Sex-based differences in immune function and responses to vaccination

Bacillus Calmette-Guerin (BCG) vaccine in children

The TB vaccine, BCG is recommended for children within the first year of life in endemic regions. The efficacy of the BCG vaccine is debated, with evidence that it protects against disseminated TB in childhood, but not adulthood.⁷ In addition to protecting children against TB, BCG vaccination offers non-specific effects by significantly reducing all causes of mortality, especially in low birth weight babies.⁸ There is some evidence to suggest that the non-specific long-term protective effects of the BCG vaccine on overall survival and reduced susceptibility to respiratory infections is greater for girls than boys.^9,10 How the BCG vaccine modifies immune function to provide non-targeted beneficial effects, especially in girls, has not been determined.

Measles, mumps and rubella (MMR) in children

The measles-mumps-rubella (MMR) vaccine consists of three virus strains that have been attenuated for virulence and replication.¹¹ While the precise vaccine schedules differ among countries, rates of MMR vaccination are reportedly similar between girls and boys in the United Kingdom,¹² but not in countries where girls have reduced access to health services compared with boys.¹³ A cross-sectional study of children over 15 years of age, who received the MMR vaccine at 12–15 months after birth, indicated that the prevalence of serum IgG antibodies against measles, mumps and rubella was significantly higher in girls than boys.¹⁴ Regression models indicate that age at the time of immunization and female sex are the two most significant predictors of antibody persistence following MMR vaccination.¹⁵ In a 9-year follow-up study of children who received two doses of the MMR vaccine, the geometric mean titers of anti-mumps antibodies were equivalent in pre-pubertal girls and boys.¹⁶ A study of pre-pubertal boys and girls who received two doses of the MMR vaccine showed that rubella virus-specific antibody responses as well as lymphocyte proliferative responses to rubella virus peptides were transiently higher in males compared to females 2–4 weeks after vaccination, but this sex difference was no longer apparent 10 weeks post-vaccination.¹⁷ At later ages (14–17 years of age), girls had higher anti-rubella IgG titers than boys, suggesting that long-term protection against rubella is greater for girls.¹⁸ Whether memory responses to the MMR vaccine differ between the sexes requires further assessment. Adverse reactions, including fever, parotitis and joint and limb pain, are higher in females than in males (6–13 years of age) up to 14 weeks after the second immunization with the MMR vaccine,^19,20 except for the risk of immune thrombocytopenic purpura, which is higher in males.²¹ It has not been reported whether higher antibody responses in girls compared to boys following receipt of the MMR vaccine results in differential protection.

The Schwarz measles vaccine is a low titer viral vaccine that is only offered to infants from 9 months of age. Administration of the Edmonston-Zagreb high titer measles vaccine to infants <9 months of age was initiated by WHO in the late 1980s, in regions of West Africa.²² A long-term follow-up study of over 3 000 children inoculated with the standard, medium and high titer measles vaccines revealed that by at least 3 years of age, mortality rates were twice as high for females than males in response to the Edmonston-Zagreb high titer measles vaccine.²² The reduced survival related to the high titer measles vaccine occurred almost solely in females and led to the termination of the vaccine trials.

Yellow fever virus (YFV) vaccine in young adults

The YFV vaccine strain, 17D, is administered subcutaneously and replicates extensively, generating a viremia but with markedly reduced clinical symptoms of infection.²³ Of considerable concern to public health officials is the severity of adverse events following 17D vaccination. An analysis of reported adverse effects following YF vaccination to the U.S. Vaccine Adverse Event Reporting System (VAERS; 2000–2006) indicated that the majority of reported adverse events occurred in adult females (61%).²⁴ The most commonly reported adverse side effects in females following YF vaccination were fever, pain, pruritus, headache, injection site erythema, urticaria, rash, nausea, dizziness, dyspnea and fatigue.²⁴ Females also reported more local inflammation than males. Because the VAERS is a passive reporting system, whether females are more likely to report adverse side effects than males must also be considered.

Sex differences in the humoral immune responses to YFV vaccination have not been reported, which is likely due to the robust nature of 17D vaccine replication in the host and the choice of high vaccine doses. Innate immune gene transcriptional profiles, including the expression of toll-like receptors (TLR) and interferon (IFN)-associated genes, immediately after vaccination with YF17D predict subsequent adaptive immune responses,^25,26 and the expression of these TLR/IFN-associated genes is significantly higher in females compared with males after vaccination.¹ Whether the efficacy of the YFV vaccine is higher in females compared to males has not been reported.

