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High-fidelity Large Eddy Simulations (LES) are performed to study the effect of hydrogen injection on a lean turbulent CH 4 /Air premixed flame. An Analytically Reduced Chemistry (ARC) mechanism is used to achieve a detailed description of CH 4 /Air-H 2 chemistry. First, a validation of this kinetic scheme against the detailed GRI-Mech 3.0 mechanism is presented considering both simplified and complex transport properties. When hydrogen is added to the mixture, large variations of the mixture Prandtl and of the N 2 Schmidt numbers are observed depending on the local species concentrations, features that are missed by simplified models. LES is then applied to study the structure and stabilization mechanisms of a lean (ϕ = 0.8) premixed CH 4 /Air swirled flame enriched with hydrogen by using different transport modeling strategies. First, the fully premixed CH 4 /Air case is considered and results are found to validate the LES approach. In agreement with experiments, a classical V-shape flame is stabilized in the low-velocity region near the flame holder created by a central recirculation zone (CRZ). Then, hydrogen enrichment is achieved injecting 2% of the CH 4 thermal power with a central fuel injection lance. Both premixed and diffusion flame branches are present in this case, impacting flame stabilization and flame angle. The flame root of the main premixed flame is stabilized by a diffusion flame kernel created by the injected hydrogen reacting with the oxygen in excess of the premixed stream. Moreover, the H 2 consumed with the remaining oxygen in burnt gases leads to the formation of a second flame branch inside the CRZ which is responsible of an increase of the flame angle. Given the high concentration of hydrogen, an impact of the molecular transport models is observed on the flame lift-off height highlighting the importance of using complex transport properties in any LES involving hydrogen combustion. [ABSTRACT FROM AUTHOR]

Skin and soft tissue infections (SSTIs) are the most common diseases caused by Staphylococcus aureus ( S. aureus ), which can progress to threatening conditions due to recurrences and systemic complications. Staphylococcal protein A (SpA) is an immunomodulator antigen of S. aureus , which allows bacterial evasion from the immune system by interfering with different types of immune responses to pathogen antigens. Immunization with SpA could potentially unmask the pathogen to the immune system, leading to the production of antibodies that can protect from a second encounter with S. aureus , as it occurs in skin infection recurrences. Here, we describe a study in which mice are immunized with a mutated form of SpA mixed with the Adjuvant System 01 (SpA mut /AS01) before a primary S. aureus skin infection. Although mice are not protected from the infection under these conditions, they are able to mount a broader pathogen-specific functional immune response that results in protection against systemic dissemination of bacteria following an S. aureus second infection (recurrence). We show that this "hidden effect" of SpA can be partially explained by higher functionality of induced anti-SpA antibodies, which promotes better phagocytic activity. Moreover, a broader and stronger humoral response is elicited against several S. aureus antigens that during an infection are masked by SpA activity, which could prevent S. aureus spreading from the skin through the blood., Competing Interests: AM was a PhD student of Università degli Studi di Siena Italy at the time of the study and supervised by GSK. GM was a student of Università di Bologna Italy at the time of the study and participated in an internship at GSK. AM is now an employee of GiGroup SpA, a contract employment organization contracted by GSK. GM is now an employee of GSK. DL and GB were employee of GSK at the time of the study. All the other authors are employee of GSK. FB, MB, GB, EC report ownership of GSK shares. FB is listed as inventor on patents concerning S. aureus vaccine development. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Mandelli, Magri, Tortoli, Torricelli, Laera, Bagnoli, Finco, Bensi, Brazzoli and Chiarot.)

