Biology subjects

Klein, S. L.

Publications and source records attributed to Klein, S. L..

14 recordsLinked to original sources

bioRxiv2026

Intrinsic T-cell programming and immune spatial organization govern sex-biased tuberculosis immunity

Biological sex can profoundly influence the susceptibility to infectious diseases, yet the mechanisms behind the sex-dependent protective immunity against tuberculosis (TB) remain poorly understood. Here we show that sexually divergent immunity during chronic Mycobacterium tuberculosis (Mtb) infection is governed by both intrinsic T cell programming and pulmonary immune spatial organization. Using the Four Core Genotype (FCG) mouse model, adoptive cell transfer, pathway-specific blockade and B cell depletion, we demonstrate that CD4 T cells from gonadal females (XXF), but not XX males (XXM), confer enhanced protection to susceptible XY male recipients, independently of sex chromosome complement. Female-derived CD4 T cells reduce Mtb burdens while promoting pulmonary Bcl6 CD4 T cell responses and limiting neutrophilic inflammation. Mechanistically, blockade of CXCR3 or CD40L abrogates female-associated protection, with CD40L signaling additionally required to maintain organized pulmonary B cell structures. Although depletion of conventional B-2 B cells did not impair bacterial control, it disrupted tertiary lymphoid organization and revealed striking sex-specific functions of pulmonary B cells. Loss of B cell follicles (BCFs) primarily remodeled adaptive T cell responses in females, whereas in males it drove inflammatory myeloid activation, exaggerated neutrophil recruitment and widespread neutrophil extracellular trap (NET) formation. Together, these findings identify two complementary layers of sex-dependent immune regulation during TB: intrinsic programming of protective female CD4 T cells, and B cell-dependent spatial organization that coordinates adaptive immunity in females while restraining pathological inflammation in males. These findings establish immune tissue organization as a key determinant of the sexually dimorphic host defense during chronic TB.

immunology↗

bioRxiv2026

Gonadal regulation of sex-specific immunity in tuberculosis: enhanced lymphocyte function in females and dysfunctional myeloid responses in males

Tuberculosis (TB), the worlds deadliest infection, shows higher prevalence and mortality in males than females (M/F ratio >1.7). Using Four Core Genotype (FCG) mice to decouple gonadal from chromosomal sex (XX, XY gonadal males and XX, XY gonadal females), we show that gonadal males develop accelerated disease driven by dysfunctional myeloid responses rather than impaired bacterial recognition. Both XX and XY males exhibited increased mortality, higher Mycobacterium tuberculosis (Mtb) burden, and severe lung pathology. Mechanistically, gonadal male susceptibility involved early myeloid priming, excessive neutrophil recruitment, CCR2 monocyte accumulation, and hyperinflammation, with enhanced neutrophil extracellular trap (NET) formation and disorganized granulomas, implicating testes and androgens as key drivers of male susceptibility. While XY gonadal females were less susceptible, XX gonadal females showed the greatest resistance, associated with coordinated T- and B-cell responses and enhanced B-cell follicle formation. Together, these findings identify gonad-driven myeloid dysregulation as a central mechanism underlying male TB susceptibility.

immunology↗

bioRxiv2026

Sex differences in vaccine-induced neuraminidase cross-recognition and protection against H5N1 in mice

