Estradiol mediates greater germinal center responses to influenza vaccination in female than male mice

Abstract

Adult females of reproductive age develop greater antibody responses to inactivated influenza vaccines (IIV) than males. How sex, age, and sex steroid concentrations impact B cells and durability of IIV-induced immunity and protection over 4 months post-vaccination (mpv) was analyzed. Vaccinated adult females had greater germinal center B cell and plasmablast frequencies in lymphoid tissues, higher neutralizing antibody responses 1-4 mpv, and better protection against live H1N1 challenge than adult males. Aged mice, regardless of sex, had reduced B cell frequencies, less durable antibody responses, and inferior protection after challenge than adult mice, which correlated with diminished estradiol among aged females. To confirm that greater IIV-induced immunity was caused by sex hormones, four core genotype (FCG) mice were used, in which the testes-determining gene, Sry, was deleted from chromosome Y (ChrY) and transferred to Chr3 to separate gonadal sex (i.e., ovaries or testes) from sex chromosome complement (i.e., XX or XY complement). Vaccinated, gonadal female FCG mice (XXF and XYF) had greater numbers of B cells, higher antiviral antibody titers, and reduced pulmonary virus titers following live H1N1 challenge than gonadal FCG males (XYM and XXM). To establish that lower estradiol concentrations cause diminished immunity, adult and aged females received either a placebo or estradiol replacement therapy prior to IIV. Estradiol replacement significantly increased IIV-induced antibody responses and reduced morbidity after the H1N1 challenge among aged females. These data highlight that estradiol is a targetable mechanism mediating greater humoral immunity following vaccination among adult females.IMPORTANCEFemales of reproductive ages develop greater antibody responses to influenza vaccines than males. We hypothesized that female-biased immunity and protection against influenza were mediated by estradiol signaling in B cells. Using diverse mouse models ranging from advanced-age mice to transgenic mice that separate sex steroids from sex chromosome complement, those mice with greater concentrations of estradiol consistently had greater numbers of antibody-producing B cells in lymphoid tissue, higher antiviral antibody titers, and greater protection against live influenza virus challenge. Treatment of aged female mice with estradiol enhanced vaccine-induced immunity and protection against disease, suggesting that estradiol signaling in B cells is critical for improved vaccine outcomes in females.

Keywords: B-cell responses; influenza vaccines; neutralizing antibodies; plasmablast; sex steroids; somatic hypermutation.

Fig 1

The frequency and number of germinal center B cells and plasmablasts are greater in the draining lymph nodes and spleens from vaccinated adults but not aged, females than males. Adult (2-3 months old) and aged (17 months old) male and female C57BL/6CR mice were vaccinated twice with inactivated 2009 H1N1 vaccine in a 3-week interval. At 35 days post-vaccination (i.e., 14 days post-boost), draining lymph nodes and spleens were collected, single-cell suspensions were prepared, and flow cytometry was performed to measure the frequencies and numbers of GC B cells and plasmablasts. (A) Representative flow plots are shown from the lymph nodes of one adult female mouse. Total numbers of lymph node cells and total B cells were quantified (B and C). Frequencies and numbers of (D and E) GC B cells and (F and G) plasmablasts in the lymph nodes were quantified. The frequencies and numbers of GC B cells in the spleen were quantified (H, I), GC B cells were sorted, and (J) mutation frequency in the J_H4 intronic region of sorted splenic GC B cells was measured. Data represent the mean ± standard error of the mean (n = 5–19/group), and asterisks (*) represent significant differences (P < 0.05) between the groups based on two-way ANOVAs followed by Tukey’s multiple comparisons tests in GraphPad Prism 10.1.0.

Fig 2

Adult, but not aged, females have higher antibody titers at 1-month post-vaccination. Adult (2-3 months old) and aged (17 months old) male and female C57BL/6CR mice were vaccinated twice with inactivated 2009 H1N1 vaccine in a 3-week interval. At 35 days post-vaccination (i.e., 1 mpv), plasma samples were collected to determine the titers of anti-2009 H1N1 influenza virus-specific (A) IgG, (B) IgG1, (C) IgG2c, and (D) virus-neutralizing antibody (nAb) titers and to measure the concentrations of (E) estradiol and (F) testosterone. Data represent the mean ± standard error of the mean (n = 5–20/group), and asterisks (*) represent significant differences (P < 0.05) between the groups based on two-way ANOVAs followed by Tukey’s multiple comparisons tests in GraphPad Prism 10.1.0.