Introduction: Tinnitus is the perception of a sound in the absence of any corresponding external sound source. Current research suggests a relationship among emotional, cognitive, and psychosomatic symptoms and the occurrence or maintenance of chronic tinnitus. This study aimed to detect the prevalence and role of psychosomatic conditions, as defined by the Diagnostic Criteria for Psychosomatic Research (DCPR), and cognitive functioning in a group of patients with tinnitus., Methods: Sixty-two patients with subjective tinnitus and 62 non-tinnitus controls were recruited from the Otorhinolaryngology Unit of the University of Bari. Pure-tone audiometry was performed in all tinnitus subjects, and sound level tolerance was evaluated. Additionally, tinnitus handicap (Tinnitus Handicap Inventory [THI]), psychopathological symptoms (Symptom Checklist-90, Revised [SCL-90-R]), anxiety (State-Trait Anxiety Inventory [STAI-Y1/2]), depression (Beck Depression Inventory [BDI]), cognitive impairment (Mini-Mental State Examination [MMSE]), executive functions (Frontal Assessment Battery [FAB]), and psychosomatic syndromes (DCPR) were evaluated. Parametric and non-parametric tests were used to detect cognitive and symptomatological differences between patients and controls. The predictivity of these factors for tinnitus severity was studied using multiple regression (Backward Elimination). All tests were considered significant at p  < 0.05 (family wise error corrected for each comparison)., Results: 69.4% tinnitus patients met multiple DCPR criteria, compared to 32.3% of controls. Tinnitus patients exhibited elevated rates of illness denial (ꭓ 2  = 9.02; p  < 0.009), demoralization (ꭓ 2  = 8.05; p  < 0.018), somatization (ꭓ 2  = 4.92; p  < 0.063) and functional symptoms (ꭓ 2  = 5.21; p  < 0.06) scoring significantly higher on the BDI, STAI-Y1, and STAI-Y2, and SCL-90-R compared to controls. Patients with tinnitus showed lower MMSE scores, compared to controls ( t  = -2.282; p  < 0.001). No association between tinnitus severity and global cognitive impairment emerged. Conversely, executive function deficits were associated to tinnitus severity. Among the cognitive and psychological factors, only trait anxiety, one or more psychosomatic syndromes, and somatization clusters were strongly correlated with tinnitus severity., Discussion: Our findings suggest a relationship between tinnitus severity, psychological, psychosomatic symptoms, and frontal impairment. Additionally, the influence of tinnitus on cognitive functions paves the way for integrated, multidisciplinary diagnostic and treatment options for patients. Although preliminary, our findings highlight the importance of early cognitive and psychological screening to improve patients' quality of life., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gasparre, Pepe, Laera, Abbatantuono, De Caro, Taurino, D’Erasmo, Fanizzi, Antonucci, Pantaleo, Cavallaro, Pontillo, Taurisano and Quaranta.)

Aluminum-based adjuvants will continue to be a key component of currently approved and next generation vaccines, including important combination vaccines. The widespread use of aluminum adjuvants is due to their excellent safety profile, which has been established through the use of hundreds of millions of doses in humans over many years. In addition, they are inexpensive, readily available, and are well known and generally accepted by regulatory agencies. Moreover, they offer a very flexible platform, to which many vaccine components can be adsorbed, enabling the preparation of liquid formulations, which typically have a long shelf life under refrigerated conditions. Nevertheless, despite their extensive use, they are perceived as relatively 'weak' vaccine adjuvants. Hence, there have been many attempts to improve their performance, which typically involves co-delivery of immune potentiators, including Toll-like receptor (TLR) agonists. This approach has allowed for the development of improved aluminum adjuvants for inclusion in licensed vaccines against HPV, HBV, and COVID-19, with others likely to follow. This review summarizes the various aluminum salts that are used in vaccines and highlights how they are prepared. We focus on the analytical challenges that remain to allowing the creation of well-characterized formulations, particularly those involving multiple antigens. In addition, we highlight how aluminum is being used to create the next generation of improved adjuvants through the adsorption and delivery of various TLR agonists.

