Improved efficiency of a Salmonella-based vaccine against human papillomavirus type 16 virus-like particles achieved by using a codon-optimized version of L1

Abstract

Cervical cancer results from cervical infection by human papillomaviruses (HPVs), especially HPV16. An effective vaccine against these HPVs is expected to have a dramatic impact on the incidence of this cancer and its precursor lesions. The leading candidate, a subunit prophylactic HPV virus-like particle (VLP) vaccine, can protect women from HPV infection. An alternative improved vaccine that avoids parenteral injection, that is efficient with a single dose, and that induces mucosal immunity might greatly facilitate vaccine implementation in different settings. In this study, we have constructed a new generation of recombinant Salmonella organisms that assemble HPV16 VLPs and induce high titers of neutralizing antibodies in mice after a single nasal or oral immunization with live bacteria. This was achieved through the expression of a HPV16 L1 capsid gene whose codon usage was optimized to fit with the most frequently used codons in Salmonella. Interestingly, the high immunogenicity of the new recombinant bacteria did not correlate with an increased expression of L1 VLPs but with a greater stability of the L1-expressing plasmid in vitro and in vivo in absence of antibiotic selection. Anti-HPV16 humoral and neutralizing responses were also observed with different Salmonella enterica serovar Typhimurium strains whose attenuating deletions have already been shown to be safe after oral vaccination of humans. Thus, our findings are a promising improvement toward a vaccine strain that could be tested in human volunteers.

FIG. 1.

Codon-optimized HPV16 L1S ORF. The nucleotide sequence of L1S is shown with the modified codons underlined; modified nucleotides are in bold.

FIG. 2.

HPV16L1 expression in PhoP^c L1 and PhoP^c L1S recombinant strains. An immunoblot of bacterial lysates with anti-HVP16 L1 monoclonal antibody is shown, and the 57-kDa protein band identified as L1 is indicated by an arrow (A). Scanning of the L1 protein bands obtained after immunoblotting of the bacterial lysates from the two recombinant strains (three independent experiments) was performed by using National Institutes of Health Image software. The results are shown as the means of pixel densities of the L1 protein bands normalized to the content in bacteria and are expressed as increases in expression (n-fold) in comparison to PhoP^c L1 (B). The amounts of VLPs determined by sandwich ELISA are shown in micrograms per 10¹¹ CFU (C).

FIG. 3.

L1 and L1S plasmid stability in vitro. The number of successive overnight cultures at 1/1000 dilution performed in LB broth without antibiotic is indicated on the horizontal axis. Each morning, bacteria were plated on LB agar in the presence or absence of antibiotic. The vertical axis represents the percentage of bacteria that have retained the plasmid. Error bars indicate the standard errors of the means. O/N, overnight cultures.

FIG. 4.

Comparison of serum anti-HPV16 VLP antibody and HPV16 neutralization titers after nasal vaccination with PhoPc L1S or PhoP^c L1. Groups of five BALB/c mice were intranasally vaccinated with 10⁶ to 10⁷ CFU of PhoP^c L1S or PhoP^c L1 as a single dose or as two doses at week 0 and week 2. Serum was sampled 4 weeks after the last immunization, and HPV16 VLP-specific IgG (A) and HPV16 neutralization (B) titers are indicated. Data are expressed as the geometric means (log₁₀) of the reciprocal serum dilutions of specific IgG (A) or reciprocal serum dilutions yielding 50% SEAP inhibition (B) from individual mice. Error bars indicate the standard errors of the means.

FIG. 5.

Anti-HPV16 VLP systemic (A) and vaginal (B) antibody titers after nasal and oral vaccination with PhoP^c L1S. Groups of five to eight BALB/c mice were immunized with 10⁶ to 10⁷ CFU by the nasal route or 10⁸ to 10⁹ CFU by the oral route. Serum and vaginal washes were sampled at the indicated weeks after immunization. Data are expressed as the geometric means (log₁₀) of the reciprocal dilutions of specific IgG from individual mice in serum (A) and specific IgG and IgA per microgram of total IgG and IgA, respectively, in secretions (B). Error bars indicate the standard errors of the means.

FIG. 6.

Serum anti-HPV16 VLP IgG and HPV16-neutralization titers after nasal or oral vaccination with χ4989, χ4990, PhoP⁻, and AroA expressing L1S or L1. Groups of four to seven BALB/c mice were immunized with ca. 10⁶ to 10⁷ CFU by the nasal route (A) or ca. 10⁸ to 10⁹ CFU by the oral route (B) with the indicated recombinant strains expressing L1S or L1. Serum was sampled 6 to 7 weeks after immunization, and HPV16 VLP-specific IgG (plain bars) or HPV16 neutralization (striped bars) titers are shown. Data are expressed as the geometric means (log₁₀) of the reciprocal serum dilutions of specific IgG (A) or reciprocal serum dilutions yielding 50% SEAP inhibition (B) from individual mice. Error bars indicate the standard errors of the means.