabbit was collected from an ear vein into heparinized tubes, diluted 1:l with RPM1 1640 (GIBCO) buffered with 10 mM HEPES, layered over a half volume of Histopaque-1077 (1.077 g ml-‘, Sigma Diagnostics, St. Louis, MO) and centrifuged at 400g for 30 min at 25°C. PBMC at the interface were harvested, washed three times at 4”C, and then resuspended to 2 x lo6 viable PBMC ml- ’ in RPM1 1640 supplemented with 10% fetal calf serum, 25 mM HEPES buffer, 2 mM L-glutamine, 1 mM Na pyruvate, 0.1 mM nonessential amino acids, 5 x 10e5 M 2-mercaptoethanol, 100 U ml-’ penicillin and lOOpug ml-’ streptomycin (“complete” RPMI, or CRPMI). Proliferation assays were performed in 96-well round bottom microtiter plates. To each well was added a 100~1 aliquot of cells (2 x 105) and 100~1 of CRPMI containing either no antigen/mitogen (background response), 6 ,ug ml- ’ of the T cell mitogen concanavalin A (Sigma, St. Louis, MO) as a positive control for T cell proliferation, or graded concentrations (0.02 to 20 pg ml- I) of either /3-galactosidase (Sigma) or CRPV VLPS~~ (the concentrations refer to the estimated concentration of the Ll protein in the VLP preparations). Cultures were incubated at 37°C in humidified 5% CO, for 3 days to assay Con A responses and 5 days to assay responses to P-gal and Ll. Eighteen hours prior to harvest, wells were pulsed with 1 &i [“Hlthymidine (sp. act. 6.7 Ci mmol-‘; ICN, Costa Mesa, CA). Triplicate wells for each dose of antigen were harvested using a semi-automated cell harvester (Skatron Instruments, Vernon, VA); filter discs were placed in vials containing Ecoscint (National Diagnostics, Atlanta, GA), and counted in a liquid scintillation counter (LKB-Wallace, Turku, Finland). Because of substantial variability in the background responses (in the absence of antigen or mitogen) of individual rabbits, which ranged from 1830 f 890 to 12720 f 3980 (mean c.p.m. f S.E.M.), the data are expressed as the stimulation index (S.I.) f S.E.M., where: S.I.=
well containing antigen c p m. of experimental . mean c.p.m. of triplicate control wells (CRPMI only).
Differences between groups unpaired Student’s t-test.
were analyzed
by use of
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Experimental production of infectious CRPV virus A cottontai1 (Sylvilagus cuniculus) rabbit was anesthetized with ketamine (15 mg kg- ‘) and inoculated at each of 35 scarified sites with 30~1 of CRPV virus. The inoculum was prepared by adding to 1.2 ml of phosphate-buffered saline 150~1 of a crude extract of a naturally occurring wild cottontail rabbit papilloma, donated by Bettie Steinberg. Papillomas formed at all sites. Three months after inoculation, biopsies of two papillomas were obtained under ketamine anesthesia, minced in DMEM medium, and implanted subcutaneously into severe combifled immunodeficient (scid) mice for further growth. Subcutaneous papillomas developed at each inoculation site and were passaged to new scid mice 2 months after implantation. Papillomas were harvested from second passage mice 1 month later. The tissue was homogenized in a tissue grinder using ca 1 ml of PBS per 100 mg of tissue and then clarified by low-speed centrifugation. An equal volume of glycerol was added and the material was stored at - 80°C. The amount of virus required to induce papillomas in New Zealand white rabbits was estimated in a pilot experiment. Challenge with CRPV virus CRPV virus was diluted in PBS to l:lO, 1:40, and 1: 160. Six weeks after the last immunizations, three sites per rabbit were infected with each dilution, by applying 30~1 to scarified skin. Papilloma formation was monitored weekly in the second month after challenge and biweekly in the third month. Neutralization of CRPV infectivity Serial tenfold dilutions of preimmune and hyperimmune serum from a Ll DNA-vaccinated rabbit were prepared in PBS containing 1% bovine serum albumin. Fifteen microliters of diluted serum was mixed with 15 ~1 of a 1:60 dilution of CRPV virus, incubated on ice for 2-3 h, and applied by scarification to two sites on a naive rabbit. Two rabbits were inoculated with the preimmune mixture and two with the immune mixture. Each rabbit was also inoculated in duplicate with CRPV virus and no serum as a control.
