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Journal of Virology, March 2004, p. 3196-3199, Vol. 78, No. 6
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.6.3196-3199.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Complete Protection from Papillomavirus Challenge after a Single Vaccination with a Vesicular Stomatitis Virus Vector Expressing High Levels of L1 Protein

Anjeanette Roberts,1 Jon D. Reuter,2 Jean H. Wilson,2 Stuart Baldwin,1 and John K. Rose1*

Department of Pathology,1 Section of Comparative Medicine, Yale University School of Medicine, New Haven, Connecticut 065102

Received 28 August 2003/ Accepted 3 November 2003


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ABSTRACT
 
We generated an attenuated, recombinant vesicular stomatitis virus (VSV) expressing high levels of the cottontail rabbit papillomavirus (CRPV) L1 protein from an upstream site in the VSV genome. Rabbits vaccinated once with this VSV-L1 recombinant produced high levels of anti-L1 antibody and were completely protected against papilloma formation after challenge with CRPV. In contrast, animals vaccinated only once with a VSV vector expressing lower levels of L1 from a downstream site in the VSV genome generated lower levels of L1 antibody and demonstrated only incomplete protection from papilloma formation after challenge. We conclude that the level of L1 protein expression is critical in generating complete immunity with a single-dose vaccine.


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INTRODUCTION
 
Vaccines based on live, replicating viruses typically activate all aspects of the immune system and evoke a balanced immune response including antibody- and cell-mediated responses. Single vaccinations with vaccines based on live viruses can provide lifelong protection from disease, and such vaccines are typically inexpensive to produce (11). When killed viruses or viral proteins are employed as vaccines, boosting is typically required to generate long-term immunity (11). A recent clinical trial has shown that multiple immunizations with the major capsid protein (L1) of human papillomavirus type 16 (HPV-16) protects against infection by HPV-16 (8). HPVs cause the vast majority of cervical cancers, and cervical cancer is the leading cause of death from cancer in women in developing countries. There are approximately 370,000 new cases of cervical cancer in the world each year, and 80% of these cases occur in developing countries (National Cervical Cancer Coalition [http://www.nccc-online.org/worldcancer.htm]). In many developing countries, multiple-injection vaccination schedules are not easily implemented or affordable. The development of an inexpensive vaccine for HPV that would require only a single inoculation for long-term immunity is highly desirable.

Attenuated, recombinant vesicular stomatitis viruses (VSVs) expressing the appropriate foreign proteins of other viruses are potent vaccine vectors and can confer long-lasting immunity after single inoculations (6, 14, 15, 17, 19). The level of foreign protein expression from VSV vectors can be controlled based on the site of gene insertion in the VSV genome (9, 16). Genes closest to the 3' end of the negative-strand RNA genome (where transcription begins) are expressed at the highest levels, and the sequential transcription of downstream genes is attenuated by about 30% at each gene junction (5). The order of VSV gene transcription is nucleocapsid (N), phosphoprotein (P), matrix (M), glycoprotein (G), and polymerase (L). In previous studies, our laboratory has reported the introduction of foreign genes between G and L (position 5) (20), between M and G (position 4) (3, 9), or between N and P (position 2) (12, 16), but most of our studies of live-attenuated VSV as a vaccine vector have used the position 5 vector, since this vector normally gives substantial protein expression and is less likely to interfere with VSV replication.

Infection of rabbits with cottontail rabbit papillomavirus (CRPV) causes papillomas that progress to carcinomas with high frequency. This system provides an excellent animal model for cancer caused by HPV in humans. In a published study, we expressed the 55-kDa major capsid protein (L1) of CRPV in a position 5 VSV vector and found that vaccination with this vector protected rabbits from disease induced by CRPV (13). However, a single vaccination with this VSV recombinant was not sufficient to fully protect rabbits from CRPV challenge. Analysis also showed that expression of the L1 protein from the L1 gene in this position 5 vector was relatively low compared to the expression of proteins from other genes previously tested in this position, and immunoprecipitation was required for the detection of L1 in lysates from infected cells. In the present study, we generated a much higher level of L1 expression in a new VSV recombinant in which the CRPV L1 gene was moved to position 2 of the VSV genome. We show here that the L1 protein expressed from position 2 was readily detected in unfractionated lysates without immunoprecipitation. Furthermore, we show that a single inoculation of rabbits with this recombinant vaccine generates a much higher antibody titer to L1 than that generated by the position 5 recombinant and provides complete protection from subsequent CRPV challenge.


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Relative expression of CRPV L1 from position 2 versus that from position 5 in VSV recombinants.
 