H5N1 vaccines have been poorly immunogenic in humans, creating a challenge for vaccine development. Seasonal influenza vaccines offer some cross-protection against H5N1, but there has been no consideration of whether protection differs between the sexes. We investigated sex differences in antibody responses following receipt of either beta-propiolactone inactivated whole virus H1N1 or H5N1 (LAIV backbone) vaccines in C57BL/6 mice. Using systems serology assays, vaccination induced strong homologous and heterologous antibody responses, with females generating greater IgG titers than males against whole virus H1N1 and H5N1, which was primarily mediated by greater IgG responses to neuraminidase (NA) than hemagglutinin (HA) protein. Cross-reactive H5N1 IgG titers were greater among H1N1-vaccinated females, and primarily mediated by greater N1-specific IgG titers. IgG2b and IgG2c were the primary antibody isotypes generated in response to these vaccines, with females having greater IgG2b titers and enhanced binding to Fc{gamma}RIV for avian and human NA than males following either homologous or heterologous vaccination. Antibody-dependent complement deposition was measured as an FcR-mediated non-neutralizing response against HA and NA and was more robust among H1N1 and H5N1 vaccinated females than their male counterparts in response to homologous HA only. Vaccinated females tended to have greater neutralizing antibody titers than males against the homologous vaccine strain, with limited cross-neutralizing antibodies detected in either sexes. Neuraminidase inhibition titers were greater in vaccinated females than males against the heterologous virus following H1N1 vaccination and against both the vaccine and heterologous viruses following H5N1 vaccination. When H1N1 and H5N1 vaccinated mice were challenged with a lethal dose of A/Texas/37/2024 H5N1, all H5N1 vaccinated mice were protected, regardless of sex. Among H1N1 vaccinated mice, while both sexes were protected against disease, H1N1 vaccinated females restricted virus to the upper respiratory tract and had lower pulmonary virus titers than males at 3 days post challenge. These findings highlight that sex differences in vaccine-induced NA-specific antibody responses are associated with differential respiratory dissemination of H5N1 and that sex should be considered in studies of vaccine-induced cross-reactive influenza immunity.

immunology↗

bioRxiv2025

Mechanisms of sex differences in acute and long COVID sequelae in mice

Males are more likely to suffer severe outcomes during acute COVID-19 and a greater proportion of females develop post-acute sequalae of COVID-19 (PASC). To identify mechanisms of PASC, mice were infected with SARS-CoV-2 and viral, inflammatory, and neurocognitive outcomes were evaluated through 84 days post-infection. Sex differences were not observed in virus replication or persistence of viral RNA in pulmonary or extrapulmonary tissues. After infectious virus clearance, females exhibited persistent neurocognitive impairments, and greater frequencies of inflammatory myeloid cells, neuroinflammation, and dysregulated T cells, including effector memory and Tregs. Sex differences in persistent inflammation and cognitive impairments during PASC were mediated by the presence of two X chromosomes and infection-induced upregulation of the X-linked genes Xist and Tlr7, which was reversed by pharmacological inhibition of TLR7 activity during the PASC phase. Sustained X-linked TLR7-mediated inflammation underlies how the burden of PASC is greater for females and provides a therapeutic target.

immunology↗

bioRxiv2025

Airway Epithelial SARS-CoV-2 Infectious and Repair Responses: Relationships to Age, Sex, and Post-COVID Pulmonary Syndromes

The long-term pulmonary sequelae of SARS-CoV-2 respiratory infections reflect infection severity, innate and adaptive immunity, and respiratory epithelial repair. This study investigated the acute and reparative responses as a function of age and sex in primary human bronchial epithelial (HBE) cultures utilizing a 14-day SARS-CoV-2 infection protocol. SARS-CoV-2 infection peaked at 3 days post-infection (dpi) with an [~] 2 log titer suppression at 14 dpi. SARS-CoV-2 infection induced interferon, interferon-induced gene, and cell damage responses. No age- or sex-dependent effects on SARS-CoV-2 infection were detected. Airway epithelia repaired to an abnormal mucus metaplastic/inflammatory state that reflected potentially beneficial and adverse consequences at 14 dpi. Repair processes were infection severity-dependent, not sex-dependent, and were more robust in young donor cultures. Analyses of long-COVID subjects with persistent pulmonary fibrosis or persistent bronchitic airway diseases exhibited expression of HBE 14 dpi failed repair gene signatures, including ISG gene signatures. Human airway epithelial repair post-SARS-CoV-2 is prolonged and incomplete in vitro over 14 days, and persistently abnormal repair may contribute to phenotypes of people with long-COVID pulmonary syndrome.