RESULTS Construction of a CRPV Ll expression vector Plasmid pCMV-Ll was constructed to transcribe the full length Ll gene of CRPV from the CMV immediate early promoter. Its ability to express Ll protein was verified in transiently transfected CHO cells by immunofluorescent staining with a CRPV Ll antibody’. pCMV-Ll transfected cells expressed Ll protein in the nucleus 36 h after transfection (Figure 1). Cells transfected with the parental pCMV vector and stained with the Ll antibody were negative. Replicate wells stained with a non-immune rabbit serum also were negative. DNA vaccination and induction of humoral immunity to conformational epitopes Rabbits were vaccinated by gene gun inoculation of skin with DNA plasmids bound to gold beads. The experimental vaccine pCMV-Ll expressed CRPV Ll
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Figure 1 Ll expression induced by pCMV-Ll transfection of CHO cells. The top panel shows immunofluorescent staining of cells transfected with pCMV-Ll. The bottom panel shows cells transfected in parallel with the parental pCMV vector and stained
120,000 100,ooo
80,000 60,000 40,000 20,000 0 Serum:
Ls A
t
Pre Bl
B2 B3
B
Pre Bl
B2 B3
Vaccine:
pCMV-CRLl
pCMV-P
Antigen:
VLP
Pgd
Figure 2 Antibody development in DNA-vaccinated rabbits. Sera obtained prior to vaccination (Pre), and after the first (Bl), second (B2) and third (83) boosts were tested. Control antibody to p-gal reacted at 1:6,400 in the p-gal ELISA, and control antiserum to Ll reacted at 1:120000 in the CRPV VLP ELISA. Arithmetic mean titers and their standard deviations are shown
protein. Plasmid pCMV$ expressed P-galactosidase Cpgal) and served as a positive control for induction of humoral and cellular immune responses. pCMV-/? and pCMV-N served as negative controls for protection from CRPV infection. Five of five rabbits vaccinated with pCMV-Ll and three of three rabbits vaccinated with pCMV$ developed specific humoral immune responses to CRPV VLPs or P-gal, respectively. Antibody titers were high after the first boost and increased after the second boost to a mean of 1:96000 in rabbits vaccinated with pCMV$ and 1: 104000 in rabbits vaccinated with pCMV-Ll (Figure 2). Increases after the third boost were minimal. Western blot analyses confirmed the ELISA findings and provided further documentation of the specificity of the humoral immune responses (Figure 3). Rabbits vaccinated with pCMV-P
lntracutaneous
#l
#2
#3
#4
#5
c
Rabbit Figure 3 Specificity of antibody responses in DNA-vaccinated rabbits. Western blots contained 1 pg of CRPV VLPs (A) or P-galactosidase (B) and were probed with preimmune (P) or immune sera (I). (A) Probes are antiserum from rabbits immunized with pCMV-Ll (Nos l-5), control antiserum to CRPV Ll (Ll), or antiserum from a rabbit immunized with pCMV$ (B). Molecular weight markers are 106, 80, 49.5, 32.5, 27.5, and 18.5 kDa. (B) Probes are antiserum from rabbits immunized with pCMV-8 (Nos 6-8) control antibody to /I-gal (8). or antiserum from a rabbit immunized with pCMV-Ll (Ll). Molecular weight markers are 205, 116.5. 80, 49.5, and 32.5 kDa
developed antibodies that recognized P-gal but not CRPV VLPs. Similarly, rabbits vaccinated with pCMV-Ll developed antibodies to the Ll protein in the CRPV VLPs but not to P-gal. The nature of the Ll epitopes recognized by antisera from the Ll DNA-vaccinated rabbits was characterized by ELISAs using VLPs that had been denatured by boiling for 10 min as well as VLPs in the native conformation. In each case, the titer to denatured epitopes was about an order of magnitude lower than the titer to conformational epitopes (Figure 4). In the same assay, stronger reactivity to denatured than native VLPs was observed by a control rabbit antiserum to disrupted CRPV VLPs. We therefore conclude that gene gun