Greater Breadth of Vaccine-Induced Immunity in Females than Males Is Mediated by Increased Antibody Diversity in Germinal Center B Cells.

Inactivated influenza vaccines induce greater antibody responses in females than males among both humans and mice. To test the breadth of protection, we used recombinant mouse-adapted A/California/2009 (maA/Cal/09) H1N1 viruses containing mutations at one (1M), two (2M), or three (3M) antigenic sites, in addition to a virus containing the 1M mutation and a substitution of the Ca2 antigenic site (Sub) with one derived from an H5 hemagglutinin (HA) to challenge mice of both sexes. Following maA/Cal/09 vaccination, females produced greater virus-specific, class-switched total IgG and IgG2c antibodies against the vaccine and all mutant viruses, and antibodies from females recognized a greater number of unique, linear HA epitopes than did antibodies from males. While females had greater neutralizing antibody titers against the vaccine virus, both sexes showed a lower neutralization capacity against mutant viruses. After virus challenge, vaccinated females had lower pulmonary virus titers and reduced morbidity than males for the 1M and 2M viruses, but not the Sub virus. Females generated greater numbers of germinal center (GC) B cells containing superior somatic hypermutation (SHM) frequencies than vaccinated males. Deletion of activation-induced cytidine deaminase (Aicda) eliminated female-biased immunity and protection against the 2M virus. Harnessing methods to improve GC B cell responses and frequencies of SHM, especially in males, should be considered in the development of universal influenza vaccines. IMPORTANCE Adult females develop greater antibody responses to influenza vaccines than males. We hypothesized that female-biased immunity and protection would be dependent on the extent of virus diversity as well as molecular mechanisms in B cells which constrain the breadth of epitope recognition. We developed a panel of mouse-adapted (ma) A/Cal/09 viruses that had mutations in the immunodominant hemagglutinin. Following vaccination against maA/Cal/09, females were better able to neutralize maA/Cal/09 than males, but neutralization of mutant maA/Cal/09 viruses was equally poor in both sexes, despite vaccinated females being better protected against these viruses. Vaccinated females benefited from the greater production of class-switched, somatically hypermutated antibodies generated in germinal center B cells, which increased recognition of more diverse maA/Cal/09 hemagglutinin antigen epitopes. Female-biased protection against influenza infection and disease after vaccination is driven by differential mechanisms in males versus females and should be considered in the design of novel vaccine platforms.

Sex-specific effects of age and body mass index on antibody responses to seasonal influenza vaccines in healthcare workers.

Healthcare institutions with mandatory influenza vaccination policies have over 90% vaccination rates among healthcare workers (HCWs) resulting in a population that has received the influenza vaccine in many, consecutive years. This study explored the impact of sex and other host factors in pre- and post-vaccination neutralizing antibody (nAb) titers and seroconversion against the H1N1 and H3N2 influenza A viruses (IAVs) among HCWs enrolled into a cross-sectional serosurvey during the annual Johns Hopkins Hospital employee vaccination campaign in the 2017-18 and 2018-19 seasons. The study enrolled 111 participants (male = 38, female = 73) in 2017-18 and 163 (male = 44, female = 119) in 2018-19. Serum samples were collected immediately prior to vaccination and approximately 28 days later and nAb titers to vaccine strains determined. An intersectional approach was used to disaggregate the combined effects of sex with age and body mass index (BMI) in the nAb response. Differences between the pre- or post-vaccination geometric mean nAb titers between male and female HCWs were not observed. Male HCWs were 2.86 times more likely to seroconvert compared to female HCWs in 2017-2018, but the same trend was not observed in the following year. When data were disaggregated by age and sex, older female HCWs had higher H1N1 pre- and post-vaccination nAb titers compared to male HCWs in the same age group for both vaccination campaign seasons. In both years, the decline in H3N2 pre-vaccination titers with increasing BMI was greater in female than male HCW. The sex-specific effects of age and BMI on nAb responses to seasonal influenza vaccines require greater consideration.

Characterizing Emerging Canine H3 Influenza Viruses.

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned.

Erratum: Author Correction: Age-associated changes in the impact of sex steroids on influenza vaccine responses in males and females.

[This corrects the article DOI: 10.1038/s41541-019-0124-6.].

