Human antibody signatures towards the Chlamydia trachomatis major outer membrane protein after natural infection and vaccination.

BACKGROUND: Chlamydia trachomatis (CT) Major Outer Membrane Protein (MOMP) holds a neutralising epitope in the Variable Domain 4 (VD4), and this region's immune dominance during infection is well known. This study aimed to assess the antibody response induced after infection and compare it for specificity and functionality to the response following vaccination with the vaccine CTH522, which contains VD4's from serovars D, E, F, and G. METHODS: We assessed the antibody epitopes in MOMP by a high density peptide array. Furthermore, the role of the VD4 epitope in neutralisation was explored by competitive inhibition experiments with a fusion protein holding the neutralising VD4 linear epitope. This was done in two independent groups: 1) MOMP seropositive individuals infected with CT (n = 10, from case-control study) and 2) CTH522/CAF®01-vaccinated females (n = 14) from the CHLM-01 clinical trial. FINDINGS: We identified the major antigenic regions in MOMP as VD4 and the conserved region just before VD3 in individuals infected with CT. The same regions, with the addition of VD1, were identified in vaccine recipients. Overall, the VD4 peptide responses were uniform in vaccinated individuals and led to inhibition of infection in vitro in all tested samples, whereas the VD4 responses were more heterogenous in individuals infected with CT, and only 2 out of 10 samples had VD4-mediated neutralising antibody responses. INTERPRETATION: These data provide insights into the role of antibodies against MOMP VD4 induced after infection and vaccination, and show that their functionality differs. The induction of functional VD4-specific antibodies in vaccine recipients mimics previous results from animal models. FUNDING: This work was supported by the European Commission through contract FP7-HEALTH-2011.1.4-4-280873 (ADITEC) and Fonden til Lægevidenskabens Fremme.

An investigation of trachoma vaccine regimens by the chlamydia vaccine CTH522 administered with cationic liposomes in healthy adults (CHLM-02): a phase 1, double-blind trial.