cell biology↗

bioRxiv2025

Vertical Transmission of Bovine H5N1 during Pregnancy and Lactation in Mice

Avian influenza H5N1 has pandemic potential and historically has caused more severe disease in pregnant women than the general population. With increasing transmission of H5N1 detected among placental mammals, animal models are necessary for testing countermeasures, including during pregnancy. Pregnant outbred mice were infected with a contemporary strain of bovine H5N1 during the second or third trimester equivalent. Second trimester infection caused in utero transmission, with infectious virus detected in the uterus, placenta, and fetus. Birth following third trimester infection resulted in offspring with decreased size, neurodevelopmental delays, and adolescent behavioral impairments, with infectious virus detected in the neonatal milk ring and lungs, as well as mammary tissues. H5N1 viral protein colocalized with trophoblast cells in the placenta and epithelial cells in mammary tissue that spatially overlapped with lectins for 2,3-linked SA. With the pandemic potential of H5N1, our vertical transmission model in placental mammals is essential for understanding viral spread and evaluating treatments during pregnancy.

microbiology↗

bioRxiv2024

A Third COVID-19 Vaccine Dose in Kidney Transplant Recipients Induces Antibody Response to Vaccine and Omicron Variants but Shows Limited Ig Subclass Switching

Solid organ transplant recipients (SOTRs) suffer more frequent and more severe infections due to their compromised immune responses resulting from immunosuppressive treatments designed to prevent organ rejection. Pharmacological immunosuppression can adversely affect immune responses to vaccination. A cohort of kidney transplant recipients (KTRs) received their third dose of ancestral, monovalent COVID-19 vaccine in the context of a clinical trial and antibody responses to the vaccine strain, as well as to Omicron variants BA.1 and BA.5 were investigated and compared with healthy controls. Total IgG and live virus neutralizing antibody titers were reduced in KTRs compared to controls for all variants. KTRs displayed altered IgG subclass switching, with significantly lower IgG3 antibodies. Responses in KTRs were also very heterogeneous, with some individuals showing strong responses but a significant number showing no Omicron-specific neutralizing antibodies. Taken together, immune responses after COVID-19 vaccination in KTRs were not only lower than healthy controls but highly variable, indicating that simply increasing the number of vaccine doses alone may not be sufficient to provide greater protection in this population. ImportanceThis study addresses the challenges faced by kidney transplant recipients (KTRs) in mounting effective immune responses against COVID-19. By evaluating the antibody responses to a third dose of monovalent mRNA COVID-19 vaccine and its effectiveness against Omicron subvariants (BA.1 and BA.5), this study reveals significant reductions in both binding and neutralizing antibodies in KTRs compared to healthy controls. The research highlights altered IgG subclass switching and heterogeneous responses within the KTR population. Reduced recognition of variants, coupled with differences in IgG subclasses, decreases both the quality and quantity of protective antibodies after vaccination in KTRs. These findings underscore the need for tailored vaccination strategies for immunosuppressed populations such as KTRs. Alternative formulations and doses of COVID-19 vaccines should be considered for people with severely compromised immune systems, as more frequent vaccinations may not significantly improve the response, especially regarding neutralizing antibodies.

immunology↗

bioRxiv2024

ZFAND6 is a subunit of a TRAF2-cIAP E3 ubiquitin ligase complex essential for mitophagy

The A20 ubiquitin-editing enzyme is a critical negative regulator of NF-{kappa}B signaling and inflammation. While the mechanisms by which A20 restricts inflammation have been extensively studied, the physiological functions of other A20-like proteins are largely unknown. Here, we report a previously unknown function of the A20 family member ZFAND6 as a novel regulator of mitophagy. Deletion of ZFAND6 in bone marrow-derived macrophages (BMDMs) promotes the upregulation of reactive oxygen species (ROS) and the accumulation of damaged mitochondria due to impaired mitophagy. Consequently, mitochondrial DNA (mtDNA) is released into the cytoplasm, triggering the spontaneous expression of interferon-stimulated genes (ISGs) in a cGAS-STING dependent manner, which leads to enhanced viral resistance in vitro. However, mice lacking ZFAND6 exhibit increased morbidity and mortality upon challenge with a sublethal dose of influenza A virus (IAV) due to impaired myeloid cell activation and diminished type I IFN signaling. Mechanistically, ZFAND6 bridges a TRAF2-cIAP1 interaction, which is required for the initiation of ubiquitin-dependent mitophagy. Our results suggest that ZFAND6 is a subunit of a TRAF2-cIAP E3 ligase complex that promotes the clearance of damaged mitochondria by mitophagy to maintain mitochondrial homeostasis.

