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

Plasmid construction and bacterial strains used.In plasmid pFS14nsd HPV16-L1S (5), the ampicillin resistance coding sequence was replaced by a kanamycin resistance coding sequence as follows. A SacII-XbaI fragment containing the kanamycin coding sequence and promoter was generated by PCR using pET-9a (Novagen) plasmid DNA as template. The primers used were a 25-mer primer located 54 nucleotides upstream from the first ATG of kanamycin and containing a SacII restriction site (underlined), 5′-GGGCCGCGGTGGTCATGAACAATAA-3′, and a 28-mer primer containing an XbaI restriction site (underlined), 5′-GGGTCTAGAAGCTGTCAAACATGAGAAT-3′. Another large SacII-XbaI fragment containing the entire pFS14nsd HPV16-L1S plasmid sequence, without the ampicillin resistance gene, was generated by inverse PCR with Expand High Fidelity PCR (Roche Molecular Biochemicals) with the following primers: a 28-mer primer located 92 nucleotides upstream from the ATG of ampicillin and containing a SacII site (underlined), 5′-GGGCCGCGGTTTGTAGAAACGCAAAAAG-3, and a 28-mer primer containing a XbaI site (underlined) and the stop codon of ampicillin (boldface type), 5′-GGGTCTAGATCCTAACTGTCAGACCAAG-3′. The two SacII-XbaI fragments were ligated together to generate plasmid pFS14nsd-kan³-HPV16-L1S. A correct nucleotide sequence was confirmed by sequencing the full plasmid. The new plasmid was introduced by electroporation (46) into the attenuated Salmonella enterica serovar Typhimurium PhoP^c (CS022) (32), PhoP⁻ (CS015) (31) (both kindly provided by John Mekalanos, Boston, MA), and ΔaroA (SL7207) (20) (kindly provided by Irene Corthésy-Theulaz, Lausanne, Switzerland) strains and into the attenuated Salmonella enterica serovar Typhi Ty800 (19) (kindly provided by the Virus Research Institute, Cambridge, MA), CVD908-htrA (48) (kindly provided by Myron Levine, Baltimore, MD), and Ty21a (17) (Berna Biotech, Switzerland) strains. The original pFS14nsd HPV16-L1S plasmid was also introduced in the three attenuated Salmonella enterica serovar Typhi vaccine strains for comparison purposes.

HPV16 L1 expression and analysis of assembly state.Expression of L1 in Salmonella lysates was analyzed by Western blotting as previously described (35) by using the anti-HPV16 L1 monoclonal antibody CAMVIR-1 (Anawa). The content in HPV16 VLP equivalents was measured by a sandwich enzyme-linked immunosorbent assay (ELISA) as previously described (6) by using two monoclonal antibodies (H16E70 or H161A and H16V5) (kindly provided by N. D. Christensen, Hershey, PA), which recognize conformational epitopes on HPV16 VLPs (11).

The L1 assembly state was analyzed by electron microcopy after OptiPrep separation as follows. Ty21a kanL1S bacteria grown to mid-log phase (optical density at 600 nm [OD₆₀₀] of ≤0.7) were harvested by centrifuging at 3,300 × g. The bacteria were lysed in ice-cold B-Per II reagent (Pierce). Halt protease inhibitor (Pierce), EDTA, Plasmid Safe, Benzonase, and MgCl₂ were added to final concentrations of 1%, 5 mM, 0.2%, 0.2%, and 9.5 mM, respectively. The lysates were incubated at room temperature for 10 min and centrifuged at 10,000 × g to obtain the pellet and supernatant fractions. For OptiPrep centrifugation, NaCl was added to the lysate to a final concentration of 0.8 M, and the lysate was clarified at 16,300 × g for 15 min at 4°C. Supernatants were loaded onto 27%, 33%, and 39% OptiPrep-Dulbecco's phosphate-buffered saline-0.8 M NaCl step gradients in polyallomer tubes (1.2 ml each; gradient diffused 1 to 2 h at room temperature before use). The OptiPreps were run at 50,000 rpm for 3 h 30 min at 16°C in a SW55Ti rotor. In some experiments, L1-positive fractions were further purified over 2% agarose columns (http://home.ccr.cancer.gov/lco/gelfiltration.htm ). L1-positive fractions were stained with uranyl acetate and examined by electron microscopy.