BACKGROUND: There is no vaccine against the major global pathogen Chlamydia trachomatis; its different serovars cause trachoma in the eye or chlamydia in the genital tract. We did a clinical trial administering CTH522, a recombinant version of the C trachomatis major outer membrane molecule, in different dose concentrations with and without adjuvant, to establish its safety and immunogenicity when administered intramuscularly, intradermally, and topically into the eye, in prime-boost regimens. METHODS: CHLM-02 was a phase 1, double-blind, randomised, placebo-controlled trial at the National Institute for Health Research Imperial Clinical Research Facility, London, UK. Participants were healthy men and non-pregnant women aged 18-45 years, without pre-existing C trachomatis genital infection. Participants were assigned into six groups by the electronic database in a pre-prepared randomisation list (A-F). Participants were randomly assigned (1:1:1:1:1) to each of the groups A-E (12 participants each) and 6 were randomly assigned to group F. Investigators were masked to treatment allocation. Groups A-E received investigational medicinal product and group F received placebo only. Two liposomal adjuvants were compared, CAF01 and CAF09b. The groups were intramuscular 85 µg CTH522-CAF01, or placebo on day 0 and two boosters or placebo at day 28 and 112, and a mucosal recall with either placebo or CTH522 topical ocularly at day 140 (A); intramuscular 85 µg CTH522-CAF01, two boosters at day 28 and 112 with additional topical ocular administration of CTH522, and a mucosal recall with either placebo or CTH522 topical ocularly at day 140 (B); intramuscular 85 µg CTH522-CAF01, two boosters at day 28 and 112 with additional intradermal administration of CTH522, and a mucosal recall with either placebo or CTH522 topical ocularly at day 140 (C); intramuscular 15 µg CTH522-CAF01, two boosters at day 28 and 112, and a mucosal recall with either placebo or CTH522 topical ocularly at day 140 (D); intramuscular 85 µg CTH522-CAF09b, two boosters at day 28 and 112, and a mucosal recall with either placebo or CTH522 topical ocularly at day 140 (E); intramuscular placebo (F). The primary outcome was safety; the secondary outcome (humoral immunogenicity) was the percentage of trial participants achieving anti-CTH522 IgG seroconversion, defined as four-fold and ten-fold increase over baseline concentrations. Analyses were done as intention to treat and as per protocol. The trial is registered with ClinicalTrials.gov, NCT03926728, and is complete. FINDINGS: Between Feb 17, 2020 and Feb 22, 2022, of 154 participants screened, 65 were randomly assigned, and 60 completed the trial (34 [52%] of 65 women, 46 [71%] of 65 White, mean age 26·8 years). No serious adverse events occurred but one participant in group A2 discontinued dosing after having self-limiting adverse events after both placebo and investigational medicinal product doses. Study procedures were otherwise well tolerated; the majority of adverse events were mild to moderate, with only seven (1%) of 865 reported as grade 3 (severe). There was 100% four-fold seroconversion rate by day 42 in the active groups (A-E) and no seroconversion in the placebo group. Serum IgG anti-CTH522 titres were higher after 85 µg CTH522-CAF01 than 15 µg, although not significantly (intention-to-treat median IgG titre ratio groups A-C:D=5·6; p=0·062), with no difference after three injections of 85 µg CTH522-CAF01 compared with CTH522-CAF09b (group E). Intradermal CTH522 (group C) induced high titres of serum IgG anti-CTH522 neutralising antibodies against serovars B (trachoma) and D (urogenital). Topical ocular CTH522 (group B) at day 28 and 112 induced higher total ocular IgA compared with baseline (p<0·001). Participants in all active vaccine groups, particularly groups B and E, developed cell mediated immune responses against CTH522. INTERPRETATION: CTH522, adjuvanted with CAF01 or CAF09b, is safe and immunogenic, with 85 µg CTH522-CAF01 inducing robust serum IgG binding titres. Intradermal vaccination conferred systemic IgG neutralisation breadth, and topical ocular administration increased ocular IgA formation. These findings indicate CTH522 vaccine regimens against ocular trachoma and urogenital chlamydia for testing in phase 2, clinical trials. FUNDING: The EU Horizon Program TRACVAC.

Immune signature of Chlamydia vaccine CTH522/CAF®01 translates from mouse-to-human and induces durable protection in mice.

The clinical development of an effective Chlamydia vaccine requires in-depth understanding of how well protective pre-clinical immune signatures translate to humans. Here, we report a comparative immunological characterization of CTH522/CAF®01 in female mice and humans. We find a range of immune signatures that translate from mouse to human, including a Th1/Th17 cytokine profile and antibody functionality. We identify vaccine-induced T cell epitopes, conserved among Chlamydia serovars, and previously found in infected individuals. Using the mouse model, we show that the common immune signature protected against ascending infection in mice, and vaccine induced antibodies could delay bacterial ascension to the oviduct, as well as development of pathology, in a T cell depleted mouse model. Finally, we demonstrate long-lasting immunity and protection of mice one year after vaccination. Based on the results obtained in the present study, we propose to further investigate CTH522/CAF®01 in a phase IIb study.

A systems serology approach to the investigation of infection-induced antibody responses and protection in trachoma.

Background: Ocular infections with Chlamydia trachomatis serovars A-C cause the neglected tropical disease trachoma. As infection does not confer complete immunity, repeated infections are common, leading to long-term sequelae such as scarring and blindness. Here, we apply a systems serology approach to investigate whether systemic antibody features are associated with susceptibility to infection. Methods: Sera from children in five trachoma endemic villages in the Gambia were assayed for 23 antibody features: IgG responses towards two C. trachomatis antigens and three serovars [elementary bodies and major outer membrane protein (MOMP), serovars A-C], IgG responses towards five MOMP peptides (serovars A-C), neutralization, and antibody-dependent phagocytosis. Participants were considered resistant if they subsequently developed infection only when over 70% of other children in the same compound were infected. Results: The antibody features assayed were not associated with resistance to infection (false discovery rate < 0.05). Anti-MOMP SvA IgG and neutralization titer were higher in susceptible individuals (p < 0.05 before multiple testing adjustment). Classification using partial least squares performed only slightly better than chance in distinguishing between susceptible and resistant participants based on systemic antibody profile (specificity 71%, sensitivity 36%). Conclusions: Systemic infection-induced IgG and functional antibody responses do not appear to be protective against subsequent infection. Ocular responses, IgA, avidity, or cell-mediated responses may play a greater role in protective immunity than systemic IgG.