vaccination with the Ll gene induced humoral immunity to conformational epitopes predominantly. In vitro proliferative responses Data summarizing the day 5 proliferative responses of PBMC from the eight vaccinated rabbits in this study are presented in Table 1. Each rabbit’s cells demonstrated a vigorous response on day 3 to the T cell mitogen Con A (data not shown). PBMC from each of the five rabbits vaccinated with pCMV-Ll exhibited significantly greater responses to Ll than to P-gal, and while their mean proliferative response to B-gal was not significantly higher than their background response to media alone (mean S.I.=l.2&0.4, mean c.p.m.= 11030& 2770), their mean response to Ll was highly significant (mean S.I.=3.3 f 0.6, mean c.p.m.=32890& 2180, PcO.005). In contrast, PBMC from control rabbit No. 6 vaccinated with pCMV+gal proliferated significantly in response to P-gal but not to Ll (S.I. 5.6& 1 vs 0.9&012), while control rabbits Nos 7 and 8 vaccinated with pCMV-N exhibited minimal responses either to Ll (S.I. 1.2-1.9) or to /?-gal (S.I. 1.5-1.6).
vaccination with papillomavirus
L 1 gene: I? Sundaram et al.
Protection against challenge with CRPV virus following DNA vaccination Effective vaccination should protect naive animals from infection. The ability of vaccinated rabbits to resist CRPV-induced papilloma formation was evaluated by virus challenge at three sites each with l:lO, 1:40, and 1: 160 dilutions of CRPV. Papillomas developed at 27 of 27 infected sites in three rabbits vaccinated with pCMV-/I or pCMV-N (Table 2). In contrast, 44 of the 45 sites (98%) in the pCMV-Ll-vaccinated rabbits remained free of disease for 3 months after challenge (the duration of the experiment) (Table 2). Furthermore, the single papilloma that formed was at all times smaller than any in the control rabbits (Figure 5). Fifteen weeks after infection it was 10 times smaller than control papillomas: 0.64 cm3 vs 9.6 f 5.1 cm3 (mean f SD.), indeed it was smaller than every one of the control lesions. These results demonstrate clearly that i.c. DNA vaccination with pCMV-Ll elicited strong prophylactic immunity. Neutralizing antibody to CRPV Previous studies have shown that prophylactic immunity to papillomavirus infection correlates with the presence of neutralizing antibody. We used an in vivo assay to show that CRPV virus was neutralized by immune serum at dilutions of I 10e3 but not by preimmune serum at the same dilutions (Table 3). This difference was highly significant (one-tailed Student’s r-test, two-sample unequal variance, P
DISCUSSION This study demonstrates that Ll antigen-specific humoral immunity and highly prophylactic immunity against CRPV infection can be achieved by i.c. vaccination of rabbits with the CRPV Ll capsid gene. Rabbits vaccinated with pCMV-Ll developed high titered antisera that reacted specifically with Ll antigens (Figure 2 and Figure 3), recognized predominantly conformational Ll epitopes (Figure 4), and neutralized CRPV (Table 3). Furthermore, Ll DNA-vaccinated rabbits were protected from papilloma formation at 44 of 45 sites (97.8%) challenged with CRPV virus, whereas control rabbits developed papillomas at all infected sites (Table 2). Moreover, the single papilloma in a Ll DNA-vaccinated rabbit was more than ten times smaller than the mean size of control papillomas (Figure 5). Interestingly, this rabbit had the lowest titer to native VLPs, suggesting that protection requires a titer of 2 1:60000, as measured by ELISA to native VLPs. While our work was in progress, Donnelly et al. published a Ll DNA vaccination study in the CRPVrabbit model using an i.m. route of vaccination26. The most important difference between our study design and
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667
lntracutaneous
L 1 gene: t? Sundaram et al.