Immunization of mice, analysis of anti-HPV16 VLP antibodies, and recovery of Salmonella.Six-week-old female BALB/c mice (Iffa Credo, France) were used in all experiments according to ethical directives of Swiss veterinary authorities. Salmonella enterica serovar Typhi and Salmonella enterica serovar Typhimurium strains were grown in Luria-Bertani broth or agar, except for CVD908-htrA, which was grown in Aro broth or agar (21). Ampicillin (Sigma) and kanamycin (Fluka) were used at 50 μg/ml when indicated. For preparation of the Salmonella inoculum, the bacteria were grown to mid-log phase (OD₆₀₀ of ≤0.7). For Ty21a kanL1S, 0.1% galactose was added at an OD₆₀₀ of 0.3 when indicated. Twenty microliters of bacterial inoculum was administered orally (10⁹ CFU) or intranasally (i.n.) (10⁷ to 10⁹ CFU) under anesthesia as previously described (22, 35). Purified baculovirus-expressed HPV16 VLPs were produced as described previously (18, 22a) and were administered subcutaneously (s.c.) (1-μg doses) or i.n. in anesthetized mice (5-μg doses) as previously described (4, 34). The latter protocol corresponds to an aerosol-like administration with inhalation of the VLP inoculum into the lung (3, 33). However, while the parenteral protocol is optimal, as three 5-μg s.c. VLP doses induced similarly high anti-HPV16 VLP titers (4, 34), the aerosol-like administration is not optimized, but higher VLPs doses cannot be used, and higher anti-HVP16 VLPs responses can be achieved only when mucosal adjuvants are added (4, 34, 42a). Sampling of blood and vaginal washes was performed before and after vaccination throughout a 2- to 10-month period depending on the experiments (see figure legends for detailed schedules).

Sampling of blood and vaginal washes as well as determination of specific antibody end-point titers were performed by ELISA as reported previously (22, 35). Briefly, for determinations of anti-HPV16 VLPs, anti-lipopolysaccharide (LPS), or anti-flagellin antibodies, ELISA plates were coated with 50 ng of HPV16 VLPs in phosphate-buffered saline with Salmonella serovar Typhi LPS (Sigma) coupled with methylated bovine serum albumin (Sigma) in carbonate buffer as previously described (37) or with flagellin purified from Ty21a (50 ng/well) in carbonate buffer, respectively. For total immunoglobulin G (IgG) or IgA determinations, plates were coated with 100 ng of sheep anti-mouse Ig (Boehringer) in carbonate buffer. Total or specific IgA or IgG antibodies were detected with biotinylated goat anti-mouse IgA (Kirkegaard & Perry Laboratories) or IgG (Amersham Pharmacia) as secondary antibodies, respectively. End-point dilutions of all samples were carried out. The specific IgA or IgG titers were expressed as the reciprocal of the highest dilutions that yielded an OD₄₉₂ that was four times that of preimmune samples. These titers were normalized to the amount of total IgA or IgG in vaginal washes (22).

Recovery of Salmonella enterica serovar Typhimurium or Salmonella enterica serovar Typhi was determined using organs from euthanized mice by plating onto agar plates with and without antibiotics as previously described (35).

Neutralization assays.Neutralization assays were performed with secreted alkaline phosphatase (SEAP) HPV16 pseudoviruses as described in detail previously (41). Briefly, OptiPrep-purified SEAP HPV16 pseudoviruses diluted 3,000-fold were incubated on ice for 1 h with twofold serial serum or vaginal wash dilutions, and the pseudovirus-antibody mixtures were used to infect 293TT cells for 3 days. The SEAP content in 10 μl of clarified cell supernatant was determined using the Great ESCAPE SEAP chemiluminescence kit (BD Clontech). Neutralization titers were defined as the reciprocal of the highest serum or vaginal wash dilution that caused at least a 50% reduction in SEAP activity (100% SEAP activity ranging from 30 to 150 relative light units). The lowest serum and vaginal wash dilutions tested were 1:10 and 1:5, respectively, which correspond, when mixed with the pseudovirions, to final dilutions in the assay of 1:50 and 1:25, respectively. Preimmune vaginal washes at this dilution were not neutralizing. Vaginal washes of immunized mice that were not neutralizing at this dilution were attributed an arbitrary value of 5 (log₁₀ = 0.7), which corresponded to the lower limit of detection in this assay.

