Beta7-integrin-independent enhancement of mucosal and systemic anti-HIV antibody responses following combined mucosal and systemic gene delivery

Abstract

Vaccination strategies that can block or limit heterosexual human immunodeficiency virus (HIV) transmissions to local and systemic tissues are the goal of much research effort. Herein, in a mouse model, we aimed to determine whether the enhancement of antibody responses through mucosal and systemic immunizations, previously observed with protein-based vaccines, applies to immunizations with DNA- or RNA-based vectors. Intranasal (i.n.) followed by intramuscular (i.m.) immunizations (i.n./i.m.) with polylactide-coglycolide (PLG)-DNA microparticles encoding HIV-gag (PLG-DNA-gag) significantly enhanced serum antibody responses, compared with i.m., i.n. or i.m. followed by i.n. (i.m./i.n.) immunizations. Moreover, while i.n./i.m., i.n. or i.m./i.n. immunizations with PLG-DNA-gag resulted in genital tract antibody responses, i.m. immunizations alone failed to do so. Importantly, beta7-deficient mice developed local and systemic antibody responses following i.n./i.m. immunization, or immunization via any other route, similar to those of wild-type mice. To compare the DNA with an RNA delivery system, immunizations were performed with VEE/SIN-gag replicon particles, composed of Venezuelan equine encephalitis virus (VEE) replicon RNA and Sindbis surface structure (SIN). i.n./i.m., compared with any other immunizations, i.n./i.m. immunization with VEE/SIN-gag resulted in enhanced genital tract but not serum antibody responses. These data show for the first time that mucosal followed by systemic immunizations with gene delivery systems enhance B-cell responses independent of the mucosal homing receptors alpha4beta7 and alphaEbeta7.

Figure 1

Intranasal (i.n.) followed by intramuscular (i.m.) immunizations with polylactide-coglycolide DNA microparticles encoding human immunodeficiency virus (HIV)-gag (PLG-DNA-gag) induced significantly higher serum antibody responses compared with i.n. alone, i.m. alone, or i.m. followed by i.n. immunization. Groups of female BALB/c mice were immunized i.n., i.m. or through combinations of i.n. and i.m. routes at 2-week intervals. Sera were collected at 2 weeks after each of the first three immunizations (Post 1st, 2nd and 3rd, respectively) and 7 days after the final immunization (Post 4th) and anti-gag antibody responses were measured by enzyme-linked immunosorbent assay (ELISA). The data are presented as mean serum anti-gag immunoglobulin G1 (IgG1) or IgG2a titres for individual mice from two independent experiments, with six or 10 mice in each experiment, plus the standard error of the mean; each group of mice is shown at the same relative position in the histograms at the different time-points. Numbers followed by ‘i.n.’ or ‘i.m.’ indicate the numbers of each type of immunization experienced by the group of mice at that time-point. The P-values between groups with statistically significant differences are shown between the compared groups.

Figure 2

Intranasal (i.n.) immunizations alone, or in combination with intramuscular (i.m.) immunization, with polylactide-coglycolide DNA microparticles encoding human immunodeficiency virus (HIV)-gag (PLG-DNA-gag) induced gag-specific antibody-secreting cells (ASCs) in the cervicovaginal mucosa. Groups of female BALB/c mice were immunized i.n., i.m. or through combinations of i.n. and i.m. routes with PLG-DNA-gag at 2-week intervals. The mice were killed 7 days after the final immunization. Single-cell suspensions were prepared from spleen (SP) and vaginal/uterine mucosa (VUM) from pools of three to five mice, and the cells were enriched by magnetic antibody cell sorting (MACS) for the populations expressing or not expressing the mucosal homing receptor α4β7. The α4β7 negatively and positively enriched populations were then used in an enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) to detect anti-gag ASCs. The data are presented as average number of anti-gag immunoglobulin A (IgA)- or IgG-secreting cells per million α4β7 negatively (α4β7–) or positively (α4β7+) enriched mononuclear cells (MNCs) from two subgroups of three to five mice per experiment and two independent experiments, plus standard deviation. Numbers followed by ‘i.n.’ or ‘i.m.’ indicate the numbers of each type of immunization.

Figure 3

The enhancement of serum antibody responses measured after intranasal (i.n.) followed by intramuscular (i.m.) immunizations with polylactide-coglycolide DNA microparticles encoding human immunodeficiency virus (HIV)-gag (PLG-DNA-gag) is independent of the expression of the mucosal homing receptor α4β7 or α_Eβ7. Groups of female mice genetically deficient in β7 expression (β7^–/–) and wild-type C57BL/6 (WT) mice were immunized i.n., i.m. or through combinations of i.n. and i.m. routes at 2-week intervals. Sera were collected at 7 days after the final immunization and anti-gag antibody responses were measured by enzyme-linked immunosorbent assay (ELISA). The data are presented as mean serum anti-gag immunoglobulin G1 (IgG1) or IgG2a titres for six individual mice, plus the standard error of the mean. Numbers followed by ‘i.n.’ or ‘i.m.’ indicate the numbers of each type of immunization. The P-values between groups with statistically significant differences are shown between the compared groups.

