Andrew J. Olive

A mucosal vaccine against Chlamydia trachomatis generates two waves of protective memory T cells

The right combination for protection Despite its prevalence, no vaccine exists to protect against infection with the sexually transmitted bacterium Chlamydia trachomatis. Stary et al. now report on one potential vaccine candidate (see the Perspective by Brunham). Vaccinating with an ultraviolet light-inactivated C. trachomatis linked to adjuvant-containing charged nanoparticles protected female conventional and humanized mice against C. trachomatis infection. The vaccine conferred protection only when delivered through mucosal routes. Protection relied on targeting the bacteria to a particular population of immunogenic dendritic cells and inducing memory T cells that resided in the female genital tract. Science, this issue 10.1126/science.aaa8205; see also p. 1322 A nanoparticle-based vaccine protects mice against infection with Chlamydia trachomatis. [Also see Perspective by Brunham] INTRODUCTION Administering vaccines through nonmucosal routes often leads to poor protection against mucosal pathogens, presumably because such vaccines do not generate memory lymphocytes that migrate to mucosal surfaces. Although mucosal vaccination induces mucosa-tropic memory lymphocytes, few mucosal vaccines are used clinically; live vaccine vectors pose safety risks, whereas killed pathogens or molecular antigens are usually weak immunogens when applied to intact mucosa. Adjuvants can boost immunogenicity; however, most conventional mucosal adjuvants have unfavorable safety profiles. Moreover, the immune mechanisms of protection against many mucosal infections are poorly understood. RATIONALE One case in point is Chlamydia trachomatis (Ct), a sexually transmitted intracellular bacterium that infects >100 million people annually. Mucosal Ct infections can cause female infertility and ectopic pregnancies. Ct is also the leading cause of preventable blindness in developing countries and induces pneumonia in infants. No approved vaccines exist to date. Here, we describe a Ct vaccine composed of ultraviolet light–inactivated Ct (UV-Ct) conjugated to charge-switching synthetic adjuvant nanoparticles (cSAPs). After immunizing mice with live Ct, UV-Ct, or UV-Ct–cSAP conjugates, we characterized mucosal immune responses to uterine Ct rechallenge and dissected the underlying cellular mechanisms. RESULTS In previously uninfected mice, Ct infection induced protective immunity that depended on CD4 T cells producing the cytokine interferon-γ, whereas uterine exposure to UV-Ct generated tolerogenic Ct-specific regulatory T cells, resulting in exacerbated bacterial burden upon Ct rechallenge. In contrast, mucosal immunization with UV-Ct–cSAP elicited long-lived protection. This differential effect of UV-Ct–cSAP versus UV-Ct was because the former was presented by immunogenic CD11b+CD103– dendritic cells (DCs), whereas the latter was presented by tolerogenic CD11b–CD103+ DCs. Intrauterine or intranasal vaccination, but not subcutaneous vaccination, induced genital protection in both conventional and humanized mice. Regardless of vaccination route, UV-Ct–cSAP always evoked a robust systemic memory T cell response. However, only mucosal vaccination induced a wave of effector T cells that seeded the uterine mucosa during the first week after vaccination and established resident memory T cells (TRM cells). Without TRM cells, mice were suboptimally protected, even when circulating memory cells were abundant. Optimal Ct clearance required both early uterine seeding by TRM cells and infection-induced recruitment of a second wave of circulating memory cells. CONCLUSIONS Mucosal exposure to both live Ct and inactivated UV-Ct induces antigen-specific CD4 T cell responses. While immunogenic DCs present the former to promote immunity, the latter is instead targeted to tolerogenic DCs that exacerbate host susceptibility to Ct infection. By combining UV-Ct with cSAP nanocarriers, we have redirected noninfectious UV-Ct to immunogenic DCs and achieved long-lived protection. This protective vaccine effect depended on the synergistic action of two memory T cell subsets with distinct differentiation kinetics and migratory properties. The cSAP technology offers a platform for efficient mucosal immunization that may also be applicable to other mucosal pathogens. Protection against C. trachomatis infection after mucosal UV-Ct–cSAP vaccination. Upon mucosal vaccination, dendritic cells carry UV-Ct–cSAP to lymph nodes and stimulate CD4 T cells. Effector T cells are imprinted to traffic to uterine mucosa (first wave) and establish tissue-resident memory cells (TRM cells). Vaccination also generates circulating memory T cells. Upon genital Ct infection, local reactivation of uterine TRM cells triggers the recruitment of the circulating memory subset (second wave). Optimal pathogen clearance requires both waves of memory cells. Genital Chlamydia trachomatis (Ct) infection induces protective immunity that depends on interferon-γ–producing CD4 T cells. By contrast, we report that mucosal exposure to ultraviolet light (UV)–inactivated Ct (UV-Ct) generated regulatory T cells that exacerbated subsequent Ct infection. We show that mucosal immunization with UV-Ct complexed with charge-switching synthetic adjuvant particles (cSAPs) elicited long-lived protection in conventional and humanized mice. UV-Ct–cSAP targeted immunogenic uterine CD11b+CD103– dendritic cells (DCs), whereas UV-Ct accumulated in tolerogenic CD11b–CD103+ DCs. Regardless of vaccination route, UV-Ct–cSAP induced systemic memory T cells, but only mucosal vaccination induced effector T cells that rapidly seeded uterine mucosa with resident memory T cells (TRM cells). Optimal Ct clearance required both TRM seeding and subsequent infection-induced recruitment of circulating memory T cells. Thus, UV-Ct–cSAP vaccination generated two synergistic memory T cell subsets with distinct migratory properties.