Influenza vaccine in adults and aged individuals

The intention of receiving either pandemic or avian influenza vaccines is reportedly 2–3 times lower for females than males, even among healthcare providers.^27–30 Receipt of seasonal trivalent influenza vaccines in the United States and in several European countries is consistently lower among both young and older adult females compared with their male counterparts.^31–34 A systematic review revealed that during the 2009 H1N1 pandemic (pH1N1), receipt of the monovalent vaccine was higher in males than females worldwide.⁴

Among both younger (18–64 years) and older (>65 years) adults, females have higher hemagglutination inhibition (HAI) antibody titers than males following seasonal trivalent influenza vaccine (TIV). Receipt of either a full or half dose of seasonal TIV in adults 18–49 years of age results in HAI antibody titers that are at least twice as high in females compared to males.³⁵ Among older adults, receipt of TIV results in significantly higher HAI titers among older females compared to males.⁶ Among older adults that received the standard seasonal TIV, higher HAI titers were associated with lower rates of hospitalization and mortality in females compared to males, suggesting that the efficacy of TIV in older adults might be higher for females.^36,37 Among older adults who receive the high-dose TIV, sex differences in HAI titers are still apparent, in which antibody responses are significantly higher in older females compared to males against each of the three influenza antigens.³⁸ Older females were also reported to have higher HAI antibody titers against the monovalent pH1N1 inactivated vaccine than males, resulting in a 2–3 times higher seroprotection and seroconversion rate in females compared to males.³⁹ Although older females produced higher antibody responses to the pH1N1 vaccine, the avidity of their antibodies after pH1N1 vaccination was significantly lower than that of older males.⁴⁰

Passive reporting of local reactions (e.g., muscle pain, redness and inflammation) to influenza vaccines is more frequent for females than males among both younger and older adults.⁴¹ Measurements of local erythema and induration, both of which are associated with inflammation, reveal that both younger and older adult females have larger (≥6 mm) injection site reactions to TIV than their male counterparts.⁴² Systemic reactions (e.g., fever, chills, nausea, headaches and body aches) to TIV also are more commonly reported by females than males.⁴³ Reports of local and systemic adverse reactions, including immediate hypersensitivity reactions, also are more frequent among adult females than males following receipt of the inactivated monovalent pH1N1 vaccine.^44–46

Sex differences in innate immune responses

Pattern recognition receptors, including the Toll-like receptors, are expressed by sentinel cells (e.g., macrophages and dendritic cells [DCs]) and detect the presence of conserved microbial and viral motifs to initiate the innate immune response not only to pathogens but also to vaccine antigens.⁴⁷ The detection of lipopolysaccharide (LPS), for example, involves TLR4, which is expressed on immune cells including monocytes, macrophages and DCs. Macrophages derived from males express higher levels of TLR4 compared to female-derived cells, both constitutively and following activation.⁴⁸ Macrophages from female mice, however, express higher levels of MyD88⁴⁹ and greater p38 MAP kinase phosphorylation and, hence, activation following LPS challenge than do macrophages from males.⁵⁰ Peripheral blood mononuclear cells (PBMCs) and plasmacytoid DCs from female patients produce higher levels of type I IFNs in response to TLR7 ligands,^51,52 but lower levels of IL-10 in response to ligands for TLR8 or TLR9⁵³ than similarly challenged male-derived cells.

Sex biases exist at the level of inflammatory cytokine production by antigen presenting cells, including macrophages and DCs. Peripheral monocytes from male human subjects as well as peritoneal macrophages from male mice produce significantly higher levels of TNF-α, IL-1β, IL-6 and CXCL10 than cells from females.^54–57 Peritoneal macrophages isolated from male rodents produce significantly lower amounts of the anti-inflammatory prostanoids following either LPS treatment⁵⁷ or adjuvant administration⁵⁸ compared to female-derived cells. Female-derived splenic macrophages also secrete higher levels of IL-10 than do cells derived from males.⁵⁶

In response to TIV, females have higher levels of inflammatory cytokines compared to males, which correlate with monocyte phosphorylated STAT3 levels.⁴ In a humanized mouse model, TLR7 ligand-induced IFN-α production by human plasmacytoid DCs is higher in female than male hosts.⁵² Treatment of postmenopausal women or mice with estradiol markedly enhances TLR7 or TLR9-mediated IFN-α production by plasmacytoid DCs in an estrogen receptor alpha (ERα)-dependent manner.⁵² Furthermore, PBMCs derived from males produce less IFN-α in response to TLR7 ligands and more IL-10 after either exposure to TLR8 or TLR9 ligands or infection with herpes simplex virus 1.⁵³ Whether sex differences in innate immune cell activity results in differences in the outcome of vaccination requires greater consideration, especially for adjuvanted vaccines.