immunology↗

bioRxiv2023

Estradiol Mediates Greater Germinal Center Responses to Influenza Vaccination in Female than Male Mice

Adult females of reproductive ages develop greater antibody responses to inactivated influenza vaccine (IIV) than males. How sex, age, and sex steroid changes 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 (GC) 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 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 was 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.

immunology↗

bioRxiv2023

Adverse outcomes in SARS-CoV-2 infected pregnant mice are gestational age-dependent and resolve with antiviral treatment

SARS-CoV-2 infection during pregnancy is associated with severe COVID-19 and adverse fetal outcomes, but the underlying mechanisms remain poorly understood. Moreover, clinical studies assessing therapeutics against SARS-CoV-2 in pregnancy are limited. To address these gaps, we developed a mouse model of SARS-CoV-2 infection during pregnancy. Outbred CD1 mice were infected at embryonic day (E) 6, E10, or E16 with a mouse adapted SARS-CoV-2 (maSCV2) virus. Outcomes were gestational age-dependent, with greater morbidity, reduced anti-viral immunity, greater viral titers, and more adverse fetal outcomes occurring with infection at E16 (3rd trimester-equivalent) than with infection at either E6 (1st trimester-equivalent) or E10 (2nd trimester-equivalent). To assess the efficacy of ritonavir-boosted nirmatrelvir (recommended for pregnant individuals with COVID-19), we treated E16-infected dams with mouse equivalent doses of nirmatrelvir and ritonavir. Treatment reduced pulmonary viral titers, decreased maternal morbidity, and prevented adverse offspring outcomes. Our results highlight that severe COVID-19 during pregnancy and adverse fetal outcomes are associated with heightened virus replication in maternal lungs. Ritonavir-boosted nirmatrelvir mitigated adverse maternal and fetal outcomes of SARS-CoV-2 infection. These findings prompt the need for further consideration of pregnancy in preclinical and clinical studies of therapeutics against viral infections.

microbiology↗

bioRxiv2022

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 IgG and IgG2c antibodies against the vaccine and all mutant viruses, and antibodies from females recognized more unique, linear HA epitopes than antibodies from males. While females had greater neutralizing antibody (nAb) titers against the vaccine virus, both sexes showed lower neutralization capacity against mutant viruses. After virus challenge, vaccinated females had lower pulmonary virus titers and reduced morbidity than males against the 1M and 2M viruses, but not the Sub virus. Females generated greater numbers of germinal center (GC) B cells containing superior somatic hypermutation 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 somatic hypermutations, especially in males, should be considered in the development of universal influenza vaccines. SummaryCompared with males, inactivated influenza vaccination of female mice causes greater production of class-switched, somatically-hypermutated antibodies and a more robust germinal center B cell response, leading to more diverse H1N1 antigen recognition and better protection against mutant influenza A viruses.

immunology↗

bioRxiv2021

A bacterial extracellular vesicle-based intranasal vaccine against SARS-CoV-2

Several vaccines have been introduced to combat the coronavirus infectious disease-2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current SARS-CoV-2 vaccines include mRNA-containing lipid nanoparticles or adenoviral vectors that encode the SARS-CoV-2 Spike (S) protein of SARS-CoV-2, inactivated virus, or protein subunits. Despite growing success in worldwide vaccination efforts, additional capabilities may be needed in the future to address issues such as stability and storage requirements, need for vaccine boosters, desirability of different routes of administration, and emergence of SARS-CoV-2 variants such as the Delta variant. Here, we present a novel, well-characterized SARS-CoV-2 vaccine candidate based on extracellular vesicles (EVs) of Salmonella typhimurium that are decorated with the mammalian cell culture-derived Spike receptor-binding domain (RBD). RBD-conjugated outer membrane vesicles (RBD-OMVs) were used to immunize the golden Syrian hamster (Mesocricetus auratus) model of COVID-19. Intranasal immunization resulted in high titers of blood anti-RBD IgG as well as detectable mucosal responses. Neutralizing antibody activity against wild-type and Delta variants was evident in all vaccinated subjects. Upon challenge with live virus, hamsters immunized with RBD-OMV, but not animals immunized with unconjugated OMVs or a vehicle control, avoided body mass loss, had lower virus titers in bronchoalveolar lavage fluid, and experienced less severe lung pathology. Our results emphasize the value and versatility of OMV-based vaccine approaches.