vaccination with papillomavirus
Rabbit:
# 1
#4
#6
#5 I
301 25 % = w .M VI
20 15 -
!J =: ‘& n”
10 5 -
o5 8 11
5 8 11
I
j 8 11
5 8 11
5 8 11
5 8 11
5 8 11
5 8 11
Weeks after challenge Figure 4 Reactivity to conformational epitopes of CRPV VLPs among antisera from pCMV-Ll vaccinated rabbits. Dark bars represent the mean titer of reactivity against native VLPs; light bars, against denatured VLPs. Rabbits Nos l-5 were vaccinated with pCMV-Ll. Rabbit “C” was immunized with denatured VLPs. The sera were diluted to 1:50000
Table 1 Day 5 proliferative responses vaccinated rabbits to Ll (VLP) and b-gal Stimulation
of PBMC
from
index (SI)&.E.M.
to:
Vaccine
Rabbit
Ll (VLP)
P-gal
pCMV-CRLl
No. No. No. No. No.
pCMV-/I pCMV-N
No. 6 No. 7 No. 8
3.7*0.3 5.1rt0.2 3.2kO.2 3.2*0.2 1.4*0.4 Mean=3.3*0.6 0.9*0.2 1.9iO.6 1.2io.4
1.5to.2 1.8kO.2 1.2*0.2 0.9*0.2 0.5*0.0 Mean=l.2*0.4 5.6&l 1.5*0.3 1.6*0.4
Table 2 Frequency with CRPV
1 2 3 4 5
of papilloma
formation
DNA-
following
challenge
Papilloma formationa Vaccine
Rabbit
Sites inoculated at: 1:lO 1:40 1:160
pCMV-CRLl
No. No. No. No. No.
o/3 o/3 o/3 o/3 o/3
o/3 o/3 o/3 o/3 o/3
o/3 o/3 o/3 113 o/3
Controlsb
No. 6 No. 7 No. 8
313 b 313 313
313 313 313
313 313 313
1 2 3 4 5
All sites
Protection W)
o/9 o/9 o/9 l/9 o/9 1145 919 919 919 27127
100 100 100 88.9 100 97.8 : :
sNo. of papilloma-forming siteslNo. of challenge sites; bRabbit No. 6 was vaccinated with pCMV-/I; rabbits Nos 7 and 8 with pCMV-N
theirs is the route of DNA delivery, although other differences-the sequences of the Ll constructs and the sources of baculovirus-derived VLPs and CRPV virus stocks-may also have influenced the results. Bearing in mind that the i.c. and i.m. routes were not directly compared, the following observations can nevertheless be made. In both studies, Ll-vaccinated rabbits developed neutralizing antibodies and were completely protected from papilloma formation at 97.8% (Table 2)
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or 96.7%26 of CRPV challenge sites. However, since papillomas formed at c 100% (28/30=940/o) of their control sites, our challenge appears to have been more stringent. Moreover, our CRPV virus stock (used here at dilutions of I 1: 160) is sufficient to induce papillomas at 100% of challenge sites infected with a 1:270 dilution (data not shown). In addition, the specific antibody titers induced by Ll DNA-vaccination appeared to be higher in our study: ca 1: 100000 (Figure 2) vs I 1:ll 000Z6, as measured by ELISA using 0.1 pug VLP per well here or 0.5pg VLP per well in Ref. 26. Since i.c. routes of DNA vaccination, when compared directly to i.m. routes, induce superior humoral immune responses’ *.19, our results should help guide the development of a Ll-based HPV DNA vaccine. Transient assays of pCMV$ expression in rabbit skin22.23 strongly suggest that, following vaccination, the CRPV Ll gene product was expressed in epidermal keratinocytes and epidermal Langerhans cells or other i.c. dendritic cells. Ll protein expressed by the vaccine gene localized to the nucleus of transfected cells and, in vivo, may have spontaneously assembled into empty CRPV capsids, as suggested by experimental studies in insect and mammalian ce11s24.27,28.Capsids may have assembled in highly differentiated keratinocytes, as in natural infection, or in other cells. Release of Llcontaining empty CRPV capsids would be expected to result in their phagocytosis or endocytosis by dendritic cells, followed by endosomal/lysosomal processing into peptides for presentation, in the context of MHC class II molecules, to the CD4+ subset of rabbit T cells that provide help for B cells to produce antigen specific IgG antibodies that recognize conformational (threedimensional) epitopes29.30. Indeed, PBMCs from Ll DNA-vaccinated rabbits demonstrated antigen specific proliferative responses in vitro to CRPV Ll-containing VLPs; similar responses were not observed in control animals vaccinated with other DNA preparations (Table 1). These responses were virtually certainly those of T lymphocytes, as documented previously in cells from rabbits bearing virus-induced papillomas or carcinostudies using antibody-mediated mas31. Additional depletion of the CD4 subset of rabbit T cells32 will be
lntracutaneous
#2
vaccination with papillomavirus
#4
#3
#5
#6
L 1 gene: I? Sundaram et al.