Age-associated changes in the impact of sex steroids on influenza vaccine responses in males and females.

Vaccine-induced immunity declines with age, which may differ between males and females. Using human sera collected before and 21 days after receipt of the monovalent A/Cal/09 H1N1 vaccine, we evaluated cytokine and antibody responses in adult (18-45 years) and aged (65+ years) individuals. After vaccination, adult females developed greater IL-6 and antibody responses than either adult males or aged females, with female antibody responses being positively associated with concentrations of estradiol. To test whether protection against influenza virus challenge was greater in females than males, we primed and boosted adult (8-10 weeks) and aged (68-70 weeks) male and female mice with an inactivated A/Cal/09 H1N1 vaccine or no vaccine and challenged with a drift variant A/Cal/09 virus. As compared with unvaccinated mice, vaccinated adult, but not aged, mice experienced less morbidity and better pulmonary viral clearance following challenge, regardless of sex. Vaccinated adult female mice developed antibody responses that were of greater quantity and quality and more protective than vaccinated adult males. Sex differences in vaccine efficacy diminished with age in mice. To determine the role of sex steroids in vaccine-induced immune responses, adult mice were gonadectomized and hormones (estradiol in females and testosterone in males) were replaced in subsets of animals before vaccination. Vaccine-induced antibody responses were increased in females by estradiol and decreased in males by testosterone. The benefit of elevated estradiol on antibody responses and protection against influenza in females is diminished with age in both mice and humans.

Biological sex affects vaccine efficacy and protection against influenza in mice.

Biological sex affects adaptive immune responses, which could impact influenza infection and vaccine efficacy. Infection of mice with 2009 H1N1 induced antibody responses, CD4+ T cell and CD8+ T cell memory responses that were greater in females than males; both sexes, however, were equally protected against secondary challenge with an H1N1 drift variant virus. To test whether greater antibody in females is sufficient for protection against influenza, males and females were immunized with an inactivated H1N1 vaccine that induced predominantly antibody-mediated immunity. Following vaccination, females had greater antibody responses and protection against challenge with an H1N1 drift variant virus than males. Antibody derived from vaccinated females was better at protecting both naïve males and females than antibody from males, and this protection was associated with increased antibody specificity and avidity to the H1N1 virus. The expression of Tlr7 was greater in B cells from vaccinated females than males and was associated with reduced DNA methylation in the Tlr7 promoter region, higher neutralizing antibody, class switch recombination, and antibody avidity in females. Deletion of Tlr7 reduced sex differences in vaccine-induced antibody responses and protection following challenge and had a greater impact on responses in females than males. Taken together, these data illustrate that greater TLR7 activation and antibody production in females improves the efficacy of vaccination against influenza.

The evolution of greater humoral immunity in females than males: implications for vaccine efficacy.

Males and females differ in their effector and memory immune responses to foreign and self-antigens. The difference in antibody responses (i.e., humoral immunity), in particular, is one of the most well conserved sex differences in immunology. Certain sex differences in humoral immunity are present throughout life, whereas others are only apparent after puberty and prior to reproductive senescence, suggesting that both genes and hormones are involved. Importantly, these sex-based differences in humoral immunity contribute to variation in the responses to vaccines and may explain some disparities in vaccine efficacy between the sexes. Elevated humoral immunity in females compared with males is phylogenetically well conserved, suggesting an adaptive advantage of elevated antibody for reproductive success, including for the transfer of protective antibodies to offspring.

Production of amphiregulin and recovery from influenza is greater in males than females.