Although aluminium-based vaccines have been used for almost over a century, their mechanism of action remains unclear. It is established that antigen adsorption to the adjuvant facilitates delivery of the antigen to immune cells at the injection site. To further increase our understanding of aluminium-based vaccines, it is important to gain additional insights on the interactions between the aluminium and antigens, including antigen distribution over the adjuvant particles. Immuno-assays can further help in this regard. In this paper, we evaluated how established formulation strategies (i.e., sequential, competitive, and separate antigen addition) applied to four different antigens and aluminium oxyhydroxide, lead to formulation changes over time. Results showed that all formulation samples were stable, and that no significant changes were observed in terms of physical-chemical properties. Antigen distribution across the bulk aluminium population, however, did show a maturation effect, with some initial dependence on the formulation approach and the antigen adsorption strength. Sequential and competitive approaches displayed similar results in terms of the homogeneity of antigen distribution across aluminium particles, while separately adsorbed antigens were initially more highly poly-dispersed. Nevertheless, the formulation sample prepared via separate adsorption also reached homogeneity according to each antigen adsorption strength. This study indicated that antigen distribution across aluminium particles is a dynamic feature that evolves over time, which is initially influenced by the formulation approach and the specific adsorption strength, but ultimately leads to homogeneous formulations.

Purified vaccine antigens offer important safety and reactogenicity advantages compared with live attenuated or whole killed virus and bacterial vaccines. However, they require the addition of adjuvants to induce the magnitude, duration and quality of immune response required to achieve protective immunity. Aluminium salts have been used as adjuvants in vaccines for almost a century. In the literature, they are often referred to as aluminium-based adjuvants (ABAs), or aluminium salt-containing adjuvants or more simply "alum". All these terms are used to group aluminium suspensions that are very different in terms of atomic composition, size, and shape. They differ also in stability, antigen-adsorption, and antigen-release kinetics. Critically, these parameters also have a profound effect on the character and magnitude of the immune response elicited. Recent findings suggest that, by reducing the size of aluminium from micro to nanometers, a more effective adjuvant is obtained, together with the ability to sterile filter the vaccine product. However, the behaviour of aluminium nanoparticles in vaccine formulations is different from microparticles, requiring specific formulation strategies, as well as a more detailed understanding of how formulation influences the immune response generated. Here we review the current state of art of aluminium nanoparticles as adjuvants, with a focus on their immunobiology, preparation methods, formulation optimisation and stabilisation., (Copyright © 2021. Published by Elsevier Ltd.)

Background: Substance Use Disorder (SUD) causes a great deal of personal suffering for patients. Recent evidence highlights how defenses and emotion regulation may play a crucial part in the onset and development of this disorder. The aim of this study was to investigate potential differences in the defensive functioning between SUD patients and non-clinical controls. Secondly, we aimed at investigating the relationships between alexithymia and maladaptive/assimilation defenses., Methods: The authors assessed defensive functioning (Response Evaluation Measure-71, REM-71), personality (MMPI-II), and alexithymia (TAS-20) of 171 SUD patients (17% female; mean age = 36.5), compared to 155 controls. Authors performed a series of ANOVAs to investigate the defensive array in SUD patients compared to that of non-clinical controls. Student t test for indipendent samples was used to compare clinical characteristics between the SUD group and the controls. To investigate the role of single defenses in explaining alexithimia's subscores, stepwise multiple regression analysis were carried out on socio-demographic characteristics of participants (gender, age, and years of education), with REM-71 defenses as predictors., Results: SUD patients presented a more maladaptive/assimilation (Factor 1) defensive array (p < .001). Among SUD sub-groups, Alcohol Use Disorder patients showed more disfuncional defenses. Factor 1 defenses were related to a worse psychological functioning. In addition, alexyhimia (particularly DIF) was strongly related to Factor 1 defenses, expecially Projection (38% of variance explained, β = .270, p < .001)., Conclusion: The REM-71 and the TAS-20 might be useful screening instruments among SUD patients.

