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, 14 (10), 1285-95

Salmonella Enterica Serovar Typhi Ty21a Expressing Human Papillomavirus Type 16 L1 as a Potential Live Vaccine Against Cervical Cancer and Typhoid Fever

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Salmonella Enterica Serovar Typhi Ty21a Expressing Human Papillomavirus Type 16 L1 as a Potential Live Vaccine Against Cervical Cancer and Typhoid Fever

Dominique Fraillery et al. Clin Vaccine Immunol.

Abstract

Human papillomavirus (HPV) vaccines based on L1 virus-like particles (VLPs) can prevent HPV-induced genital neoplasias, the precursors of cervical cancer. However, most cervical cancers occur in developing countries, where the implementation of expensive vaccines requiring multiple injections will be difficult. A live Salmonella-based vaccine could be a lower-cost alternative. We previously demonstrated that high HPV type 16 (HPV16)-neutralizing titers are induced after a single oral immunization of mice with attenuated Salmonella enterica serovar Typhimurium strains expressing a codon-optimized version of HPV16 L1 (L1S). To allow the testing of this type of vaccine in women, we constructed a new L1-expressing plasmid, kanL1S, and tested kanL1S recombinants of three Salmonella enterica serovar Typhi vaccine strains shown to be safe in humans, i.e., Ty21a, the actual licensed typhoid vaccine, and two highly immunogenic typhoid vaccine candidates, Ty800 and CVD908-htrA. In an intranasal mouse model of Salmonella serovar Typhi infection, Ty21a kanL1S was unique in inducing HPV16-neutralizing antibodies in serum and genital secretions, while anti-Salmonella responses were similar to those against the parental Ty21a vaccine. Electron microscopy examination of Ty21a kanL1S lysates showed that L1 assembled in capsomers and capsomer aggregates but not well-ordered VLPs. Comparison to the neutralizing antibody response induced by purified HPV16 L1 VLP immunizations in mice suggests that Ty21a kanL1S may be an effective prophylactic HPV vaccine. Ty21a has been widely used against typhoid fever in humans with a remarkable safety record. These finds encourage clinical testing of Ty21a kanL1S as a combined typhoid fever/cervical cancer vaccine with the potential for worldwide application.

Figures

FIG. 1.
FIG. 1.
Comparison of serum anti-HPV16 VLP antibody titers after oral vaccination with PhoPc kanL1S, PhoP kanL1S, and ΔAroA kanL1S. Groups of five BALB/c mice were orally immunized with 109 CFU of PhoPc kanL1S (black bars), PhoP kanL1S (white bars), and AroA kanL1S (striped bars). Anti-HPV16 VLP ELISA titers are shown 8 weeks after immunization in serum (A) and in vaginal washes (B). Data are expressed as the geometric means (log10) of the reciprocal dilutions of specific IgG from individual mice in serum and specific IgG and IgA per microgram of total IgG (IgA) in vaginal washes. Error bars indicate the standard errors of the means (SEM).
FIG. 2.
FIG. 2.
Comparison of serum anti-HPV16 VLP antibody titers after nasal vaccination with Ty21a kanL1S, Ty800 kanL1S, and CVD908-htrA kanL1S. Groups of five BALB/c mice were nasally vaccinated at weeks 0 and 4 with 109 CFU of Ty21a kanL1S (plain line) and CVD908-htrA kanL1S (pointed line) and with 107 CFU of Ty800 kanL1S (dashed line). Anti-HPV16 VLP (A), as well as anti-LPS and anti-flagellin (B) IgG ELISA titers in serum were determined every 2 weeks. Comparisons of serum IgG titers induced after immunization with Ty21a kan L1S (black bars) and Ty21a (white bars) are also indicated (C). Data are expressed as the geometric means (log10) of the reciprocal dilutions of specific IgG from individual mice. Error bars indicate the SEM.
FIG. 3.
FIG. 3.
Comparison of HPV16 VLP and flagellin-specific CD4+ T-cell proliferations. Groups of five BALB/c mice were nasally vaccinated at weeks 0 and 4 with 109 CFU of Ty21a kanL1S (black bars) and CVD908-htrA kanL1S (punctated bar) and with 107 CFU of Ty800 kanL1S (striped bars). HPV16 VLP (A)- and flagellin (B)-specific CD4+ T-cell proliferations are shown at week 8. Data are expressed as mean stimulation indices of triplicate cell cultures (cpm in presence of antigen/cpm in absence of antigen). CD4+ T-cell proliferation was also determined in naïve mice (white bars), and statistical analysis was performed with one-way analysis of variance and a Bonferroni posttest using GraphPad Prism software.
FIG. 4.
FIG. 4.
In vitro stability of the kanL1S and L1S plasmids in different Salmonella enterica serovar Typhi strains. The number of successive cultures at a 1/100 dilution performed overnight (ON) in medium without antibiotic is indicated on the horizontal axis. Each morning, bacteria (plain line, kanL1S; dashed line, L1S) were plated onto agar in the presence or absence of antibiotic. The vertical axis represents the percentage of bacteria that have retained the plasmid. Error bars indicate SEM.
FIG. 5.
FIG. 5.
Electron microscopy analysis of Ty21a kanL1S lysate. (A) Clarified lysate was separated on a 27%, 33%, or 39% OptiPrep step gradient. Sixteen fractions were collected from the bottom of the tube. The first 11 fractions were analyzed by Western blotting with CAMVIR-1 monoclonal antibody, which recognizes denatured 16L1. Molecular mass markers (in kilodaltons) are depicted on the right. The location of full-length monomeric L1 is indicated by the arrow. (B) Fractions that were 16L1 positive in CAMVIR-1 Western blots were pooled and further purified on a 2% agarose column. Sixteen L1 Western blot-positive fractions were pooled and examined by transmission electron microscopy at a magnification of ×26,000. Capsomer aggregates (large arrows), 17-nm-diameter “stars” (small arrows), and 12-nm capsomers (arrowheads) are indicated.
FIG. 6.
FIG. 6.
Anti-HPV16 VLPs and HPV16-neutralizing antibodies in serum and genital secretions of mice immunized with Ty21a kanL1S or the prototype HPV16 VLP vaccine. Groups of five mice were immunized with two i.n. doses of Ty21a kanL1S (ca. 109 CFU) at weeks 0 and 4 (squares); three s.c. doses of 1 μg VLPs at weeks 0, 4, and 25 (triangles); three i.n.(aerosol-like [aer]) doses of 5 μg VLPs at weeks 0, 1, and 2 (circles); or a combination of VLPs (two s.c. 1-μg doses at weeks 0 and 4) and Ty21a kanL1S (a single i.n. boost at week 8) (diamonds). Anti-HPV16 VLP and HPV16-neutralizing antibody titers were determined in serum and genital secretions by ELISA and the SEAP HPV16 pseudovirion neutralization assay, respectively, at short term (4 to 5 weeks after the last immunization) (A, C, and E) and at long term (18 to 24 weeks after the last immunization) (B, D, and F). Data are expressed as the log10 of the reciprocal dilutions of specific IgG in serum and specific IgG and IgA per microgram of total IgG or IgA, respectively, in secretions for the ELISA titer or reciprocal dilutions yielding 50% SEAP inhibition for the neutralizing titers. Between-group differences were analyzed with a Student's t test (GraphPad Prism), and significant differences are indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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