Proliferation assays.Splenocytes were isolated by mechanical dissociation as previously described (3). CD4⁺ T cells were purified by magnetic antibody cell sorting using anti-CD4⁺-coated microbeads (Miltenyi Biotec, Gladbach, Germany) according to the manufacturer's instructions. CD4⁺ T cells from naïve or immunized mice were incubated for 3 days with HPV16 VLPs (2 μg/ml [GMP preparation from Novavax]) (36) and flagellin (10 μg/ml, purified from Ty21a). Incubations with medium alone or concanavalin A (2.5 μg/ml) were used as negative and positive controls, respectively. [³H]thymidine (0.5 μCi; Amersham) was added overnight, and incorporation was measured as counts per minute.

Expression of HPV16 L1S and plasmid stability in a kanamycin-resistant PhoP^c strain.A kanamycin-resistant plasmid expressing HPV16 L1S was constructed by replacing the ampicillin-resistant gene in the original pFSnsdHPV16 L1S (5) by a kanamycin-selectable gene. An inverse PCR strategy was used to amplify the entire plasmid flanking the ampicillin-resistant gene sequence, and the resulting fragment was ligated into a kanamycin resistance-encoding sequence (see Materials and Methods for details). The resulting plasmid was designated pFS14nsd-kan³-HPV16 L1S and electroporated into PhoP^c to yield PhoP^c kanL1S (Table 1). In vitro expression of neutralizing antibody-reactive HPV16 L1 in this new strain was similar to that measured in the ampicillin-resistant PhoP^c L1S strain (ca. 10 μg VLPs/10¹¹ CFU) (5). Similar growth rates were also observed in the two strains, with about 7 h to reach the mid-log phase, and plasmid stability was very high, with almost 100% of the bacteria still harboring the plasmid after four consecutive cultures performed overnight in antibiotic-free medium. In addition, the stability of the kanL1S plasmid was increased in vivo compared to the ampicillin plasmid, with all bacteria harboring the kanL1S plasmid in all organs examined 2 weeks after oral immunization (Table 2).

Anti-HPV16 VLP humoral responses after oral immunization with PhoP^c, PhoP⁻, and AroA strains carrying the kanL1S plasmid.The kanL1S plasmid was then introduced into two attenuated Salmonella enterica serovar Typhimurium strains, the PhoP⁻ and AroA strains (Table 1), carrying attenuating mutations in Salmonella enterica serovar Typhi vaccine strains that were previously shown to be safe for humans. Three groups of mice received a single oral dose of these three recombinant Salmonella enterica serovar Typhimurium strains, and HPV16 VLP-specific antibody responses in serum and vaginal washes were analyzed at 8 weeks postimmunization (Fig. 1). Interestingly, the serum anti-VLP antibody titers induced by the AroA kanL1S strain were as high as those induced by the PhoP^c kanL1S strain (Fig. 1A) and by the former PhoP^c L1S strain (5). In contrast, the anti-VLP antibody titers induced by PhoP⁻ kanL1S were 2 logs lower, as previously reported for PhoP⁻ L1S (5). HPV16 VLP-specific IgG and IgA were also induced in genital secretions of mice immunized with the PhoP^c kanL1S and AroA kanL1S strains, while none were detected in mice receiving the PhoP⁻ kanL1S strain (Fig. 1B). All serum titers remained stable for at least 6 months, while some decrease in genital titers were observed, as found previously with PhoP^c L1S (5; data not shown). These data obtained in mice using oral immunization suggest that recombinant Salmonella enterica serovar Typhi harboring aro deletions may be more immunogenic than those harboring phoP/phoQ deletions.

• Open in new tab
• Download powerpoint

FIG. 1.