Introduction: Chronic pain (CP) is a complex multidimensional experience severely affecting individuals' quality of life. Multiple cognitive, affective, emotional, and interpersonal factors play a major role in CP. Furthermore, the psychological, social, and physical circumstances leading to CP show high inter-individual variability, thus making it difficult to identify core syndrome characteristics. In a biopsychosocial perspective, we aim at identifying a pattern of psycho-physical impairments that can reliably discriminate between CP individuals and healthy controls (HC) with high accuracy and estimated generalizability using machine learning., Methods: A total of 118 CP and 86 HC were recruited. All individuals were administered several scales assessing quality of life, physical and mental health, personal functioning, anxiety, depression, beliefs about medical treatments, and cognitive ability. These features were trained to separate CP from HC using support vector classification and repeated nested cross-validation., Results: Our psycho-physical classifier was able to discriminate CP from HC with 86.5% balanced accuracy and significance (p = 0.0001). The most reliable features characterizing CP were anxiety and depression scores, and belief of harm from prolonged pharmacological treatments; for HP, the most reliable features were physical and occupational functioning, and vitality levels., Conclusion: Our findings suggest that, using psychological and physical assessments, it is possible to classify CP from HC with high reliability and estimated generalizability via (i) a pattern of psychological symptoms and cognitive beliefs characteristic of CP, and (ii) a pattern of intact physical functioning characteristic of HC. We think that our algorithm enables novel insights into potential individualized targets for CP-related early intervention programs.

Adjuvants are necessary to enable vaccine development against a significant number of challenging pathogens for which effective vaccines are not available. We engineered a novel small-molecule immune potentiator, a benzonaphthyridine agonist targeting toll-like receptor 7 (TLR7), as a vaccine adjuvant. TLR7 agonist (TLR7a) was engineered to be adsorbed onto aluminum hydroxide (AlOH), and the resulting AlOH/TLR7a was evaluated as a vaccine adjuvant. AlOH/TLR7a exploits the flexibility of AlOH formulations, has an application in many vaccine candidates, and induced good efficacy and safety profiles against all tested antigens (bacterial- and viral-derived protein antigens, toxoids, glycoconjugates, and so forth) in many animal models, including nonhuman primates. In this article, we describe the outcome of the physicochemical characterization of AlOH/TLR7a. Reverse-phase ultra performance liquid chromatography, confocal microscopy, flow cytometry, zeta potential, and phosphophilicity assays were used as tools to demonstrate the association of TLR7a to AlOH and to characterize this novel formulation. Raman spectroscopy, nuclear magnetic resonance, and mass spectroscopy were also used to investigate the interaction between TLR7a and AlOH (data not shown). This pivotal work paved the way for AlOH/TLR7a to progress into the clinic for evaluation as an adjuvant platform for vaccines against challenging preventable diseases., (Copyright © 2018 American Pharmacists Association®. All rights reserved.)

Staphylococcus aureus is the major cause of human septic arthritis and osteomyelitis, which deserve special attention due to their rapid evolution and resistance to treatment. The progression of the disease depends on both bacterial presence in situ and uncontrolled disruptive immune response, which is responsible for chronic disease. Articular and bone infections are often the result of blood bacteremia, with the knees and hips being the most frequently infected joints showing the worst clinical outcome. We report the development of a hematogenous model of septic arthritis in murine knees, which progresses from an acute to a chronic phase, similarly to what occurs in humans. Characterization of the local and systemic inflammatory and immune responses following bacterial infection brought to light specific signatures of disease. Immunization of mice with the vaccine formulation we have recently described (4C-Staph), induced a strong antibody response and specific CD4+ effector memory T cells, and resulted in reduced bacterial load in the knee joints, a milder general inflammatory state and protection against bacterial-mediated cellular toxicity. Possible correlates of protection are finally proposed, which might contribute to the development of an effective vaccine for human use., Competing Interests: This work was sponsored by Novartis Vaccines and Diagnostics Srl, now acquired by the GSK group of companies. All authors have declared the following interests: all authors were permanent employees of Novartis Vaccines at the time of the study. Following the acquisition of Novartis Vaccines by the GSK group of companies in March, 2015, all but A.C. and T.S. are now permanent employees of the GSK group of companies. E.D.G., F.B., A.S. and S.B. report ownership of GSK shares and/or restricted GSK shares. A.C., F.B., G.B. and E.C. are listed as inventors on patents owned by the GSK group of companies.

A rapidly acting, single dose vaccine against Staphylococcus aureus would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent S. aureus candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4+ T cells not only reduced survival but also increased S. aureus load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by in vivo neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against S. aureus through the combined actions of antibodies, CD4+ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units.