Sex differences in adaptive immune responses

Generally, females exhibit elevated humoral and cell-mediated immune responses to vaccination compared to males.⁵⁹ Clinical studies reveal that males have lower absolute CD3+ cell counts, absolute numbers of CD4+ T cells, CD4+:CD8+ cell ratios and helper T cell type 1 (Th1) responses.^60–63 Studies in mice further reveal that cytokine responses of CD4+ T cells often differ between males and females.^64–66 For example, female mice have been shown to produce higher Th2 cytokine responses (e.g., IL-4, IL-9 and IL-13) than males, at least following viral and parasitic infections.^67,68 Polyclonal activation of PBMCs with an exogenous antigen results in higher Th2 responses in female-derived cells compared to male-derived cells.⁶⁹ Female mice have higher proportions of regulatory T cells (Tregs) than males⁷⁰ that may underlie tolerance of fetal antigens in women during pregnancy.⁷¹

Females exhibit higher cytotoxic T-lymphocyte activity than males, including upregulated expression of antiviral and proinflammatory genes in T cells isolated from women as compared with T cells isolated from men.⁷² Treatment-naïve women chronically infected with HIV-1 show higher levels of CD8+ T-cell activation than men when adjusted for viral load.⁵¹ Several non-specific indicators of cell-mediated immunity are also elevated in females, with females having higher mitogen-stimulated lymphocyte proliferation, faster wound healing, and increased immunological intolerance to foreign substances than males.^73–75

In general, females demonstrate greater antibody responses than males.^1,4,76,77 Both basal levels of immunoglobulin,⁷⁸ as well as antibody responses to vaccines, are consistently higher in females than males.⁷⁷ There are, however, reports of higher humoral responses in males than females following some vaccinations, which seem to depend on the vaccine type, age and societal status of the recipient.⁷⁷

Sex steroid hormones

Estradiol, progesterone and testosterone affect the effector functions of immune cells. The estrogen receptors, ERα and ERβ, for example, are expressed in various immune cells including T and B cells, natural killer cells, macrophages, DCs and neutrophils indicating responsiveness to estrogens. Activation of their cognate receptors by progesterone and testosterone also modulates the functions of T and B cells, DCs, macrophages and natural killer cells. The effects of these sex hormones are dose-dependent. This is especially relevant for estrogen and progesterone where their concentrations vary during different stages of the menstrual cycle, during pregnancy and after menopause. For example, low doses of estrogen during the luteal phase of the menstrual cycle influences Tbet expression, associated with a Th1 response, namely increased IFN-γ production, whereas higher doses of estrogen, such as during the follicular phase, down-regulate IRF1 expression, leading to Th2 polarization and IL-4, IL-5 and IL-10 cytokine production.⁵⁸ Estrogens drive expansion of Tregs and increase their suppressive effects, most notably early during pregnancy, whereas increasing progesterone levels in the second trimester lead to diminished Treg numbers.⁷⁹ Dose-dependent estrogen effects on Tregs contribute to down-regulation of the pro-inflammatory effects of Th17 cells. Recent studies have identified that human T cells exhibit a sex difference in IFN-γ and IL-17A production, i.e., Th1 vs Th17 bias, based on androgen effects on peroxisome proliferator-activated receptors (PPAR)α and γ, with androgens increasing PPARα and decreasing PPARγ, thereby limiting IFN-γ production and increasing IL-17A production.⁸⁰

Estrogens and progesterone/androgens likewise exert opposing effects on B cells, with estrogens implicated in promoting antibody production by B cells, mediated by Th2 associated cytokines IL-4 and IL-5 driving B cell proliferation and differentiation to plasma cells, and progesterone and androgens exerting inhibitory effects on antibody production by B cells.^4,81

Both estradiol and testosterone are associated with enhanced functional responses by DCs,^82,83 whereas progesterone exerts immunosuppressive effects on DCs.⁸⁴ The estrogen/progesterone balance differentially affects macrophage TNF-α and NO production and estradiol exerts anti-inflammatory effects on macrophages in vitro but pro-inflammatory effects on macrophages in vivo.⁸⁵ Accumulating evidence suggests that testosterone suppresses pro-inflammatory cytokine secretion by macrophages.⁸⁶