Comparison of serum anti-HPV16 VLP antibody titers after oral vaccination with PhoP^c kanL1S, PhoP⁻ kanL1S, and ΔAroA kanL1S. Groups of five BALB/c mice were orally immunized with 10⁹ CFU of PhoP^c 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).

Humoral and cellular immune responses induced by Salmonella enterica serovar Typhi vaccine strains expressing HPV16 L1 in the i.n. murine model.We introduced the kanL1S plasmid into three Salmonella enterica serovar Typhi vaccine strains that were available to us, i.e., Ty21a, the licensed typhoid vaccine strain (ΔgalE with undefined attenuating mutations) (17); Ty800 (ΔphoP/phoQ) (19); and CVD908-htrA (ΔaroC ΔaroD htrA) (48) (Table 1). Salmonella enterica serovar Typhi strains are not invasive in mice by the oral route. However, mice can be transiently infected if bacteria are administered at high doses by the nasal route, as shown with recombinant CVD908-htrA (40, 42). The immunogenicity of the three recombinant Salmonella enterica serovar Typhi strains was therefore evaluated in BALB/c mice after administration of two i.n. doses (10⁹ CFU for Ty21a and CVD908-htrA and 10⁷ CFU for Ty800 recombinant strains), with the first dose given on day 1 and the second dose given 1 month later. The 10⁹-CFU doses and schedule were chosen according to previously reported experiments performed by other groups using the nasal murine model with Ty21a (16, 23) and CVD908-htrA (27). Administration of Ty800 to mice was not previously reported. A lower dose (10⁷ CFU) was used because higher inocula were lethal to the mice. Our observation is in agreement with a previously described higher mortality of such PhoPQ-deleted bacteria after intraperitoneal injection with hog gastric mucin in mice (2). The IgG titers against the heterologous HPV16 VLP antigen, as well as against two homologous antigens, LPS and flagellin, were measured in serum over an 8-week period (Fig. 2A). Interestingly, high anti-VLP IgG titers were induced only after immunization with Ty21a kanL1S, while low or barely detectable titers were induced by the two other strains. In contrast, Ty800 kanL1S and CVD908-htrA kanL1S induced higher anti-LPS and anti-flagellin IgG titers than Ty21a kanL1S, indicating that the two former recombinant strains had successfully infected mice and induced humoral responses against the bacterial antigens (Fig. 2B). However, the anti-LPS and anti-flagellin titers induced by Ty21a kan L1S and the parental Ty21a strain were indistinguishable (Fig. 2C). This result suggests that the parental and recombinant Ty21a strains may have a similar potential to protect against typhoid fever.

• Open in new tab
• Download powerpoint

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.

CD4⁺ T-helper cells participate in the generation and maintenance of high antibody titers; therefore, cell-mediated immune responses against flagellin and HPV16 VLPs were also examined. Eight weeks after immunization, mice were sacrificed, and antigen-specific proliferation was measured using CD4⁺ T cells purified from the corresponding spleen (Fig. 3). HPV16 VLP stimulation of CD4⁺ T cells was strongly induced after i.n. vaccination with Ty21a kanL1S (P < 0.001) (Fig. 3A), in agreement with the induction of anti-HPV16 VLP antibodies. In contrast, weak CD4⁺ T-cell proliferation was observed in mice immunized with the other two strains (Ty800 kanL1S and CVD908-htrA kanL1S). Only immunization with Ty800 kanL1S induced significant flagellin-specific stimulation of CD4⁺ T cells (P < 0.001) (Fig. 3B).

• Open in new tab
• Download powerpoint

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.