Induction of persistent protective immune responses is a key attribute of a successful vaccine formulation. MF59 adjuvant, an oil-in-water emulsion used in human vaccines, is known to induce persistent high-affinity functional Ab titers and memory B cells, but how it really shapes the Ag-specific B cell compartment is poorly documented. In this study, we characterized the Ab- and Ag-specific B cell compartment in wild-type mice immunized with HlaH35L, a Staphylococcus aureus Ag known to induce measurable functional Ab responses, formulated with MF59 or aluminum salts, focusing on germinal centers (GC) in secondary lymphoid organs. Taking advantage of single-cell flow cytometry analyses, HlaH35L-specific B cells were characterized for the expression of CD38 and GL-7, markers of memory and GC, respectively, and for CD80 and CD73 activation markers. We demonstrated that immunization with MF59-, but not aluminum salt-adjuvanted HlaH35L, induced expanded Ag-specific CD73(+)CD80(-) GC B cells in proximal- and distal-draining lymph nodes, and promoted the persistence of GC B cells, detected up to 4 mo after immunization. In addition to increasing GC B cells, MF59-adjuvanted HlaH35L also increased the frequency of T follicular helper cells. This work extends previous knowledge regarding adaptive immune responses to MF59-adjuvanted vaccines, and, to our knowledge, for the first time an adjuvant used in human licensed products is shown to promote strong and persistent Ag-specific GC responses that might benefit the rational design of new vaccination strategies., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Staphylococcus aureus is a human bacterial pathogen causing a variety of diseases. The occurrence of multidrug-resistant strains of Staphylococcus aureus underlines the need for a vaccine. Defining immune correlates of protection may support the design of an effective vaccine. We used a murine Staphylococcus aureus infection model, in which bacteria were inoculated in an air pouch generated on the back of the animal. Analysis of the air-pouch content in mice immunized or not with an adjuvanted multiantigen vaccine formulation, four-component S. aureus vaccine (4C-Staph), prior to infection allowed us to measure bacteria, cytokines, and 4C-Staph-specific antibodies and to analyze host immune cells recruited to the infection site. Immunization with 4C-Staph resulted in accumulation of antigen-specific antibodies in the pouch and mitigated the infection. Neutrophils were the most abundant cells in the pouch, and they showed the upregulation of Fcγ receptor (FcγR) following immunization with 4C-Staph. Reduction of the infection was also obtained in mice immunized with 4C-Staph and depleted of neutrophils; these mice showed an increase in monocytes and macrophages. Upregulation of the FcγR and the presence of antigen-specific antibodies induced by immunization with 4C-Staph may contribute to increase bacterial opsonophagocytosis. Protection in neutropenic mice indicated that an effective vaccine could activate alternative protection mechanisms compensating for neutropenia, a condition often occurring in S. aureus-infected patients., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Adjuvants increase vaccine potency largely by activating innate immunity and promoting inflammation. Limiting the side effects of this inflammation is a major hurdle for adjuvant use in vaccines for humans. It has been difficult to improve on adjuvant safety because of a poor understanding of adjuvant mechanism and the empirical nature of adjuvant discovery and development historically. We describe new principles for the rational optimization of small-molecule immune potentiators (SMIPs) targeting Toll-like receptor 7 as adjuvants with a predicted increase in their therapeutic indices. Unlike traditional drugs, SMIP-based adjuvants need to have limited bioavailability and remain localized for optimal efficacy. These features also lead to temporally and spatially restricted inflammation that should decrease side effects. Through medicinal and formulation chemistry and extensive immunopharmacology, we show that in vivo potency can be increased with little to no systemic exposure, localized innate immune activation and short in vivo residence times of SMIP-based adjuvants. This work provides a systematic and generalizable approach to engineering small molecules for use as vaccine adjuvants., (Copyright © 2014, American Association for the Advancement of Science.)

