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Impact of inhibitors and L2 antibodies upon the infectivity of diverse alpha and beta human papillomavirus types.

Kwak K, Jiang R, Wang JW, Jagu S, Kirnbauer R, Roden RB - PLoS ONE (2014)

Bottom Line: Residues 11-88 of minor capsid protein L2 contain cross-protective epitopes, and vaccination with concatamers of this region derived from as many as eight alpha HPV (L2 α11-88x8) is being developed as an alternative prophylactic vaccine with potentially broader efficacy.These findings suggest that the distinct tropism of mucosal and cutaneous HPV may reflect distinct cell surface receptor interactions, but a common uptake mechanism dependent upon furin and γ-secretase proteolytic activities.Vaccination with an L2 multimer derived exclusively from alpha papillomavirus sequences induced antibodies that broadly neutralized PsV of all 34 HPVs from within both the alpha and beta families, suggesting each displays conserved L2 neutralizing epitopes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The Johns Hopkins University, Baltimore, Maryland, United States of America.

ABSTRACT
The licensed human papillomavirus (HPV) vaccines elicit type-restricted immunity but do not target cutaneous HPV types of the beta genus that are associated with non-melanoma skin cancer in immune-compromised patients, and it is unclear if these diverse types share a common mechanism of infection. Residues 11-88 of minor capsid protein L2 contain cross-protective epitopes, and vaccination with concatamers of this region derived from as many as eight alpha HPV (L2 α11-88x8) is being developed as an alternative prophylactic vaccine with potentially broader efficacy. There is also interest in developing broadly protective topical microbicides, such as carrageenan or heparin that block HPV receptor interactions, or small molecule inhibitors of infection. Here we have examined several inhibitors of HPV infection and antisera to L2 α11-88x8 for their breadth of activity against infection by 34 HPV types from within both the alpha and beta families using pseudovirions (PsV) carrying a luciferase reporter as surrogates for native virus. We observed that both heparin and carrageenan prevented infection by mucosatropic HPV types, but surprisingly PsV of several epidermotropic alpha4 and beta HPV types exhibited increased infectivity especially at low inhibitor concentrations. Furin and γ-secretase inhibitors and L2 α11-88x8 antiserum blocked infection by all HPV PsV types tested. These findings suggest that the distinct tropism of mucosal and cutaneous HPV may reflect distinct cell surface receptor interactions, but a common uptake mechanism dependent upon furin and γ-secretase proteolytic activities. Carrageenan, which is being tested as a vaginal microbicide, broadly inhibited infection by the high-risk mucosatropic HPV PsV, but not most skin tropic alpha and beta HPV. Vaccination with an L2 multimer derived exclusively from alpha papillomavirus sequences induced antibodies that broadly neutralized PsV of all 34 HPVs from within both the alpha and beta families, suggesting each displays conserved L2 neutralizing epitopes.

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Assessment of infectivity, L2 incorporation, and reporter DNA encapsidation by PsV preparations of 34 HPV types.(A) 250 million particles (as estimated based on L1 concentration) of HPV PsV were transferred to 293TT cells, incubated at 37°C for 72 hours before cell lysis. Luciferase activity was measured in cell lysate (n = 4). (B) Western blot analysis detecting L2 in 100 ng of each HPV PsV preparation using rabbit antiserum to L2 α11-88x8. (C) Reporter plasmid copy number of each PsV preparations for each HPV type sample was measured by quantitative real time PCR. Black and blue bars represent alpha and beta type, respectively, and mean ± standard error plotted.
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pone-0097232-g001: Assessment of infectivity, L2 incorporation, and reporter DNA encapsidation by PsV preparations of 34 HPV types.(A) 250 million particles (as estimated based on L1 concentration) of HPV PsV were transferred to 293TT cells, incubated at 37°C for 72 hours before cell lysis. Luciferase activity was measured in cell lysate (n = 4). (B) Western blot analysis detecting L2 in 100 ng of each HPV PsV preparation using rabbit antiserum to L2 α11-88x8. (C) Reporter plasmid copy number of each PsV preparations for each HPV type sample was measured by quantitative real time PCR. Black and blue bars represent alpha and beta type, respectively, and mean ± standard error plotted.

Mentions: We first tested infectivity of each HPV PsV type normalized to 250 million particles (as estimated based on L1 concentration) by measuring luciferase activity (relative light units, RLU) 72 h post-infection in 293TT cells. The HPV PsV infected cells showed dramatic differences in reporter expression (Figure 1A). Overall, the cutaneous HPV type PsV showed lower infectivity compared to mucosal tropic types (p<0.0001), and the PsVs derived from alpha species HPV showed greater infectivity that those derived from beta HPV types (p = 0.01). However, even for the weakest PsV types, the signal observed was still more than 100-fold higher than background signal. Furthermore, pre-incubation of the PsV with rabbit antiserum raised against the cognate L1 VLP prevented reporter delivery, consistent with antibody-mediated neutralization of an infection rather than a non-specific transfection. Rabbit antiserum to L1 VLP was not available for a number of HPV types. Instead, sera was harvested from mice two weeks after electroporation i.m. three times at two week intervals with the an HPV L1 expression vector [47]. Pre-incubation of the HPV PsV with these mouse antisera similarly inhibited reporter transfer (Figure S1).