Stability and expression of the kanL1S plasmid in three Salmonella enterica serovar Typhi vaccine strains and L1 assembly status in Ty21a kanL1S.In order to gain insights into the higher immune responses against VLP observed with the Ty21a strain, plasmid stability and expression in these three recombinant Salmonella enterica serovar Typhi strains were compared. The stability of the kanL1S plasmid in the three vaccine strains in the absence of antibiotics was first examined in vitro (Fig. 4). In contrast to the above-described results for Salmonella enterica serovar Typhimurium strains, a lower stability of the kanL1S plasmid was observed in all Salmonella enterica serovar Typhi strains tested. A slow loss of plasmid occurred after the second culture performed overnight, but after five consecutive cultures performed overnight, the plasmid was retained in 10, 6, and 20% of the bacteria in Ty21a, Ty800, and CVD908-htrA, respectively. To examine whether this plasmid instability was linked to the Salmonella serovar Typhi bacterial host or to the kanamycin selection marker, the original L1S plasmid was introduced into the three Salmonella enterica serovar Typhi vaccines strains, and its stability was determined in vitro (Fig. 4). After five consecutive cultures performed overnight, the stability of the original L1S plasmid containing the ampicillin selection marker was rather similar to that of the kanL1S plasmid in Ty21a and CVD908-htrA (14% and 8% of bacteria retaining the plasmid, respectively). In contrast, the L1S plasmid was highly unstable in Ty800 (0.0002% of bacteria retaining the plasmid). Therefore, plasmid instability can be linked to either strain or resistance marker and remains unpredictable.

• Open in new tab
• Download powerpoint

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.

Salmonella enterica serovar Typhi vaccine strains will undergo only limited rounds of replication in humans. To estimate how the plasmid instability observed in vitro may translate in vivo, plasmid stability in Salmonella cells recovered from the lung and spleen of mice was examined 1 week after i.n. immunization with 10⁹ CFU of Ty21a kanL1S and CVD908-htrA kanL1S or 10⁷ CFU of Ty800 kanL1S. Despite the use of a 2-log-lower dose of Ty800, the total number of bacteria recovered from the lung was higher with this strain, suggesting a higher invasiveness (Table 3). In agreement with this finding, bacteria were recovered only from the spleen of mice immunized with Ty800 recombinant strains (data not shown). Interestingly, all bacteria recovered from the lung after Ty21a kanL1S immunization harbored the plasmid, while after Ty800 kanL1S and CVD908-htrA kanL1S immunizations, few (15.8 and 1.7%, respectively) harbored the kanL1S plasmid (Table 3). Comparison after immunization with the strains harboring the original L1S plasmid also revealed a higher plasmid stability in Ty21a (Table 3). Our data thus indicate that the L1S-expressing plasmids are more stable in Ty21a than in the other Salmonella enterica serovar Typhi vaccine strains tested in the nasal murine model.

In vitro expression levels of neutralizing antibody-reactive HPV16 L1 were similar in the three Salmonella serovar Typhi strains (20 to 30 μg VLP/10¹¹ CFU), suggesting that anti-VLP immunogenicity did not correlate to in vitro expression levels. The sandwich ELISA used to quantify conformationally correct L1 does not discriminate between L1 capsomers, capsomer aggregates, or VLPs, since all are presumably recognized by H16 E70 and H16 V5, the antibodies used in this assay (11). We therefore further evaluated the L1 assembly state in Ty21a extracts. Exponentially growing cultures were lysed and subjected to centrifugation at 10,000 × g. L1, as assessed by Western blotting with an L1-specific monoclonal antibody, was distributed approximately equally between the pellet and supernatant (data not shown), suggesting that a considerable fraction of L1 was in a high-molecular-weight complex, higher than expected for individual VLPs. The supernatant was subjected to OptiPrep step gradient centrifugation under conditions that separate VLPs from capsomers (8). Western blots reveled L1 in both high- and low-molecular-weight fractions (Fig. 5A). Separate fractions or pools of L1-positive fractions were examined by electron microscopy after negative staining. Structures resembling pentamers and various-sized amorphous aggregates of pentamers were seen, but no well-ordered VLPs were visible in any of the fractions (Fig. 5B). Star-shaped objects (Fig. 5B) were detected in the intermediate-molecular-weight fractions. Examination of these fractions after the addition of purified HPV16 L1 VLPs or pentamers confirmed that these objects were intermediate in size (data not shown), with a diameter of 17 nm, compared to 12 nm and 60 nm for pentamers and VLPs, respectively. However, they are clearly not T = 1 capsids, which can spontaneously assemble in vitro from Escherichia coli-derived N-terminally-deleted HPV16 L1, but are reported to be 30 nm (10). The star-shaped objects remained after rabbit HPV16 L1 VLP antiserum-mediated immunodepletion of all Western blot-detectable L1 (data not shown). Consequently, it is unlikely that these structures are L1 derived. We were concerned that the lysis buffer used to disrupt the cells (B-Per II reagent) might have caused a disaggregation of preformed VLPs after lysis. However, incubation of purified insect cell-derived HPV16 L1 VLPs with B-Per II reagent did not lead to the disassembly of the VLPs, as assessed by electron microscopic examination of negatively stained preparations (data not shown).