To determine the level of expression of the CRPV L1 protein that could be obtained from gene position 2 in a recombinant VSV vector, the 1.5-kb coding sequence for the L1 gene was obtained from the pVSV-CRPVL1 vector (13). The L1 gene was excised directly from pVSV-CRPVL1 with XhoI and NheI and cloned into the full-length VSV vector designated pVSV-XSN, with the expression site for the foreign gene being located between the VSV N and P genes (12). A recombinant VSV expressing L1 was then obtained by using standard VSV recovery techniques (10, 20). Figure 1A shows the relative positions of the L1 gene in the VSV recombinants. Expression of the L1 protein from the recombinant was analyzed in infected BHK cells initially by indirect immunofluorescence with rabbit polyclonal antiserum specific for CRPV-L1. These studies confirmed the expression of L1 and revealed substantially more expression than was seen in cells infected with the position 5 L1 vector (data not shown).



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  		FIG. 1. Diagram of VSV-L1 recombinants and relative expression of L1 proteins. (A) Schematic representation of gene order in wild-type VSV and in the two recombinant VSVs expressing CRPV-L1 proteins. Gene order is presented in the 3'-to-5' orientation on the negative-strand genome. (B) Metabolically labeled proteins from crude lysates of BHK cells infected with the indicated viruses (lanes 1 to 3) or immunoprecipitates of the same lysates with a rabbit polyclonal antibody to L1 protein (lanes 4 to 6).

To quantitate relative levels of L1 protein expression from the two vectors, we infected dishes of 4 x 105 BHK cells at an multiplicity of infection of 10 with each vector and with wild-type VSV, and we labeled cells between 4 and 5 h postinfection with [35S]methionine. Lysates were then analyzed directly by polyacrylamide gel electrophoresis (Fig. 1B, lanes 1 to 3) or immunoprecipitated (13) with rabbit anti-L1 antibody and then analyzed by polyacrylamide gel electrophoresis (Fig. 1B, lanes 4 to 6). Quantitation of the gels showed that L1 protein was expressed at a 4.3-fold higher level from position 2 than from position 5 (Fig. 1B, lanes 2 and 3). This is a reasonable result, since the theoretical increase would be 2.9-fold based on 30% transcription attenuation at each gene junction.

The major capsid proteins of papillomaviruses are able to form viruslike particles (VLPs) in the absence of any other papillomavirus protein. These VLPs can be detected in the supernatants of infected cells in tissue culture. Previously, L1 was not detected in supernatants from BHK cells infected with position 5 recombinants expressing L1 (13). It is likely that the L1 protein was not expressed from this recombinant at high enough levels for release into or detection in the supernatant. In contrast, the L1 protein expressed from the position 2 vector was easily detectable in the supernatant at 4 h following infection (data not shown).


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Increased immune responses to CRPV L1.
 
We reasoned that the increased expression of L1, as well as its release into the medium, might enhance L1 presentation to the immune system of a vaccinated animal. To determine whether L1-specific immune responses and the protection of animals were enhanced with the position 2 recombinant, we inoculated three New Zealand White rabbits with VSVL1-2 and three with VSVL1-5 at 3.25 x 106 PFU/250 µl/rabbit. Two control rabbits received 250 µl of medium alone. Each rabbit was bled prior to immunization, and each received a single intramuscular inoculation on day 0. We used intramuscular vaccination rather than intranasal vaccination because it gives more reproducible results with VSV vectors. Rabbits were bled again at 3 and 5 weeks postinoculation. Both plasma and sera were screened for antibodies to VSV and L1 by serum neutralization assays and enzyme-linked immunosorbent assay (ELISA), respectively.

All rabbits immunized with recombinant VSVs had measurable titers of antibodies that neutralize VSV at 5 weeks postimmunization (Table 1). As in previous experiments (13), titers of antibodies that neutralize VSV differed greatly from one rabbit to another, a result typical of responses in outbred animals. We found no significant differences in titers of antibodies that neutralize VSV between the group vaccinated with VSVL1-2 and the group vaccinated with VSVL1-5. The two control rabbits had no measurable titers of antibodies that neutralize VSV at the lowest dilution assayed (1:8).


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  		TABLE 1. Antibody responses to CRPV VLP-L1 detected by ELISA

Importantly, the immune responses of the two groups to L1 were dramatically different. L1-specific antibody was produced in all immunized rabbits (Table 1), but the L1 antibody titers as determined by VLP-L1 ELISA were consistently high for VSVL1-2-immunized rabbits (average titer, 1:10666) and low for VSVL1-5-immunized rabbits (average titer, 1:633) (P = 0.005, two-sample t test).


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Protection from CRPV challenge.
 
Five weeks after immunization, rabbits were challenged with high doses (3 sites/rabbit) and low doses (3 sites/rabbit) of CRPV as previously described (13). Rabbits were observed weekly for 10 weeks following CRPV challenge. Total papilloma volumes for each rabbit are reported in Table 1.

All rabbits immunized with VSVL1-2 were completely protected against CRPV-induced papilloma formation (Fig. 2). Immunization with VSVL1-5 offered partial protection, but all rabbits immunized with VSVL1-5 or medium alone developed papillomas.