Impact of inhibitors and L2 antibodies upon the infectivity of diverse alpha and beta human papillomavirus types.

Kwak K, Jiang R, Wang JW, Jagu S, Kirnbauer R, Roden RB - PLoS ONE (2014)

Assessment of infectivity, L2 incorporation, and reporter DNA encapsidation by PsV preparations of 34 HPV types.(A) 250 million particles (as estimated based on L1 concentration) of HPV PsV were transferred to 293TT cells, incubated at 37°C for 72 hours before cell lysis. Luciferase activity was measured in cell lysate (n = 4). (B) Western blot analysis detecting L2 in 100 ng of each HPV PsV preparation using rabbit antiserum to L2 α11-88x8. (C) Reporter plasmid copy number of each PsV preparations for each HPV type sample was measured by quantitative real time PCR. Black and blue bars represent alpha and beta type, respectively, and mean ± standard error plotted.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016295&req=5

pone-0097232-g001: Assessment of infectivity, L2 incorporation, and reporter DNA encapsidation by PsV preparations of 34 HPV types.(A) 250 million particles (as estimated based on L1 concentration) of HPV PsV were transferred to 293TT cells, incubated at 37°C for 72 hours before cell lysis. Luciferase activity was measured in cell lysate (n = 4). (B) Western blot analysis detecting L2 in 100 ng of each HPV PsV preparation using rabbit antiserum to L2 α11-88x8. (C) Reporter plasmid copy number of each PsV preparations for each HPV type sample was measured by quantitative real time PCR. Black and blue bars represent alpha and beta type, respectively, and mean ± standard error plotted.
Mentions: We first tested infectivity of each HPV PsV type normalized to 250 million particles (as estimated based on L1 concentration) by measuring luciferase activity (relative light units, RLU) 72 h post-infection in 293TT cells. The HPV PsV infected cells showed dramatic differences in reporter expression (Figure 1A). Overall, the cutaneous HPV type PsV showed lower infectivity compared to mucosal tropic types (p<0.0001), and the PsVs derived from alpha species HPV showed greater infectivity that those derived from beta HPV types (p = 0.01). However, even for the weakest PsV types, the signal observed was still more than 100-fold higher than background signal. Furthermore, pre-incubation of the PsV with rabbit antiserum raised against the cognate L1 VLP prevented reporter delivery, consistent with antibody-mediated neutralization of an infection rather than a non-specific transfection. Rabbit antiserum to L1 VLP was not available for a number of HPV types. Instead, sera was harvested from mice two weeks after electroporation i.m. three times at two week intervals with the an HPV L1 expression vector [47]. Pre-incubation of the HPV PsV with these mouse antisera similarly inhibited reporter transfer (Figure S1).

Bottom Line: Residues 11-88 of minor capsid protein L2 contain cross-protective epitopes, and vaccination with concatamers of this region derived from as many as eight alpha HPV (L2 α11-88x8) is being developed as an alternative prophylactic vaccine with potentially broader efficacy.These findings suggest that the distinct tropism of mucosal and cutaneous HPV may reflect distinct cell surface receptor interactions, but a common uptake mechanism dependent upon furin and γ-secretase proteolytic activities.Vaccination with an L2 multimer derived exclusively from alpha papillomavirus sequences induced antibodies that broadly neutralized PsV of all 34 HPVs from within both the alpha and beta families, suggesting each displays conserved L2 neutralizing epitopes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The Johns Hopkins University, Baltimore, Maryland, United States of America.

ABSTRACT
The licensed human papillomavirus (HPV) vaccines elicit type-restricted immunity but do not target cutaneous HPV types of the beta genus that are associated with non-melanoma skin cancer in immune-compromised patients, and it is unclear if these diverse types share a common mechanism of infection. Residues 11-88 of minor capsid protein L2 contain cross-protective epitopes, and vaccination with concatamers of this region derived from as many as eight alpha HPV (L2 α11-88x8) is being developed as an alternative prophylactic vaccine with potentially broader efficacy. There is also interest in developing broadly protective topical microbicides, such as carrageenan or heparin that block HPV receptor interactions, or small molecule inhibitors of infection. Here we have examined several inhibitors of HPV infection and antisera to L2 α11-88x8 for their breadth of activity against infection by 34 HPV types from within both the alpha and beta families using pseudovirions (PsV) carrying a luciferase reporter as surrogates for native virus. We observed that both heparin and carrageenan prevented infection by mucosatropic HPV types, but surprisingly PsV of several epidermotropic alpha4 and beta HPV types exhibited increased infectivity especially at low inhibitor concentrations. Furin and γ-secretase inhibitors and L2 α11-88x8 antiserum blocked infection by all HPV PsV types tested. These findings suggest that the distinct tropism of mucosal and cutaneous HPV may reflect distinct cell surface receptor interactions, but a common uptake mechanism dependent upon furin and γ-secretase proteolytic activities. Carrageenan, which is being tested as a vaginal microbicide, broadly inhibited infection by the high-risk mucosatropic HPV PsV, but not most skin tropic alpha and beta HPV. Vaccination with an L2 multimer derived exclusively from alpha papillomavirus sequences induced antibodies that broadly neutralized PsV of all 34 HPVs from within both the alpha and beta families, suggesting each displays conserved L2 neutralizing epitopes.

Show MeSH
Related in: MedlinePlus