• Open in new tab
• Download powerpoint

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.

Induction of anti-HPV16 VLPs and HPV16-neutralizing antibodies in serum and genital secretions of mice immunized with Ty21a kanL1S or the prototype HPV16 VLP vaccine.In order to evaluate the potential of Ty21a kanL1S as a cervical cancer prophylactic vaccine, we compared its immunogenicity to that of purified HPV16 VLPs. Groups of five mice were immunized with two i.n. doses of Ty21a kanL1S (ca. 10⁹ CFU) at weeks 0 and 4 (Fig. 6, squares); three s.c. doses of 1 μg VLPs at weeks 0, 4, and 25 (Fig. 6, triangles); or three i.n. (aerosol-like) doses of 5 μg VLPs at weeks 0, 1, and 2 (Fig. 6, circles). The parenteral and aerosol-like protocols using purified VLPs were chosen according to our previous experience (4, 34) and to mimic vaccination of humans (18, 33). 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) was also tested (Fig. 6, diamonds). Anti-HPV16 VLP and HPV16-neutralizing antibody titers were determined in serum and vaginal washes by ELISA and the SEAP HPV16 pseudovirion neutralization assay, respectively, at short term (4 to 5 weeks after the last immunization) (Fig. 6A, C, and E) and at long term (18 to 24 weeks after the last immunization) (Fig. 6B, D, and F). Our data show that immunization with Ty21a kanL1S induced VLP-specific and HPV16-neutralizing antibodies not only in serum (Fig. 6A and B, squares) but also in genital secretions (Fig. 6C, D, E, and F, squares), which may be critical for protection against genital HPV infection. The immune responses at short term (Fig. 6A, C, and E, squares) are representative of three independent experiments.

• Open in new tab
• Download powerpoint

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.

Immunization with the prototype s.c. VLP vaccine (Fig. 6, triangles) induced significantly higher VLP-specific and HPV16-neutralizing titers in serum than the mucosal vaccines, i.e., aerosol vaccination with Ty21a kanL1S or VLPs (Fig. 6A and B, triangles versus squares and circles). However, this was not exactly reflected in genital secretions, where the differential induction of VLP-specific IgG and IgA by parenteral versus mucosal protocols of vaccination complicated the situation. While at short term, there was no statistical differences in the normalized titers of VLP-specific IgG induced in the four groups (Fig. 6C), at long term, the mice vaccinated with the prototype s.c. VLP vaccine exhibited significantly higher VLP-specific IgG titers than the mucosal vaccines (Fig. 6D). However, the opposite was true when the IgA tiers were examined: both at short term and long term, the prototype s.c. VLP vaccine induced significantly lower VLP-specific IgA titers than the mucosal vaccines (Fig. 6C and D). To have more definitive insights into the protective potential of Ty21a kanL1S compared to the prototype VLP vaccine, HPV16-neutralizing titers of vaginal washes were examined. Our data show that the protective potential of Ty21a kanL1S is not lower than those of VLP vaccines at either short or long term (Fig. 6E and F), despite the fact that only two doses were administered, suggesting that this is a promising vaccine to test in women. Evaluation of the combined VLP-Ty21a kanL1S protocol raised the possibility that the third dose of the s.c. prototype VLP vaccine may be replaced by Ty21a kanL1S, as no statistical differences were observed in serum or genital secretions with the complete s.c. VLP protocols. However, the VLP/Ty21a kanL1s prime-boost strategy generated a lower geometric mean neutralizing titer than the two-dose Ty21a kanL1S vaccination, at least at short term (Fig. 6E).