BACKGROUND: Amphiregulin (AREG) is an epidermal growth factor that is a significant mediator of tissue repair at mucosal sites, including in the lungs during influenza A virus (IAV) infection. Previous research illustrates that males of reproductive ages experience less severe disease and recover faster than females following infection with IAV. METHODS: Whether males and females differentially produce and utilize AREG for pulmonary repair after IAV infection was investigated using murine models on a C57BL/6 background and primary mouse and human epithelial cell culture systems. RESULTS: Following sublethal infection with 2009 H1N1 IAV, adult female mice experienced greater morbidity and pulmonary inflammation during the acute phase of infection as well as worse pulmonary function during the recovery phase of infection than males, despite having similar virus clearance kinetics. As compared with females, AREG expression was greater in the lungs of male mice as well as in primary respiratory epithelial cells derived from mouse and human male donors, in response to H1N1 IAVs. Internalization of the epidermal growth factor receptor (EGFR) was also greater in respiratory epithelial cells derived from male than female mice. IAV infection of Areg knock-out (Areg-/-) mice eliminated sex differences in IAV pathogenesis, with a more significant role for AREG in infection of male compared to female mice. Deletion of Areg had no effect on virus replication kinetics in either sex. Gonadectomy and treatment of either wild-type or Areg-/- males with testosterone improved the outcome of IAV as compared with their placebo-treated conspecifics. CONCLUSIONS: Taken together, these data show that elevated levels of testosterone and AREG, either independently or in combination, improve resilience (i.e., repair and recovery of damaged tissue) and contribute to better influenza outcomes in males compared with females.

The Confluence of Sex Hormones and Aging on Immunity.

The immune systems of post-pubescent males and females differ significantly with profound consequences to health and disease. In many cases, sex-specific differences in the immune responses of young adults are also apparent in aged men and women. Moreover, as in young adults, aged women develop several late-adult onset autoimmune conditions more frequently than do men, while aged men continue to develop many cancers to a greater extent than aged women. However, sex differences in the immune systems of aged individuals have not been extensively investigated and data addressing the effectiveness of vaccinations and immunotherapies in aged men and women are scarce. In this review, we evaluate age- and sex hormone-related changes to innate and adaptive immunity, with consideration about how this impacts age- and sex-associated changes in the incidence and pathogenesis of autoimmunity and cancer as well as the efficacy of vaccination and cancer immunotherapy. We conclude that future preclinical and clinical studies should consider age and sex to better understand the ways in which these characteristics intersect with immune function and the resulting consequences for autoimmunity, cancer, and therapeutic interventions.

Sex Reporting in Preclinical Microbiological and Immunological Research.

Both sex (i.e., biological construct of male and female) and gender (i.e., social construct of masculine and feminine) impact the pathogenesis of diseases, including those caused by microbial infections. Following the 2015 NIH policy for consideration of sex as a biological variable in preclinical research, in 2018, authors of papers published in primary-research American Society for Microbiology (ASM) journals will be asked to report the sex of the research subjects and animals and of materials derived directly from them. To address the need for sex reporting in ASM journals, we systematically reviewed 2,928 primary-research articles published in six primary-research ASM journals (Antimicrobial Agents and Chemotherapy, Clinical and Vaccine Immunology, Infection and Immunity, Journal of Bacteriology, Journal of Virology, and mBio) in 2016. Approximately 37% of animal studies and 9% of primary cell culture papers published in 2016 would have been affected by the new sex-reporting policy. For animal studies (i.e., studies with any nonhuman vertebrate hosts), most published papers either did not report the sex of the animals or used only female animals, and a minority used only males or both sexes. For published studies using primary cells from diverse animal species (i.e., humans and nonhuman vertebrates), almost all studies failed to report the sex of donors from which the cells were isolated. We believe that reporting the sex of animals and even of the donors of derived cells could improve the rigor and reproducibility of research conducted in microbiology and immunology and published in ASM journals.

Sex and Gender Differences in the Outcomes of Vaccination over the Life Course.

Both sex (i.e., biological differences) and gender (i.e., social or cultural influences) impact vaccine acceptance, responses, and outcomes. Clinical data illustrate that among children, young adults, and aged individuals, males and females differ in vaccine-induced immune responses, adverse events, and protection. Although males are more likely to receive vaccines, following vaccination, females typically develop higher antibody responses and report more adverse effects of vaccination than do males. Human, nonhuman animal, and in vitro studies reveal numerous immunological, genetic, hormonal, and environmental factors that differ between males and females and contribute to sex- and gender-specific vaccine responses and outcomes. Herein, we address the impact of sex and gender variables that should be considered in preclinical and clinical studies of vaccines.

Dengue virus specific IgY provides protection following lethal dengue virus challenge and is neutralizing in the absence of inducing antibody dependent enhancement.

Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are severe disease manifestations that can occur following sequential infection with different dengue virus serotypes (DENV1-4). At present, there are no licensed therapies to treat DENV-induced disease. DHF and DSS are thought to be mediated by serotype cross-reactive antibodies that facilitate antibody-dependent enhancement (ADE) by binding to viral antigens and then Fcγ receptors (FcγR) on target myeloid cells. Using genetically engineered DENV-specific antibodies, it has been shown that the interaction between the Fc portion of serotype cross-reactive antibodies and FcγR is required to induce ADE. Additionally, it was demonstrated that these antibodies were as neutralizing as their non-modified variants, were incapable of inducing ADE, and were therapeutic following a lethal, antibody-enhanced infection. Therefore, we hypothesized that avian IgY, which do not interact with mammalian FcγR, would provide a novel therapy for DENV-induced disease. We demonstrate here that goose-derived anti-DENV2 IgY neutralized DENV2 and did not induce ADE in vitro. Anti-DENV2 IgY was also protective in vivo when administered 24 hours following a lethal DENV2 infection. We were also able to demonstrate via epitope mapping that both full-length and alternatively spliced anti-DENV2 IgY recognized different epitopes, including epitopes that have not been previously identified. These observations provide evidence for the potential therapeutic applications of goose-derived anti-DENV2 IgY.

Progesterone-Based Contraceptives Reduce Adaptive Immune Responses and Protection against Sequential Influenza A Virus Infections.

In addition to their intended use, progesterone (P4)-based contraceptives promote anti-inflammatory immune responses, yet their effects on the outcome of infectious diseases, including influenza A virus (IAV) infection, are rarely evaluated. To evaluate their impact on immune responses to sequential IAV infections, adult female mice were treated with placebo or one of two progestins, P4 or levonorgestrel (LNG), and infected with a mouse-adapted H1N1 (maH1N1) virus. Treatment with P4 or LNG reduced morbidity but had no effect on pulmonary virus titers during primary H1N1 infection compared to placebo treatment. In serum and bronchoalveolar lavage fluid, total anti-IAV IgG and IgA titers and virus-neutralizing antibody titers but not hemagglutinin stalk antibody titers were lower in progestin-treated mice than placebo-treated mice. Females were challenged 6 weeks later with either an maH1N1 drift variant (maH1N1dv) or maH3N2 IAV. The level of protection following infection with the maH1N1dv was similar among all groups. In contrast, following challenge with maH3N2, progestin treatment reduced survival as well as the numbers and activity of H1N1- and H3N2-specific memory CD8+ T cells, including tissue-resident cells, compared with placebo treatment. In contrast to primary IAV infection, progestin treatment increased the titers of neutralizing and IgG antibodies against both challenge viruses compared with those achieved with placebo treatment. While the immunomodulatory properties of progestins protected immunologically naive female mice from the severe outcomes from IAV infection, it made them more susceptible to secondary challenge with a heterologous IAV, despite improving their antibody responses against a secondary IAV infection. Taken together, the immunomodulatory effects of progestins differentially regulate the outcome of infection depending on exposure history.IMPORTANCE The impact of hormone-based contraceptives on the outcome of infectious diseases outside the reproductive tract is rarely considered. Using a mouse model, we have made the novel observation that treatment with either progesterone or a synthetic analog found in hormonal contraceptives, levonorgestrel, impacts sequential influenza A virus infection by modulating antibody responses and decreasing the numbers and activity of memory CD8+ T cells. Progestins reduced the antibody responses during primary H1N1 virus infection but increased antibody titers following a sequential infection with either an H1N1 drift variant or an H3N2 virus. Following challenge with an H3N2 virus, female mice treated with progestins experienced greater mortality with increased pulmonary inflammation and reduced numbers and activity of CD8+ T cells. This study suggests that progestins significantly affect adaptive immune responses to influenza A virus infection, with their effect on the outcome of infection depending on exposure history.

Sex and Gender Impact Immune Responses to Vaccines Among the Elderly.

In response to the recommended vaccines in older-aged individuals, sex differences occur in response to those that protect against influenza, tetanus, pertussis, shingles, and pneumococcal infections. The efficacy of vaccines recommended for older-aged adults is consistently greater for females than for males. Gender differences as well as biological sex differences can influence vaccine uptake, responses, and outcome in older-aged individuals, which should influence guidelines, formulations, and dosage recommendations for vaccines in the elderly.