Both systemic and mucosal LCMV immunization generate robust viral-specific IgG in mucosal secretions, but elicit poor LCMV-specific IgA

Abstract

Immunoglobulins in secretions play a critical role in protection at mucosal surfaces. We examined the generation of viral-specific IgG and IgA in plasma and mucosal secretions of mice following systemic or mucosal immunization with lymphocytic choriomeningitis virus (LCMV), a widely used experimental model of viral infection. While there are early differences in humoral responses depending on the route of viral entry, we show that both routes generate comparably robust viral-specific IgG in plasma, vaginal, lung, and nasal secretions of immune mice. In contrast, LCMV elicited poor viral-specific IgA responses. Mice that were infected IN showed elevated viral-specific IgA in nasal and lung washes compared to IP-infected mice; however, LCMV-specific IgG overwhelmingly contributed to the humoral response in all mucosal secretions examined. Thus similarly to HIV-1, and several other mucosally-encountered microbial infections, these data suggest that LCMV infection fails to induce vigorous viral-specific IgA responses.

FIG. 1.

LCMV-specific humoral responses in plasma and vaginal washes at day 8 p.i. In each experiment groups of female mice were infected with 2 × 10⁵ pfu LCMV-Armstong IP or IN, and LCMV-specific antibody responses were determined by ELISA at day 8 p.i. (A) LCMV-specific IgG, LCMV-specific IgA, and LCMV-specific IgM in plasma. (B) LCMV-specific IgG and IgA in vaginal washes. (C) LCMV-specific IgG isotypes in plasma: IgG_2c, IgG₃, IgG_2b, and IgG₁. (D) Viral titers from indicated tissues at day 3 p.i. Dotted lines indicate the limit of detection for each assay. Each symbol represents one mouse. Results from several similar independent experiments were combined, with 3–4 female mice per group in each experiment. Dark circles represent mice infected IP, white circles represent mice infected IN, and dark triangles represent naïve, uninfected mice. Unpaired Student's t-tests were performed to determine statistical significance (***p < 0.001; **p < 0.01; *p < 0.5; ILN, iliac nodes; CLN, cervical lymph nodes).

FIG. 1.

LCMV-specific humoral responses in plasma and vaginal washes at day 8 p.i. In each experiment groups of female mice were infected with 2 × 10⁵ pfu LCMV-Armstong IP or IN, and LCMV-specific antibody responses were determined by ELISA at day 8 p.i. (A) LCMV-specific IgG, LCMV-specific IgA, and LCMV-specific IgM in plasma. (B) LCMV-specific IgG and IgA in vaginal washes. (C) LCMV-specific IgG isotypes in plasma: IgG_2c, IgG₃, IgG_2b, and IgG₁. (D) Viral titers from indicated tissues at day 3 p.i. Dotted lines indicate the limit of detection for each assay. Each symbol represents one mouse. Results from several similar independent experiments were combined, with 3–4 female mice per group in each experiment. Dark circles represent mice infected IP, white circles represent mice infected IN, and dark triangles represent naïve, uninfected mice. Unpaired Student's t-tests were performed to determine statistical significance (***p < 0.001; **p < 0.01; *p < 0.5; ILN, iliac nodes; CLN, cervical lymph nodes).

FIG. 2.

LCMV-specific humoral responses in plasma and vaginal washes at memory. Groups of female mice were infected with 2 × 10⁵ pfu LCMV-Armstong IP or IN, and LCMV-specific antibody responses in vaginal washes were determined by ELISA at day 44 or 140 p.i. (memory includes data from days 44 and 140). (A) LCMV-specific IgG and IgA in plasma. (B) LCMV-specific IgG and IgA in vaginal washes. (C) Total IgG in vaginal washes comparing IP and IN immune mice to uninfected mice. (D) Total IgA at memory comparing IP and IN immune mice to uninfected mice. (E) LCMV-specific IgG_2c, IgG₃, IgG_2b, and IgG₁ in plasma. Each symbol represents one mouse. Dark circles represent mice infected IP, white circles represent mice infected IN, and dark triangles represent naïve, uninfected mice. Results from several independent experiments with 3–4 female mice per group were combined. Unpaired Student's t-tests were performed to determine statistical significance (***p < 0.001; **p < 0.01; *p < 0.5; ns, not significant).

FIG. 3.

LCMV-specific humoral responses at day 8. Groups of C57BL/6 female mice that did not receive transgenic T cells were infected with 2 × 10⁵ pfu LCMV-Armstrong IP or IN, and LCMV-specific antibody responses in plasma, vaginal, lung, and nasal washes were determined by ELISA on day 8. (A) LCMV-specific IgG, and (B) LCMV-specific IgA in the indicated tissues at day 8 p.i. (C) LCMV-specific IgM, IgG_2c, IgG₃, IgG_2b, and IgG₁ in plasma. (D) Frequency of LCMV-specific IgG, IgA, and IgM ASCs were determined in spleens using ELISPOT. Each symbol represents one mouse, with dark circles representing mice infected IP, white circles representing mice infected IN, and dark triangles representing naïve, uninfected mice. Results were combined from two independent experiments. Dashed lines indicate the limit of detection for each assay. Unpaired Student's t-tests were performed to determine statistical significance (***p < 0.001; **p < 0.01; *p < 0.5; ns, not significant).

FIG. 3.

LCMV-specific humoral responses at day 8. Groups of C57BL/6 female mice that did not receive transgenic T cells were infected with 2 × 10⁵ pfu LCMV-Armstrong IP or IN, and LCMV-specific antibody responses in plasma, vaginal, lung, and nasal washes were determined by ELISA on day 8. (A) LCMV-specific IgG, and (B) LCMV-specific IgA in the indicated tissues at day 8 p.i. (C) LCMV-specific IgM, IgG_2c, IgG₃, IgG_2b, and IgG₁ in plasma. (D) Frequency of LCMV-specific IgG, IgA, and IgM ASCs were determined in spleens using ELISPOT. Each symbol represents one mouse, with dark circles representing mice infected IP, white circles representing mice infected IN, and dark triangles representing naïve, uninfected mice. Results were combined from two independent experiments. Dashed lines indicate the limit of detection for each assay. Unpaired Student's t-tests were performed to determine statistical significance (***p < 0.001; **p < 0.01; *p < 0.5; ns, not significant).

FIG. 4.

LCMV-specific humoral responses at memory. Groups of C57BL/6 female mice that did not receive transgenic T cells were infected with 2 × 10⁵ pfu LCMV-Armstrong IP or IN, and LCMV-specific antibody responses in plasma, vaginal, lung, and nasal washes were determined by ELISA at memory (days 44 and 102 p.i.). (A) LCMV-specific IgG, and (B) LCMV-specific IgA in the indicated tissues of immune mice. (C) LCMV-specific IgG and IgA from A and B were added for total LCMV-specific antibody, and the percentage of LCMV-specific IgA for each site is shown. (D) LCMV-specific IgG and IgA ASCs were determined by ELISPOT analysis in spleen and bone marrow (BM) of immune mice. (E) Total GP61-80-specific CD4 T cells and percentage of IFN-γ⁺ IL-2⁺ co-expressing cells of immune mice as determined by intracellular cytokine staining. Each symbol represents one mouse, and dark symbols represent IP mice and white symbols represent IN mice. The results were combined from two independent experiments, with 3 female mice per group in each experiment, except for vaginal wash, for which only one experiment is shown. Dashed lines indicate the limit of detection for each assay. Unpaired Student's t-tests were performed to determine statistical significance (***p < 0.001; **p < 0.01; *p < 0.5; ns, not significant).