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  		FIG. 2. Protection from CRPV challenge with VSVL1-2 and VSVL1-5 vectors. (A) Average percentages of papilloma-free sites for rabbits immunized with a single dose of VSVL1-2 or VSVL1-5 vaccine or for unvaccinated controls. The error bars represent standard errors of the means. (B and C) Average high-dose (B) and low-dose (C) papilloma volumes (natural log) following vaccination. Filled circle, VSVL1-5; , VSVL1-2; diamond, unvaccinated control.

For the vaccinated rabbits developing papillomas, the time to papilloma onset was delayed, with papillomas appearing at 35 days post-CRPV challenge in the VSVL1-5 group and at 21 days postchallenge in control rabbits (P < 0.039, single-factor analysis of variance). The number of papilloma-free sites was significantly greater in the VSVL1-2 group (18 of 18 rabbits) than in the VSVL1-5 group (1 of 18 rabbits) and the control group (0 of 12 rabbits). Papillomas were absent in the VSVL1-2 group, while total papilloma volume per rabbit in the VSVL1-5-immunized rabbits was 2,848 mm3, compared with 5,993 mm3 in control rabbits (P < 0.046, single-factor analysis of variance). From these data we conclude that increased expression of the L1 protein in VSVL1-2 significantly enhanced protection against CRPV challenge.

The studies reported here indicate that the level of foreign antigen expression from attenuated VSV-based vectors correlates very well with the strength of the antibody response to that antigen. By increasing the expression of L1 4.3-fold in the upstream vector, we obtained an increase in the average ELISA L1 antibody titer of 16.8-fold (Table 1). This increased expression and the corresponding increase in immunogenicity are obviously critical in promoting complete protection against challenge.

We do not know why the L1 protein was expressed relatively poorly in the position 5 vector. We used the natural L1 gene sequence rather than a codon-optimized gene, and it is possible that an optimized gene might be expressed at higher levels. However, it is our experience that codon-optimized genes encoding other proteins are not expressed well from the cytoplasmic VSV vectors. Others have had similar experiences with a codon-optimized human immunodeficiency virus type 1 gag gene expressed from another cytoplasmic vector, vaccinia virus (7). In that study, the authors concluded that the same codon optimization that allowed greater expression from DNA in the nucleus did not allow greater expression in the cytoplasm from vaccinia virus. They concluded that codon optimization probably affected not mRNA translation efficiency but rather some other aspect of RNA transport or metabolism. Since VSV is a cytoplasmic vector, codon optimization is unlikely to improve the expression of L1 in this vector.

Other investigators have obtained good protection against CRPV infection by using VLPs composed of CRPV L1 and L2 proteins (1, 2). However, these studies used multiple immunizations to generate the level of protective immunity reported here for a single inoculation with our VSVL1-2 recombinant. A vaccine vector like the VSV-CRPVL1-2 recombinant that could generate protection against human papillomaviruses in a single-dose inoculation is obviously quite attractive. Recombinant VSVs can be grown easily in large quantities and grow to high titers in cell lines such as the Vero line that are approved for vaccine production. In addition, relatively low doses of vaccine virus induce strong immune responses. Live attenuated recombinant VSVs given by an intranasal route are highly effective at stimulating humoral and cellular immunity in mice (4, 18) and in rhesus macaques (Michael A. Egan, Siew Yen Chong, Nina F. Rose, Shakuntala Megati, Kevin J. Lopez, Eva B. Schadeck, J. Erik Johnson, Amjed Masood, Priscilla Piacente, Robert E. Druilhet, Paul W. Barras, Dana L. Hasselschwert, Patricia Reilly, Eric M. Mishkin, David C. Montefiori, Mark G. Lewis, David K. Clarke, R. Michael Hendry, Preston A. Marx, John H. Eldridge, Stephen A. Udem, Zimra R. Israel, and John K. Rose, submitted for publication). Vaccination against genital HPV infection by the intranasal route is particularly attractive, since it is likely to generate greater mucosal immunity at the sites where natural HPV infection occurs. Recombinant VSVs are also especially attractive as vaccine vectors for use in developing countries because they can be delivered by the intranasal route without the need for injections.


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ACKNOWLEDGMENTS
 
We thank Janet Brandsma for providing the challenge virus stock and antibody to L1 and for excellent suggestions on the manuscript.

Anjeanette Roberts was supported by a Cancer Research Institute Fellowship. This work was funded in part by American Cancer Society grant IRG 58-012-42 to J.D.R. and NIH grant R01AI24345 to J.K.R.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pathology, BML 342, 310 Cedar St., New Haven, CT 06510. Phone: (203) 785-6184. Fax: (203) 785-7467. E-mail: john.rose{at}yale.edu. Back


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Journal of Virology, March 2004, p. 3196-3199, Vol. 78, No. 6
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.6.3196-3199.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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