_j
_-o 7 1115
7 1115
#8
#7
7 1115
Weeks
7 1115
7 1115
7 1115
after challenge
Figure 5 Papilloma size and growth rate in DNA-vaccinated rabbits. Each panel represents one rabbit. Rabbits Nos 1-5 were vaccinated with pCMV-Ll , rabbit No. 6 with pCMV-8, and rabbits Nos 7 and 8 with pCMV-N. Measurements obtained 7, 11, and 15 weeks after CRPV challenge are shown. Open circles represent the size of individual papillomas (nine per rabbit), and bars represent their mean size
Table 3 Neutralization DNA-vaccinated rabbit
of CRPV
infectivity
by serum from a Ll
Papilloma formation Serum
Dilution
lb
2
Both
Papilloma sizes mean+S.D.
Preimmune
10 o lo-’ 1o-* 10-S 1o-4 1o-5
212 = 212 212 212 212 212 212
212 212 212 l/2 Z 212
414 414 414 314 414 414 414
N.D. N.D. N.D. N.D. N.D. N.D. N.D.
3
4
Both
o/2 o/2 o/2 l/2 Z
l/2 o/2 l/2 212 212 212 212
114 o/4 II4 314 414 414 414
None
Immune
None
10 o 1o-’ 1o-* 1 o-3 1 o-4 1 o-5
2l2
2.1 1.7 0.2*0.1 N.D. N.D. 43.oZt9.0
=Mean size (cm3)+S.D. Measurements were obtained 7 weeks after infection; %abbit No.; ‘No. of papillomas/No. of infected sites
necessary to determine whether the proliferative response is contained exclusively within the T helper subset of T cells. DNA vaccination of skin can induce strong and long-lived cytotoxic T cell responses15-19 and may have done so in this study. Even proteins that contain nuclear localization signals33 can be presented as peptides in the context of MHC class I molecules on professional antigen presenting cells (APCs), such as Langerhans cells, to activate naive CD8+ T cells that then can lyse target cells expressing the appropriate peptide:MHC class I complexes (reviewed in Ref. 34). If the Ll DNA vaccine induced specific cytotoxicity and if Ll gene functions are required for papilloma formation, the vaccine might be able to prevent the induction or maintenance of papillomas. Genetic experiments suggest that Ll gene functions are required for papilloma formation in rabbits, since CRPV mutant genomes with a deletion in Ll do not induce papillomas35 (and our unpublished data). Ll expression in CPRV papillomas is low but appropriately localized in differentiated kera-
tinocytes36 (and our unpublished data) and sufficient for most rabbits to mount Ll-specific antibody37 and T cell 24 months after infection. No studies to responses3’ date have addressed the question of whether cytotoxic T cells do indeed recognize foreign antigens (such as ones derived from Ll) on differentiated keratinocytes in vivo (in other words, whether T cell-mediated immune surveillance extends into the spinous layers of differentiating keratinocytes in papillomas). On the other hand, the presence of lymphocytes within the epidermis immediately adjacent to differentiating keratinocytes is a regularly observed histopathologic phenomenon in a variety of inflammatory skin lesions, including spontaneously that there is no a regressing papillomas 38,39, indicating priori reason to assume that cytotoxic cells cannot gain access to this site. T cell responses were not evaluated in the study by Donnelly et CII.