Fig 3

Adult female mice maintain higher titers of influenza vaccine-induced antibodies up to 4 months post-vaccination, which is mitigated with aging. Adult (2-3 months old) and aged (17 months old) male and female C57BL/6CR mice were vaccinated twice with inactivated 2009 H1N1 vaccine at a 3-week interval. Plasma samples were collected each month until 4 mpv, and anti-2009 H1N1 influenza virus-specific (A) IgG, (B) IgG2c, and (C) virus-neutralizing antibody (nAb) titers were measured. Data represent the mean ± standard error of the mean (n = 15–20/group), and significant differences between the groups are denoted by asterisks (*P < 0.05) based on repeated measures two-way ANOVAs followed by Tukey’s multiple comparisons tests in GraphPad Prism 10.1.0.

Fig 4

Female-biased vaccine-induced protection against infection, but not disease, is maintained for up to 4 months post-vaccination (mpv) among adult, but not aged, animals. Adult (2-3 months old) and aged (17 months old) male and female C57BL/6CR mice were vaccinated twice with inactivated 2009 H1N1 vaccine at a 3-week interval. At 1 or 4 mpv, vaccinated mice were challenged with 10⁵ TCID₅₀ of a drift variant of the 2009 H1N1 virus. (A and B) Replicating virus titers in the lungs were measured in a subset of mice at 5 days post-challenge (dpc), and (C and D) changes in body mass over a period of 14 dpc were measured in another subset of mice to compare protection from severe disease. Data represent the mean ± standard error of the mean (n = 5–10/group), and significant differences between the groups are denoted by asterisks (*P < 0.05) based on two-way ANOVAs or repeated measures two-way ANOVAs followed by Tukey’s multiple comparisons tests in GraphPad Prism 10.1.0.

Fig 5

Gonadal sex more than sex chromosomal complement mediates influenza vaccine-induced immunity and protection. Eight- to 10-week-old four core genotype C57BL/6 J mice were vaccinated twice with the inactivated 2009 H1N1 vaccine at a 3-week interval. Plasma samples were collected at 28 days post-vaccination, and concentrations of (A) estradiol and (B) testosterone along with 2009 H1N1 influenza virus-specific (C) IgG, (D) IgG2c, and (E) virus-neutralizing antibody (nAb) titers were measured. At 35 dpv (i.e., 14 days post-boost), popliteal and inguinal lymph nodes were collected, single-cell suspensions were prepared, and the numbers of (F) germinal center B cells and (G) plasmablasts were quantified using flow cytometry. At 42 dpv, mice were challenged with 10⁵ TCID₅₀ of a drift variant of the 2009 H1N1 virus, and (H) replicating virus titers in the lungs were measured in a subset of mice at 5 days post-challenge, and (I) the percentage change in body mass over a period of 14 dpc was measured in another subset of mice to evaluate protection from severe disease. Data represent the mean ± standard error of the mean (n = 5–27/group), and significant differences between the groups are denoted by asterisks (*P < 0.05) based on two-way ANOVAs or repeated measures two-way ANOVAs followed by Tukey’s multiple comparisons tests in GraphPad Prism 10.1.0.

Fig 6

Estradiol replacement improves influenza vaccine-induced antibody responses and protection in aged female mice. Adult (2-3 months old) or aged (17 months old) female C57BL/6CR mice were subcutaneously implanted either with placebo or estradiol (E2)-loaded capsules. One week after capsule implantation, mice were vaccinated with inactivated 2009 H1N1 vaccine and boosted after 3 weeks. At 35 days post-vaccination, plasma samples were collected, and anti-2009 H1N1 influenza virus-specific (A) IgG, (B) IgG2c, and (C) virus-neutralizing antibody (nAb) titers were measured. At 42 dpv, vaccinated mice were challenged with 10⁵ TCID₅₀ of a drift variant of the 2009 H1N1 virus. (D) Replicating virus titers in the lungs were measured in a subset of mice at 3 days post-challenge, and (E) changes in body mass over a period of 14 dpc were measured in another subset of mice to compare protection from severe disease. Data represent the mean ± standard error of the mean (n = 7–15/group), and significant differences between the groups are denoted by asterisks (*P < 0.05) based on two-way ANOVAs or repeated measures two-way ANOVAs followed by Tukey’s multiple comparisons tests in GraphPad Prism 10.1.0.