Global Mapping of the Inc-Human Interactome Reveals that Retromer Restricts Chlamydia Infection

Chlamydia trachomatis is a leading cause of genital and ocular infections for which no vaccine exists. Upon entry into host cells, C. trachomatis resides within a membrane-bound compartment—the inclusion—and secretes inclusion membrane proteins (Incs) that are thought to modulate the host-bacterium interface. To expand our understanding of Inc function(s), we subjected putative C. trachomatis Incs to affinity purification-mass spectroscopy (AP-MS). We identified Inc-human interactions for 38/58 Incs with enrichment in host processes consistent with Chlamydias intracellular life cycle. There is significant overlap between Inc targets and viral proteins, suggesting common pathogenic mechanisms among obligate intracellular microbes. IncE binds to sorting nexins (SNXs) 5/6, components of the retromer, which relocalizes SNX5/6 to the inclusion membrane and augments inclusion membrane tubulation. Depletion of retromer components enhances progeny production, revealing that retromer restricts Chlamydia infection. This study demonstrates the value of proteomics in unveiling host-pathogen interactions in genetically challenging microbes.

A CREB3–ARF4 signalling pathway mediates the response to Golgi stress and susceptibility to pathogens

Treatment of cells with brefeldin A (BFA) blocks secretory vesicle transport and causes a collapse of the Golgi apparatus. To gain more insight into the cellular mechanisms mediating BFA toxicity, we conducted a genome-wide haploid genetic screen that led to the identification of the small G protein ADP-ribosylation factor 4 (ARF4). ARF4 depletion preserves viability, Golgi integrity and cargo trafficking in the presence of BFA, and these effects depend on the guanine nucleotide exchange factor GBF1 and other ARF isoforms including ARF1 and ARF5. ARF4 knockdown cells show increased resistance to several human pathogens including Chlamydia trachomatis and Shigella flexneri. Furthermore, ARF4 expression is induced when cells are exposed to several Golgi-disturbing agents and requires the CREB3 (also known as Luman or LZIP) transcription factor, whose downregulation mimics ARF4 loss. Thus, we have uncovered a CREB3–ARF4 signalling cascade that may be part of a Golgi stress response set in motion by stimuli compromising Golgi capacity.

CD4+ T Cells Are Necessary and Sufficient To Confer Protection against Chlamydia trachomatis Infection in the Murine Upper Genital Tract

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease in the United States. Chlamydia infections that ascend to the upper genital tract can persist, trigger inflammation, and result in serious sequelae such as infertility. However, mouse models in which the vaginal vault is inoculated with C. trachomatis do not recapitulate the course of human disease. These intravaginal infections of the mouse do not ascend efficiently to the upper genital tract, do not cause persistent infection, do not induce significant inflammation, and do not induce significant CD4+ T cell infiltration. In this article, we describe a noninvasive transcervical infection model in which we bypass the cervix and directly inoculate C. trachomatis into the uterus. We show that direct C. trachomatis infection of the murine upper genital tract stimulates a robust Chlamydia-specific CD4+ T cell response that is both necessary and sufficient to clear infection and provide protection against reinfection.