Figure 4

Local and systemic antibody responses in α4β7^–/– and wild-type (WT) mice following combinations of intranasal (i.n.) and intramuscular (i.m.) or i.m. alone or i.n. alone immunizations with polylactide-coglycolide DNA microparticles encoding human immunodeficiency virus (HIV)-gag (PLG-DNA-gag). Groups of female mice genetically deficient in α4β7 expression (α4β7^–/–) and wild-type C57BL/6 (WT) mice were immunized i.n., i.m. or through combinations of i.n. and i.m. routes at 2-week intervals. Spleens (a) and iliac lymph nodes (b) were collected at 7 days after the final immunization. Single-cell suspensions were prepared from spleen and iliac lymph nodes, which drain the vaginal mucosa, from pools of three mice, and used in an enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) to detect anti-gag immunoglobulin G (IgG)- or IgA-secreting cells. The data are presented as the average number of anti-gag IgA- or IgG-secreting cells (ASCs) per million mononuclear cells (MNCs) from two subgroups of three mice per experiment, plus standard deviation. Numbers followed by ‘i.n.’ or ‘i.m.’ indicate the numbers of each type of immunization. The P-values between groups with statistically significant differences are shown between the compared groups.

Figure 5

Intranasal (i.n.) followed by intramuscular (i.m.) immunizations with Venezuelan equine encephalitis virus (VEE)/Sindbis surface structure (SIN)-gag alphavirus-based replicon particles induced similar serum antibody responses compared with i.m. alone immunizations. Groups of female BALB/c mice were immunized i.n., i.m. or through combinations of i.n. and i.m. routes at 2-week intervals. Sera were collected at 2 weeks after each of the first three immunizations (Post 1st, 2nd and 3rd, respectively) and 7 days after the final immunization (Post 4th) and anti-gag antibody responses were measured by enzyme-linked immunosorbent assay (ELISA). The limit of detection for this assay was a titre of 5. The data are presented as mean serum anti-gag immunoglobulin G1 (IgG1) or IgG2a titres for individual mice from two independent experiments, with five mice in each experiment, plus the standard error of the mean; each group of mice is shown at the same relative position in the histograms at the different time-points. Numbers followed by ‘i.n.’ or ‘i.m.’ indicate the numbers of each type of immunization experienced by the group of mice at that time-point. The P-values between groups with statistically significant differences are shown between the compared groups.

Figure 6

Only intranasal (i.n.) followed by intramuscular (i.m.) immunizations with Venezuelan equine encephalitis virus (VEE)/Sindbis surface structure (SIN)-gag alphavirus-based replicon particles induced gag-specific antibody-secreting cells (ASCs) in the cervicovaginal mucosa. Groups of female BALB/c mice were immunized i.n., i.m. or through combinations of i.n. and i.m. routes with VEE/SIN-gag at 2-week intervals. The mice were killed 7 days after the final immunization. Single cell suspensions were prepared from spleen (SP) and vaginal/uterine mucosa (VUM) from pools of three to five mice, and the cells were enriched by magnetic antibody cell sorting (MACS) for the populations expressing or not expressing the mucosal homing receptor α4β7. The α4β7 negatively and positively enriched populations were then used in an enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) to detect anti-gag ASCs. The data are presented as average number of anti-gag immunoglobulin A (IgA)- or IgG-secreting cells per million α4β7 negatively (α4β7–) or positively (α4β7+) enriched mononuclear cells (MNCs) from groups of five mice per experiment and two independent experiments, plus standard deviation. Numbers followed by ‘i.n.’ or ‘i.m.’ indicate the numbers of each type of immunization.

Figure 7

Simultaneous intranasal (i.n.) and intramuscular (i.m.) immunizations with polylactide-coglycolide DNA microparticles encoding human immunodeficiency virus (HIV)-gag (PLG-DNA-gag) or Venezuelan equine encephalitis virus (VEE)/Sindbis surface structure (SIN)-gag alphavirus-based replicon particles. Groups of female BALB/c mice were immunized i.m. or through simultaneous combinations of i.n. and i.m. routes four times at 2-week intervals. Sera were collected at 7 days after the final immunization and anti-gag antibody responses were measured by enzyme-linked immunosorbent assay (ELISA). The data are presented as mean serum anti-gag immunoglobulin G1 (IgG1) or IgG2a titres for individual mice from two independent experiments, with five mice in each experiment, plus the standard error of the mean. Numbers followed by ‘i.n.’ or ‘i.m.’ indicate the numbers of each type of immunization. The P-values between groups with statistically significant differences are shown between the compared groups.