~~, and they did not discuss the regression of a papilloma that occurred in one of their Ll DNA-vaccinated rabbits. However, since papillomas in their control rabbits did not regress, we suggest cytotoxicity induced by the Ll vaccine as an explanation. Even if papillomas can form without Ll, progeny virus in a permissive host cannot be replicated without the major capsid protein. Ll cytotoxicity might therefore be useful for killing Ll-expressing permissive cells, inhibiting the production of infectious viral progeny, and hence interrupting viral transmission. The value of a therapeutic vaccine to prevent transmission of a sexually transmitted disease as widespread as papillomavirus cannot be overestimated. This possibility could be tested by monitoring virus production in papillomas of the permissive cottontail rabbit host after vaccination with pCMV-Ll. Having established that three inoculations of 3Opg pCMV-Ll each are effective, further studies are warranted to determine the minimal effective DNA dose. Most likely, lower doses will be sufficient, since gene gun delivery of as little as 16 ng of DNA can induce effective immune responses in mice4’. Vaccination with a viral gene eliminates the risk of infection while retaining the advantages of in vivo expression. The promoter can be engineered to control the level of antigen expression, the target cell or its state of differentiation. The vaccine gene can be engineered to inactivate potentially pathogenic
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domains. In addition, DNA vaccines, as compared to protein-based vaccines, have the advantage that they are more easily purified. Furthermore, different constructs can be combined in a single DNA vaccine4’, and any formulation issues about different types of vaccines would be eliminated. Assuming that antigenic competition will not be an impediment, DNA vaccination could offer the potential for simultaneous immunization against multiple papillomavirus strains.
16 17
18
19
ACKNOWLEDGEMENTS We are indebted to John Schiller for the CRPV VLPs, Felix Wettstein for antibody to CRPV Ll, Bettie M. Steinberg for CRPV virus, and Auragen Inc., Middleton, WI, for providing the Accell@ gene delivery device, operational protocols, gold beads and helpful discussions. Andrew Makarovskiy, Yanwen Yang, and Namitha Kumar contributed technical assistance. We thank Daniel C. DiMaio and Robert 0. Jacoby for helpful comments on the manuscript. This work was supported by Grants ROl-CA57970, POl-16038, T32CA09159, AI27404 and AR41942.
20
21
22
23
24
REFERENCES 1
2
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13
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McDonnell, W.M. and Askari, F.K. DNA vaccines. N. Engl. J. Med. 1996, 334, 42-45 Ciernik, I.F., Berzofsy, J.A. and Carbone, D.P. Induction of cytotoxic T lymphocytes and antitumor immunity with DNA vaccines expressing single T cell epitopes. J. fmmunot. 1996, 156, 2369-2375 Raz, E., Carson, D.A., Parker, S.E. et a/. lntradermal gene immunization: the possible role of DNA uptake in the induction of cellular immunity to viruses. Proc. NaNAcad. Sci. USA 1994, 91, 9519-9523 Pertmer, T.M., Eisenbraun, M.D., Prayaga, S.K., Fuller, D.H. and Haynes, J.R. Gene gun-based nucleic acid immunization: ellicitation of humoral and cytotoxic T lymphocyte responses following epidermal delivery of nanogram quantitites of DNA. Vaccine 1995,13, 1427-l 430 Fynan, E.F., Webster, R.G., Fuller, D.H., Haynes, J.R., Santoro, J.C. and Robinson, H.L. DNA vaccines-a novel approach to immunization. Int. J. Immunopharmacol. 1995, 17, 79-83 Sprecher, E. and Becker, Y. Role of Langerhans cells and other dendritic cells in viral diseases. Arch. Viral. 