Nasal immunization of mice with human papillomavirus type 16 virus-like particles elicits neutralizing antibodies in mucosal secretions

Abstract

To specifically induce a mucosal antibody response to purified human papillomavirus type 16 (HPV16) virus-like particles (VLP), we immunized female BALB/c mice orally, intranasally, and/or parenterally and evaluated cholera toxin (CT) as a mucosal adjuvant. Anti-HPV16 VLP immunoglobulin G (IgG) and IgA titers in serum, saliva, and genital secretions were measured by enzyme-linked immunosorbent assay (ELISA). Systemic immunizations alone induced HPV16 VLP-specific IgG in serum and, to a lesser extent, in genital secretions but no secretory IgA. Oral immunization, even in the presence of CT, was inefficient. However, three nasal immunizations with 5 microgram of VLP given at weekly intervals to anesthetized mice induced high (>10(4)) and long-lasting (>15 weeks) titers of anti-HPV16 VLP antibodies in all samples, including IgA and IgG in saliva and genital secretions. CT enhanced the VLP-specific antibody response 10-fold in serum and to a lesser extent in saliva and genital secretions. Nasal immunization of conscious mice compared to anesthetized mice was inefficient and correlated with the absence of uptake of a marker into the lung. However, a 1-microgram VLP systemic priming followed by two 5-microgram VLP intranasal boosts in conscious mice induced both HPV16 VLP-specific IgG and IgA in secretions, although the titers were lower than in anesthetized mice given three intranasal immunizations. Antibodies in serum, saliva, and genital secretions of immunized mice were strongly neutralizing in vitro (50% neutralization with ELISA titers of 65 to 125). The mucosal and systemic/mucosal HPV16 VLP immunization protocols that induced significant titers of neutralizing IgG and secretory IgA in mucosal secretions in mice may be relevant to genital HPV VLP-based human vaccine trials.

FIG. 1

Anti-HPV16 VLP systemic and mucosal antibody responses after intranasal immunization with purified HPV16 VLP in the presence or absence of the CT adjuvant. Two groups of four 6-week-old BALB/c anesthetized mice were immunized three times weekly with 5 μg of HPV16 VLP alone or mixed with 5 μg of CT by the intranasal route. Data are expressed as the geometric means (log₁₀) of the reciprocal dilutions of specific IgG in serum and specific IgA per microgram of total IgA or specific IgG per microgram of total IgG in secretions. Error bars indicate the standard errors of the means.

FIG. 2

Fate of an inoculum 15 min after intranasal immunization of anesthetized or conscious mice. Two groups of three 6-week-old BALB/c mice were immunized intranasally with 10⁸ CFU of S. typhimurium PhoP^c in 20 μl of PBS (25, 43) while they were under anesthesia or conscious. At 15 min later, the nasal tissue, the trachea and lungs (“lung”), the esophage, pharynx, and stomach (“stomach”), and the intestine and rectum (“intestine”) were recovered and homogenized in a Dounce homogenizer in 2 to 4 ml of 15% sucrose in PBS (43). Data are expressed as the geometric mean (log₁₀) of recovered CFU per organ. Error bars indicate the standard errors of the means. The percentages of the recovered inoculum in each organ is also indicated.

FIG. 3

Anti-HPV16 VLP systemic and mucosal antibody responses after single or double nasal immunizations. Two groups of four 6-week-old BALB/c anesthetized mice were immunized at week 0 and at week 8 with 1 or 5 μg of HPV16 VLP by the intranasal route. Data are expressed as the geometric means (log₁₀) of the reciprocal dilutions of specific IgG in serum and specific IgA per microgram of total IgA or specific IgG per microgram of total IgG in secretions. Error bars indicate the standard errors of the means.

FIG. 4

Anti-HPV16 VLP systemic and mucosal antibody responses following systemic boosting. Three groups of mice previously immunized (Fig. 1 and conscious mice) by the intranasal route, three times weekly, with 5 μg of HPV16 VLP mixed with CT or alone under anesthesia or conscious were given subcutaneous (s.c.) booster doses at week 15 with 1 μg of HPV16 VLP. Data are expressed as the geometric means (log₁₀) of the reciprocal dilutions of specific IgG in serum and specific IgA per microgram of total IgA or specific IgG per microgram of total IgG in secretions. Error bars have been deleted to simplify reading.

FIG. 5

Anti-HPV16 VLP systemic and mucosal antibody responses in mice systemically primed. Four groups of four 6-week-old BALB/c mice were primed with 1 μg of HPV16 VLP subcutaneously and then given booster doses at weeks 2 and 10 either intranasally with 1 or 5 μg of HPV16 VLP under anesthesia or conscious or subcutaneously (s.c.) with 1 μg of HPV16 VLP. Data are expressed as the geometric means (log₁₀) of the reciprocal dilutions of specific IgG in serum and specific IgA per microgram of total IgA or specific IgG per microgram of total IgG in secretions. Error bars indicate the standard errors of the means.

FIG. 6

Direct comparison of anti-HPV16 VLP systemic and mucosal antibody responses in mice systemically primed or only intranasally immunized. Three groups of four 6-week-old BALB/c mice were either primed subcutaneously with 1 μg of HPV16 VLP and then given intranasal booster doses of 5 μg at weeks 1 and 2 under anesthesia or conscious or only intranasally immunized three times weekly with 5 μg of HPV16 VLP under anesthesia. Data are expressed as the geometric means (log₁₀) of the reciprocal dilutions of specific IgG in serum and specific IgA per microgram of total IgA or IgG per microgram of total IgG in secretions. Error bars indicate the standard errors of the means.

FIG. 7

Neutralization efficacy of anti-HPV16 IgA and/or IgG in secretion and serum. The final titers of anti-HPV16 VLP IgA and/or IgG in the samples that were used for neutralization experiments were plotted against the neutralization efficacy elicited. Best-fitting sigmoidal curves were drawn with the different groups (GraphPad, Prism). In the secretions that contained both specific IgA and IgG (Table 1), the titers of the two specific isotypes were added and the summed value is indicated.