Attachment of Chlamydia trachomatis L2 to host cells requires sulfation

Chlamydia trachomatis is a pathogen responsible for a prevalent sexually transmitted disease. It is also the most common cause of infectious blindness in the developing world. We performed a loss-of-function genetic screen in human haploid cells to identify host factors important in C. trachomatis L2 infection. We identified and confirmed B3GAT3, B4GALT7, and SLC35B2, which encode glucuronosyltransferase I, galactosyltransferase I, and the 3′-phosphoadenosine 5′-phosphosulfate transporter 1, respectively, as important in facilitating Chlamydia infection. Knockout of any of these three genes inhibits Chlamydia attachment. In complementation studies, we found that the introduction of functional copies of these three genes into the null clones restored full susceptibility to Chlamydia infection. The degree of attachment of Chlamydia strongly correlates with the level of sulfation of the host cell, not simply with the amount of heparan sulfate. Thus, other, as-yet unidentified sulfated macromolecules must contribute to infection. These results demonstrate the utility of screens in haploid cells to study interactions of human cells with bacteria. Furthermore, the human null clones generated can be used to investigate the role of heparan sulfate and sulfation in other settings not limited to infectious disease.

CXCR3 AND CCR5 ARE BOTH REQUIRED FOR T CELL MEDIATED PROTECTION AGAINST C. TRACHOMATIS INFECTION IN THE MURINE GENITAL MUCOSA

Chemokine receptors direct T lymphocytes to the site of an infection by following coordinated chemokine gradients, which allow their recruitment to specific tissues. Although identification of receptors needed for homing to some mucosal sites, such as skin and gut, have been elucidated, the receptors that direct lymphocytes to the genital mucosa remain relatively uncharacterized. In this study we identify that the chemokine receptors CXCR3 (chemokine (C-X-C motif) receptor 3) and CCR5 (chemokine (C-C motif) receptor 5) are pivotal for T-lymphocyte access to the genital tract during Chlamydia trachomatis infection. Chlamydia-specific CD4+ transgenic T cells that lack CXCR3 or CCR5 do not accumulate in the genital mucosa following infection. Loss of either CXCR3 or CCR5 impairs the protective capacity of Chlamydia-specific T cells, whereas T cells lacking both receptors are completely nonprotective. These results show that CXCR3 and CCR5 are the predominant chemokine receptors that act cooperatively to promote homing to the genital mucosa during Chlamydia infection.

Targeting HER2-positive cancer cells with receptor-redirected anthrax protective antigen

Targeted therapeutics have emerged in recent years as an attractive approach to treating various types of cancer. One approach is to modify a cytocidal protein toxin to direct its action to a specific population of cancer cells. We created a targeted toxin in which the receptor‐binding and pore‐forming moiety of anthrax toxin, termed Protective Antigen (PA), was modified to redirect its receptor specificity to HER2, a marker expressed at the surface of a significant fraction of breast and ovarian tumors. The resulting fusion protein (mPA‐ZHER2) delivered cytocidal effectors specifically into HER2‐positive tumor cells, including a trastuzumab‐resistant line, causing death of the cells. No off‐target killing of HER2‐negative cells was observed, either with homogeneous populations or with mixtures of HER2‐positive and HER2‐negative cells. A mixture of mPA variants targeting different receptors mediated killing of cells bearing either receptor, without affecting cells devoid of these receptors. Anthrax toxin may serve as an effective platform for developing therapeutics to ablate cells bearing HER2 or other tumor‐specific cell‐surface markers.