immunology↗

bioRxiv2021

Progression and Resolution of SARS-1 CoV-2 Infection in Golden Syrian Hamsters

To catalyze SARS-CoV-2 research including development of novel interventive and preventive strategies, we characterized progression of disease in depth in a robust COVID-19 animal model. In this model, male and female golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 USA-WA1/2020. Groups of inoculated and mock-inoculated uninfected control animals were euthanized at day 2, 4, 7, 14, and 28 days post-inoculation to track multiple clinical, pathology, virology, and immunology outcomes. SARS-CoV-2-inoculated animals consistently lost body weight during the first week of infection, had higher lung weights at terminal timepoints, and developed lung consolidation per histopathology and quantitative image analysis measurements. High levels of infectious virus and viral RNA were reliably present in the respiratory tract at days 2 and 4 post-inoculation, corresponding with widespread necrosis and inflammation. At day 7, when infectious virus was rare, interstitial and alveolar macrophage infiltrates and marked reparative epithelial responses (type II hyperplasia) dominated in the lung. These lesions resolved over time, with only residual epithelial repair evident by day 28 post-inoculation. The use of quantitative approaches to measure cellular and morphologic alterations in the lung provides valuable outcome measures for developing therapeutic and preventive interventions for COVID-19 using the hamster COVID-19 model.

pathology↗

bioRxiv2021

Sex differences in lung imaging and SARS-CoV-2 antibody responses in a COVID-19 golden Syrian hamster model

In the ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), more severe outcomes are reported in males compared with females, including hospitalizations and deaths. Animal models can provide an opportunity to mechanistically interrogate causes of sex differences in the pathogenesis of SARS-CoV-2. Adult male and female golden Syrian hamsters (8-10 weeks of age) were inoculated intranasally with 105 TCID50 of SARS-CoV-2/USA-WA1/2020 and euthanized at several time points during the acute (i.e., virus actively replicating) and recovery (i.e., after the infectious virus has been cleared) phases of infection. There was no mortality, but infected male hamsters experienced greater morbidity, losing a greater percentage of body mass, developing more extensive pneumonia as noted on chest computed tomography, and recovering more slowly than females. Treatment of male hamsters with estradiol did not alter pulmonary damage. Virus titers in respiratory tissues, including nasal turbinates, trachea, and lungs, and pulmonary cytokine concentrations, including IFN{beta} and TNF, were comparable between the sexes. However, during the recovery phase of infection, females mounted two-fold greater IgM, IgG, and IgA responses against the receptor-binding domain of the spike protein (S-RBD) in both plasma and respiratory tissues. Female hamsters also had significantly greater IgG antibodies against whole inactivated SARS-CoV-2 and mutant S-RBDs, as well as virus neutralizing antibodies in plasma. The development of an animal model to study COVID-19 sex differences will allow for a greater mechanistic understanding of the SARS-CoV-2 associated sex differences seen in the human population. ImportanceMen experience more severe outcomes from COVID-19 than women. Golden Syrian hamsters were used to explore sex differences in the pathogenesis of a human clinical isolate of SARS-CoV-2. After inoculation, male hamsters experienced greater sickness, developed more severe lung pathology, and recovered more slowly than females. Sex differences in disease could not be reversed by estradiol treatment in males and were not explained by either virus replication kinetics or the concentrations of inflammatory cytokines in the lungs. During the recovery period, antiviral antibody responses in the respiratory tract and plasma, including to newly emerging SARS-CoV-2 variants, were greater in females than male hamsters. Greater lung pathology during the acute phase combined with reduced antiviral antibody responses during the recovery phase of infection in males than females illustrate the utility of golden Syrian hamsters as a model to explore sex differences in the pathogenesis of SARS-CoV-2 and vaccine-induced immunity and protection. One Sentence SummaryFollowing SARS-CoV-2 infection, male hamsters experience worse clinical disease and have lower antiviral antibody responses than females.

microbiology↗