A protective, single-visit TB vaccination regimen by co-administration of a subunit vaccine with BCG.

The only licensed tuberculosis (TB) vaccine, Bacillus Calmette Guerin (BCG), fails to reliably protect adolescents and adults from pulmonary TB, resulting in ~1.6 million deaths annually. Protein subunit vaccines have shown promise against TB in clinical studies. Unfortunately, most subunit vaccines require multiple administrations, which increases the risk of loss to follow-up and necessitates more complex and costly logistics. Given the well-documented adjuvant effect of BCG, we hypothesized that BCG co-administration could compensate for a reduced number of subunit vaccinations. To explore this, we developed an expression-optimized version of our H107 vaccine candidate (H107e), which does not cross-react with BCG. In the CAF®01 adjuvant, a single dose of H107e induced inferior protection compared to three H107e/CAF®01 administrations. However, co-administering a single dose of H107e/CAF®01 with BCG significantly improved protection, which was equal to BCG co-administered with three H107e/CAF®01 doses. Importantly, combining BCG with a single H107e/CAF®01 dose also increased protection in previously BCG-primed animals. Overall, a single dose of H107e/CAF®01 with BCG induced long-lived immunity and triggered BCG-specific Th17 responses. These data support co-administration of BCG and subunit vaccines in both BCG naïve and BCG-primed individuals as an improved TB vaccine strategy with reduced number of vaccination visits.

A novel adjuvant formulation induces robust Th1/Th17 memory and mucosal recall responses in Non-Human Primates.

After clean drinking water, vaccination is the most impactful global health intervention. However, development of new vaccines against difficult-to-target diseases is hampered by the lack of diverse adjuvants for human use. Of particular interest, none of the currently available adjuvants induce Th17 cells. Here, we develop and test an improved liposomal adjuvant, termed CAF®10b, that incorporates a TLR-9 agonist. In a head-to-head study in non-human primates (NHPs), immunization with antigen adjuvanted with CAF®10b induced significantly increased antibody and cellular immune responses compared to previous CAF® adjuvants, already in clinical trials. This was not seen in the mouse model, demonstrating that adjuvant effects can be highly species specific. Importantly, intramuscular immunization of NHPs with CAF®10b induced robust Th17 responses that were observed in circulation half a year after vaccination. Furthermore, subsequent instillation of unadjuvanted antigen into the skin and lungs of these memory animals led to significant recall responses including transient local lung inflammation observed by Positron Emission Tomography-Computed Tomography (PET-CT), elevated antibody titers, and expanded systemic and local Th1 and Th17 responses, including >20% antigen-specific T cells in the bronchoalveolar lavage. Overall, CAF®10b demonstrated an adjuvant able to drive true memory antibody, Th1 and Th17 vaccine-responses across rodent and primate species, supporting its translational potential.

Multi-component prime-boost Chlamydia trachomatis vaccination regimes induce antibody and T cell responses and accelerate clearance of infection in a non-human primate model.