Compensatory T Cell Responses in IRG-Deficient Mice Prevent Sustained Chlamydia trachomatis Infections

The obligate intracellular pathogen Chlamydia trachomatis is the most common cause of bacterial sexually transmitted diseases in the United States. In women C. trachomatis can establish persistent genital infections that lead to pelvic inflammatory disease and sterility. In contrast to natural infections in humans, experimentally induced infections with C. trachomatis in mice are rapidly cleared. The cytokine interferon-γ (IFNγ) plays a critical role in the clearance of C. trachomatis infections in mice. Because IFNγ induces an antimicrobial defense system in mice but not in humans that is composed of a large family of Immunity Related GTPases (IRGs), we questioned whether mice deficient in IRG immunity would develop persistent infections with C. trachomatis as observed in human patients. We found that IRG-deficient Irgm1/m3 (-/-) mice transiently develop high bacterial burden post intrauterine infection, but subsequently clear the infection more efficiently than wildtype mice. We show that the delayed but highly effective clearance of intrauterine C. trachomatis infections in Irgm1/m3 (-/-) mice is dependent on an exacerbated CD4+ T cell response. These findings indicate that the absence of the predominant murine innate effector mechanism restricting C. trachomatis growth inside epithelial cells results in a compensatory adaptive immune response, which is at least in part driven by CD4+ T cells and prevents the establishment of a persistent infection in mice.

Integrin α4β1 Is Necessary for CD4+ T Cell–Mediated Protection against Genital Chlamydia trachomatis Infection

Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection in the United States and a significant health burden worldwide. Protection from Chlamydia infection in the genital mucosa is dependent on IFN-γ derived from CD4+ Th1 cells. These CD4+ T cells must home successfully to the genital tract to exert their effector function and decrease C. trachomatis burden. Although adhesion receptors expressed by CD4+ T cells in the genital tract have been characterized, the integrin receptor required for Chlamydia-specific CD4+ T cell–mediated protection has not been explored. In this study, we demonstrate that C. trachomatis infection of the upper genital tract results in recruitment of Chlamydia-specific CD4+ T cells robustly expressing the integrin α4β1. Interfering with α4β1, but not α4β7, function resulted in defective CD4+ T cell trafficking to the uterus and high bacterial load. We conclude that integrin α4β1 is necessary for CD4+ T cell–mediated protection against C. trachomatis infection in the genital mucosa. By identifying homing molecules required for successful CD4+ T cell trafficking to C. trachomatis–infected tissues, we will be better equipped to design vaccines that elicit sterilizing, long-lasting immunity without inducing immune pathologies in the upper genital tract.

Pathology after Chlamydia trachomatis infection is driven by nonprotective immune cells that are distinct from protective populations

Significance Immunopathology after Chlamydia trachomatis infection is the major cause of human suffering associated with this pathogen, yet the immune responses that drive pathology are not well defined. We demonstrate that a mucosal influx of neutrophils and CXCR3-driven CD4+ and CD8+ T cells is required for C. trachomatis pathology and does not contribute to bacterial clearance. Our study decouples immune-mediated pathology from immune-mediated protection for C. trachomatis, which will have important translational implications for the development of vaccines and CXCR3-mediated treatments. Infection with Chlamydia trachomatis drives severe mucosal immunopathology; however, the immune responses that are required for mediating pathology vs. protection are not well understood. Here, we employed a mouse model to identify immune responses required for C. trachomatis-induced upper genital tract pathology and to determine whether these responses are also required for bacterial clearance. In mice as in humans, immunopathology was characterized by extravasation of leukocytes into the upper genital tract that occluded luminal spaces in the uterus and ovaries. Flow cytometry identified these cells as neutrophils at early time points and CD4+ and CD8+ T cells at later time points. To determine what draws these cells to C. trachomatis-infected tissue, we measured the expression of 700 inflammation-related genes in the upper genital tract and found an up-regulation of many chemokines, including a node of interaction between CXCL9/10/11 and their common receptor CXCR3. Either depleting neutrophils or reducing T-cell numbers by CXCR3 blockade was sufficient to significantly ameliorate immunopathology but had no effect on bacterial burden, demonstrating that these responses are necessary for mucosal pathology but dispensable for C. trachomatis clearance. Therapies that specifically target these host responses may therefore prove useful in ameliorating C. trachomatis-induced pathology without exacerbating infection or transmission.