Background: Outer membrane vesicles (OMVs) are spheroid particles released by all Gram-negative bacteria as a result of the budding out of the outer membrane. Since they carry many of the bacterial surface-associated proteins and feature a potent built-in adjuvanticity, OMVs are being utilized as vaccines, some of which commercially available. Recently, methods for manipulating the protein content of OMVs have been proposed, thus making OMVs a promising platform for recombinant, multivalent vaccines development., Methods: Chlamydia muridarum DO serine protease HtrA, an antigen which stimulates strong humoral and cellular responses in mice and humans, was expressed in Escherichia coli fused to the OmpA leader sequence to deliver it to the OMV compartment. Purified OMVs carrying HtrA (CM rHtrA-OMV) were analyzed for their capacity to induce antibodies capable of neutralizing Chlamydia infection of LLC-MK2 cells in vitro., Results: CM rHtrA-OMV immunization in mice induced antibodies that neutralize Chlamydial invasion as judged by an in vitro infectivity assay. This was remarkably different from what observed with an enzymatically functional recombinant HtrA expressed in, and purified from the E. coli cytoplasm (CM rHtrA). The difference in functionality between anti-CM rHtrA and anti-CM rHtrA-OMV antibodies was associated to a different pattern of protein epitopes recognition. The epitope recognition profile of anti-CM HtrA-OMV antibodies was similar to that induced in mice during Chlamydial infection., Conclusions: When expressed in OMVs HtrA appears to assume a conformation similar to the native one and this results in the elicitation of functional immune responses. These data further support the potentiality of OMVs as vaccine platform.

Flow cytometry (FC) has been widely used in biological research; however, its use for vaccine characterization has been very limited. Here we describe the development of an FC method for the direct quantification of two Neisseria meningitidis vaccine antigens, in mono- and multivalent formulations, while still adsorbed on aluminum hydroxide (AH) suspension. The antibody-based method is specific and sensitive. Because FC allows microscopic particle examination, the entire aluminum suspension carrying adsorbed antigen(s) can be analyzed directly. In addition to determining antigen concentration and identity, the assay is able to determine the distribution of the antigens on AH. High correlation coefficients (r(2)) were routinely achieved for a broad range of antigen doses from 0 to 150 μg/dose. Traditional assays for quantitative and qualitative antigen characterization on AH particles involve either complete aluminum dissolution or antigen desorption from the adjuvant. Because our direct method uses the whole AH suspension, the cumbersome steps used by traditional methods are not required. Those steps are often inefficient in desorbing the antigens and in some cases can lead to protein denaturation. We believe that this novel FC-based assay could circumvent some of the complex and tedious antigen-adjuvant desorption methods., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Natural immunity against obligate and/or facultative intracellular pathogens is usually mediated by both humoral and cellular immunity. The identification of those antigens stimulating both arms of the immune system is instrumental for vaccine discovery. Although high-throughput technologies have been applied for the discovery of antibody-inducing antigens, few examples of their application for T-cell antigens have been reported. We describe how the compilation of the immunome, here defined as the pool of immunogenic antigens inducing T- and B-cell responses in vivo, can lead to vaccine candidates against Chlamydia trachomatis. We selected 120 C. trachomatis proteins and assessed their immunogenicity using two parallel high-throughput approaches. Protein arrays were generated and screened with sera from C. trachomatis-infected patients to identify antibody-inducing antigens. Splenocytes from C. trachomatis-infected mice were stimulated with 79 proteins, and the frequency of antigen-specific CD4(+)/IFN-γ(+) T cells was analyzed by flow cytometry. We identified 21 antibody-inducing antigens, 16 CD4(+)/IFN-γ(+)-inducing antigens, and five antigens eliciting both types of responses. Assessment of their protective activity in a mouse model of Chlamydia muridarum lung infection led to the identification of seven antigens conferring partial protection when administered with LTK63/CpG adjuvant. Protection was largely the result of cellular immunity as assessed by CD4(+) T-cell depletion. The seven antigens provided robust additive protection when combined in four-antigen combinations. This study paves the way for the development of an effective anti-Chlamydia vaccine and provides a general approach for the discovery of vaccines against other intracellular pathogens.