It is of international priority to develop a vaccine against sexually transmitted Chlamydia trachomatis infections to combat the continued global spread of the infection. The optimal immunization strategy still remains to be fully elucidated. The aim of this study was to evaluate immunization strategies in a nonhuman primate (NHP) model. Cynomolgus macaques (Macaqua fascicularis) were immunized following different multi-component prime-boost immunization-schedules and subsequently challenged with C. trachomatis SvD in the lower genital tract. The immunization antigens included the recombinant protein antigen CTH522 adjuvanted with CAF01 or aluminium hydroxide, MOMP DNA antigen and MOMP vector antigens (HuAd5 MOMP and MVA MOMP). All antigen constructs were highly immunogenic raising significant systemic C. trachomatis-specific IgG responses. In particularly the CTH522 protein vaccinated groups raised a fast and strong pecificsIgG in serum. The mapping of specific B cell epitopes within the MOMP showed that all vaccinated groups, recognized epitopes near or within the variable domains (VD) of MOMP, with a consistent VD4 response in all animals. Furthermore, serum from all vaccinated groups were able to in vitro neutralize both SvD, SvE and SvF. Antibody responses were reflected on the vaginal and ocular mucosa, which showed detectable levels of IgG. Vaccines also induced C. trachomatis-specific cell mediated responses, as shown by in vitro stimulation and intracellular cytokine staining of peripheral blood mononuclear cells (PBMCs). In general, the protein (CTH522) vaccinated groups established a multifunctional CD4 T cell response, whereas the DNA and Vector vaccinated groups also established a CD8 T cells response. Following vaginal challenge with C. trachomatis SvD, several of the vaccinated groups showed accelerated clearance of the infection, but especially the DNA group, boosted with CAF01 adjuvanted CTH522 to achieve a balanced CD4/CD8 T cell response combined with an IgG response, showed accelerated clearance of the infection.

Protection against SARS-CoV-2 transmission by a parenteral prime-Intranasal boost vaccine strategy.

BACKGROUND: Licensed vaccines against SARS-CoV-2 effectively protect against severe disease, but display incomplete protection against virus transmission. Mucosal vaccines providing immune responses in the upper airways are one strategy to protect against transmission. METHODS: We administered Spike HexaPro trimer formulated in a cationic liposomal adjuvant as a parenteral (subcutaneous - s.c.) prime - intranasal boost regimen to elicit airway mucosal immune responses and evaluated this in a Syrian hamster model of virus transmission. FINDINGS: Parenteral prime - intranasal boost elicited high-magnitude serum neutralizing antibody responses and IgA responses in the upper respiratory tract. The vaccine strategy protected against virus in the lower airways and lung pathology, but virus could still be detected in the upper airways. Despite this, the parenteral prime - intranasal booster vaccine effectively protected against onward SARS-CoV-2 transmission. INTERPRETATION: This study suggests that parenteral-prime mucosal boost is an effective strategy for protecting against SARS-CoV-2 infection and highlights that protection against virus transmission may be obtained despite incomplete clearance of virus from the upper respiratory tract. It should be noted that protection against onward transmission was not compared to standard parenteral prime-boost, which should be a focus for future studies. FUNDING: This work was primarily supported by the European Union Horizon 2020 research and innovation program under grant agreement no. 101003653.

A Mycobacterium tuberculosis-specific subunit vaccine that provides synergistic immunity upon co-administration with Bacillus Calmette-Guérin.

Given the encouraging clinical results of both candidate subunit vaccines and revaccination with Bacillus Calmette-Guérin (BCG) against tuberculosis (TB), there is support for combining BCG and subunit vaccination for increased efficacy. BCG and Mycobacterium tuberculosis (Mtb) share ~98% of their genome and current subunit vaccines are almost exclusively designed as BCG boosters. The goal of this study is to design a TB subunit vaccine composed of antigens not shared with BCG and explore the advantages of this design in a BCG + subunit co-administration vaccine strategy. Eight protective antigens are selected to create an Mtb-specific subunit vaccine, named H107. Whereas traditional vaccines containing BCG-shared antigens exhibit in vivo cross-reactivity to BCG, H107 shows no cross-reactivity and does not inhibit BCG colonization. Instead, co-administering H107 with BCG leads to increased adaptive responses against both H107 and BCG. Importantly, rather than expanding BCG-primed T cells, H107 broadens the overall vaccine repertoire with new T cell clones and introduces 'adjuvant-imprinted' qualities including Th17 responses and less-differentiated Th1 cells. Collectively, these features of H107 are associated with a substantial increase in long-term protection.