1993, 132, l-28 Sundaram, P., Xiao, W. and Brandsma, J.L. Particle-mediated delivery of recombinant expression vectors to rabbit skin induces high-titered polyclonal antisera (and circumvents purification of a protein immunogen). Nucl. Acids Res. 1996, 24, 1375-l 377 Xiao, W. and Brandsma, J.L. High efficiency long term clinical expression of cottontail rabbit papillomavirus (CRPV) DNA in rabbit skin following particle-mediated DNA transfer. Nucl. Acids Res. 1996, 24,2620-2622 Kirnbauer, R., Taub, J., Greenstone, H. et al. Efficient selfassembly of human papillomavirus type 16 Ll and Ll-L2 into virus-like particles. J. Viral. 1993, 67, 6929-6936 Sundaram, P. and Brandsma, J.L. Rapid, efficient, large-scale purification of unfused, non-denatured E7 protein of cottontail rabbit papillomavirus. J. Viral. Meth. 1996, 57, 61-70 Donnelly, J.J., Martinez, D., Jansen, K.U., Ellis, R.W., Montgomery, D.L. and Liu, M.A. Protection against papillomavirus with a polynucleotide vaccine. J. Infect. Dis. 1996, 173, 314-320 Zhou, J., Sun, X.Y., Stenzel, D.J. and Frazer, I.H. Expression of vaccinia recombnant HPV16 Ll and L2 ORF proteins in epithelial cells is sufficient for assembly of HPV virion-like particles. Virology 1991, 165, 251-257 Kirnbauer, R., Booy, F., Cheng, N., Lowy, D.R. and Schiller, J.T. Papillomavirus Ll major capsid protein self-assembles into virus-like particles that are hiqhlv immunoqenic. Proc. Nat/ Acad. Sci.‘USA 1992, 69, 1218ki2184 Germain. R.N. and Marauiles. D.H. The biochemistrv and cell biology of antigen process and presentation. A. Rev.‘lmmunol. 1993, 11, 403-450 Miller, J. and Sant, A.J. MHC class II-restricted antigen presentation cells. Sem. Immunol. 1995, 7, 343-346 Selvakumar, R., Borenstein, L.A., Ahmed, R. and Wettstein, F.O. T-cell response to cottontail rabbit papillomavirus structural proteins in infected rabbits. J. Viral. 1994, 66, 4043-4048 Kotani, M., Yamamura, Y., Tamatani, T., Kitamura, F. and Miyasaka, M. Generation and characterization of monoclonal antibodies against rabbit CD4, CD5, and CDlla antigens. J. Immunol. Meth. 1993, 157, 241-252 Ulmer, J.B., Donnelly, J.J., Parker, S.E. et al. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 1993, 259, 1745-l 749 Rock, K.L. and Unanue, E.R. Antigen recognition, an editorial overview. Curr. Opinion Immunol. 1995, 7, 65-68 Nasseri, M., Meyers, C. and Wettstein, F.O. Genetic analysis of CRPV pathogenesis: the Ll open reading frame is dispensable for cellular transformation but is required for papilloma formation. Virology 1989, 165, 251-257 Zeltner, R., Borenstein, L.A., Wettstein, F.O. and Iftner, T. Chanqes in RNA expression pattern during the malignant progression of cottontail rabbit papillomavirus induced tumors in rabbits. J. Viral. 1994. 66, 3620-3630 Lin, Y.-L., Borenstein, L.A.,. Selvakumar, R., Ahmed, R. and Wettstein, F.O. Progression from papilloma to carcinoma is accompanied by changes in antibody response to papillomavirus proteins. J. Viral. 1993, 67, 382-389 Murphy G.F. Dermatopathology: A Practical Guide to Common Disorders; chapter 3, Spongiotic Dermatitis. W.B. Saunders Co. Philadelphia, 1995, pp. 49-71
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Berman, A. and Winkelmann, R.K. Involuting common warts; clinical and histopathologic findings. J. Am. Acad. Dermatol. 1980, 3,356-362 Haynes, JR, Fuller, D.H., Eisenbraun, M.D., Ford, M.J. and Pertmer, T.M. Accell particle-mediated DNA immunization elicits
vaccination with papillomavirus
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humoral, cytotoxic, and protective immune responses. AlDS Res. Hum. Retroviruses 1994, 102, S43-S45 Barry, M.A., Lai, W.C. and Johnston, S.A. Protection against mycoplasma infection using expression-library immunization. Nature 1995, 377, 632-635
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