Genetic and epigenetic regulation

In addition to hormonal influences, genetic and epigenetic factors contribute to sex-based differences in an immune response to vaccination. Specifically, the sex-based immune response differences observed between pre-pubertal boys and girls, and post-menopausal women and elderly men, suggest other contributions beyond sex hormones. A large number of immune-related genes encoding proteins, including IL-2 receptor-γ chain, IL-3 receptor-α chain, IL-13 receptor-α chains, TLR7, TLR8, GATA1, IRAK1, CD40 ligand and FOXP3 are located on the X chromosome.⁵⁹ A number of critical transcriptional and translational control effectors, which function downstream of activated cytokine receptors, are encoded on the X chromosome. The implications are that X-linked genes are determinants of immunocompetence. Given that males are XY and females XX, any damaging mutations or polymorphisms to X-linked genes are more likely to have an immune consequence in males compared to females.⁸⁷ There is evidence that X chromosome inactivation, the process whereby gene dosage is addressed in XX females, is not entirely random, but that skewed X chromosome inactivation may favor elimination of mutant genes and the persistence of intact genes by clonal maintenance.⁸⁸ The androgen receptor is encoded on the X chromosome.⁸⁹ Both estrogen and androgen receptors bind to hormone response elements upstream of target genes and recruit methyltransferase and histone acetyltransferase enzymes that regulate gene transcription. The combined effects of hormones influencing the epigenetic regulation of gene expression, and gene composition on the X chromosome potentially differing between XX females and XY males, will determine an immune response to vaccination.

Considerable attention has focused in recent years on the contributions of non-coding microRNAs (miRNAs) to the control of gene expression, by either repressing mRNA translation or triggering mRNA degradation. The X chromosome contains 10% of the approximate 800 miRNAs in the genome; whereas the Y chromosome contains only 2 miRNAs.⁹⁰ Accordingly, the prevalence of miRNAs on the X chromosome that includes a large number of immune-related genes inevitably influences sex-based differences in immune responses. Certainly, miRNAs are critical regulators in immune cell development and function.⁹¹ Most notable are the X-chromosome encoded miRNA-18 and -19, implicated as miRNAs that have a role in sex-based differences in immunity, specifically potentiating inflammatory responses through the control of repressors of NF-κB signaling.^92,93

Microbiome

Recent studies have provided insights into the relationship between human microbiota and the host's immunophenotype,⁹⁴ with supporting evidence for sex-specific relationships.⁹⁰ The human microbiota is composed of microbial communities in different habitats including the skin, gut, oral cavity, genitals that vary according to sex and time. Both diet and antibiotic use produces shifts in microbiota.⁹⁵ Age-related gastric atrophy affects the gastric microbiota, as do age-related vaginal changes pre- and post-menopause. Notably, bacteria can metabolize sex hormones, mediated by hydroxysteroid dehydrogenase enzymes that regulate the balance between active and inactive steroids.⁹⁶ Consequently, microbiome composition will directly influence an immune response, in a sex-specific manner. Antibiotics use will affect this bacteria-regulated hormonal metabolism.

Accumulating evidence indicates that hormonal status can shape microbiome composition; the onset of puberty and concomitant hormone-specific changes result in sex-specific microbiome profiles.⁹⁷ These sex-specific and hormonally directed microbiomes appear to influence the immunophenotype because in a spontaneous mouse model of type I diabetes the adoptive transfer of male-enriched gut commensals into females resulted in systemic hormonal changes and protected against disease.^98,99

Viewed together, site-specific microbiome composition will affect an immune response in a sex-specific manner. Data are accumulating that site-specific microbiota have a role in modulating immune responses both locally and systemically. The impact of the host microbiota on a vaccine response, therefore, necessitates investigation.¹⁰⁰ Differences in the immune response outcomes, i.e., efficacy of oral vaccines against poliovirus, rotavirus and cholera in different geographical locations were ascribed to differences in gut microbiota.^100,101 Other studies have shown that probiotics enhanced antibody responses to oral vaccines against rotavirus, poliovirus, Salmonella and Vibrio cholera.¹⁰¹ Likewise, probiotics improved immune responses in infants given oral vaccines against diphtheria, tetanus, Haemophilus influenza B and hepatitis virus B.¹⁰¹