Human papillomavirus type 11 recombinant L1 capsomeres induce virus-neutralizing antibodies

Abstract

The human papillomavirus type 11 (HPV-11) L1 major capsid protein can be trypsinized to generate recombinant capsomeres that retain HPV genotype-restricted capsid antigenicity (M. Li, T. P. Cripe, P. A. Estes, M. K. Lyon, R. C. Rose, and R. L. Garcea, J. Virol. 71:2988-2995, 1997). In the present study, HPV-11 virion-neutralizing monoclonal antibodies H11.F1 and H11.H3, previously characterized as recognizing two distinct HPV-11 capsid-neutralizing antigenic domains (S. W. Ludmerer, D. Benincasa, and G. E. Mark III, J. Virol. 70:4791-4794, 1996), were each found to be highly immunoreactive with trypsin-generated capsomeres in an enzyme-linked immunosorbent assay (ELISA). Capsomeres were used to generate high-titer polyclonal immune sera that demonstrated HPV genotype-restricted reactivity by ELISA. The capsomere antisera were then tested in an in vitro infectivity assay and found to neutralize HPV-11 virions. In this assay, HPV-11 capsomere polyclonal antisera exhibited neutralization titers (10(-5) to 10(-6)) comparable to those obtained with a virion-neutralizing antiserum raised previously against intact HPV-11 VLPs (R. C. Rose, R. C. Reichman, and W. Bonnez, J. Gen. Virol. 75:2075-2079, 1994). These results indicate that highly immunogenic, genotype-restricted HPV capsid-neutralizing antigenic domains are contained entirely within capsomeres. Thus, capsomeres may be viable vaccine candidates for the prevention of HPV disease.

FIG. 1

HPV-11-neutralizing antibodies bind HPV-11 capsomeres. H11.F1 and H11.H3 ascites were diluted 1:128,000 and then tested in an ELISA against trypsin-generated HPV-11 capsomeres. As a control, ascites containing an HPV-16 capsid-specific MAb (H16.V5) (8) was assayed at a dilution of 1:250. OD, optical density.

FIG. 2

HPV-11 capsomeres are highly immunogenic and display HPV genotype-restricted antigenicity. Threefold serial dilutions of rabbit capsomere polyclonal antisera R6-311 (solid symbols) and R6-312 (open symbols) were tested in an ELISA in duplicate against HPV-11 capsomeres (triangles), HPV-11 VLPs (squares), HPV-16 VLPs (circles), and HPV-18 VLPs (diamonds).

FIG. 3

HPV-11 capsomeres induce virus-neutralizing antibodies. (A) HPV-11 virions were incubated with antiserum before addition to HaCaT cells. Six days postinfection, cells were harvested, and the presence of an E1̂E4 spliced message, diagnostic of HPV-11 infection (3), was determined by RT-PCR. PCR products were separated on 2% agarose gels, stained with ethidium bromide, and examined under UV light for the presence of the ∼0.6-kb E1̂E4 band. Lane 1, HaCaT cells alone, no virus, no antibodies (negative control); lane 2, HPV-11 virions plus preimmune serum from an animal subsequently immunized with HPV-11 capsomeres (10⁻³ dilution); lane 3, HPV-11 virions plus HPV-16 L1 VLP postimmune serum (10⁻³ dilution); lanes 4 to 8, HPV-11 virions plus HPV-11 capsomere antiserum (dilutions 10⁻³ to 10⁻⁷, respectively); lanes 9 to 13, HPV-11 virions plus HPV-11 L1 VLP antiserum (dilutions 10⁻³ to 10⁻⁷, respectively). A second rabbit anti-HPV-11 capsomere antiserum tested produced comparable results (i.e., a neutralization titer of 10⁻⁵) (data not shown). (B) PCR amplification of β-actin was performed on all cDNA samples as an internal control. The expected size of the β-actin band is ∼0.6 kb. The lanes are the same as in panel A. The HPV-11_Hershey virus stock was titrated on HaCat cells on multiple occasions prior to being used in the present study. The titer of the inoculum (i.e., the last dilution which resulted in the detection of spliced E1̂E4 mRNA by RT-PCR) consistently was between 10⁻⁵ and 10⁻⁶. In the present study, the inoculum was used at a dilution of 10⁻⁴.