Adsorption of protein antigen to the cationic liposome adjuvant CAF®01 is required for induction of Th1 and Th17 responses but not for antibody induction.

The degree of antigen adsorption to adjuvants in subunit vaccines may significantly influence the immune responses they induce upon vaccination. Commonly used approaches for studying how the level of adsorption affects the induction of antigen-specific immune responses include (i) using adjuvants with different abilities to adsorb antigens, and (ii) comparing different antigens selected based on their ability to adsorb to the adjuvant. A weakness of these approaches is that not only the antigen adsorption level is varied, but also other important functional factors such as adjuvant composition and/or the B/T cell epitopes, which may affect immunogenicity. Hence, we investigated how changing the adsorption capabilities of a single antigen to an adjuvant influenced the vaccine-induced immune responses. The model antigen lysozyme, which displays a positive net charge at physiological pH due to an isoelectric point (pI) of 11, was succinylated to different extents, resulting in a reduction of the pI value to 4.4-5.9, depending on the degree of succinylation. A pronounced inverse correlation was found between the pI value of the succinylated lysozyme analogues and the degree of adsorption to a cationic liposomal adjuvant consisting of dimethyldioctadecylammonium bromide (DDA) and trehalose dibehenate (TDB) (CAF®01). Furthermore, increased adsorption to this adjuvant correlated directly with the magnitude of lysozyme-specific Th1/Th17 immune responses induced by the vaccine in mice, while there was an inverse correlation with antibody induction. However, high lysozyme-specific antibody titers were induced with an increased antigen dose, even upon vaccination with a strongly adsorbed succinylated lysozyme analogue. Hence, these data illustrate that the degree of lysozyme adsorption to CAF®01 strongly affects the quality of the resulting immune responses.

A Chlamydia trachomatis VD1-MOMP vaccine elicits cross-neutralizing and protective antibodies against C/C-related complex serovars.

Ocular and urogenital infections with Chlamydia trachomatis (C.t.) are caused by a range of different serovars. The first C.t. vaccine in clinical development (CTH522/CAF®01) induced neutralizing antibodies directed to the variable domain 4 (VD4) region of major outer membrane protein (MOMP), covering predominantly B and intermediate groups of serovars. The VD1 region of MOMP contains neutralizing B-cell epitopes targeting serovars of the C and C-related complex. Using an immuno-repeat strategy, we extended the VD1 region of SvA and SvJ to include surrounding conserved segments, extVD1A and extVD1J, and repeated this region four times. The extVD1A*4 was most immunogenic with broad cross-surface and neutralizing reactivity against representative members of the C and C-related complex serovars. Importantly, in vitro results for extVD1A*4 translated into in vivo biological effects, demonstrated by in vivo neutralization of SvA and protection/cross-protection against intravaginal challenge with both SvA and the heterologous SvIa strain.

In Vivo Antigen Expression Regulates CD4 T Cell Differentiation and Vaccine Efficacy against Mycobacterium tuberculosis Infection.

New vaccines are urgently needed against Mycobacterium tuberculosis (Mtb), which kills more than 1.4 million people each year. CD4 T cell differentiation is a key determinant of protective immunity against Mtb, but it is not fully understood how host-pathogen interactions shape individual antigen-specific T cell populations and their protective capacity. Here, we investigated the immunodominant Mtb antigen, MPT70, which is upregulated in response to gamma interferon (IFN-γ) or nutrient/oxygen deprivation of in vitro-infected macrophages. Using a murine aerosol infection model, we compared the in vivo expression kinetics of MPT70 to a constitutively expressed antigen, ESAT-6, and analyzed their corresponding CD4 T cell phenotype and vaccine protection. For wild-type Mtb, we found that in vivo expression of MPT70 was delayed compared to ESAT-6. This delayed expression was associated with induction of less differentiated MPT70-specific CD4 T cells but, compared to ESAT-6, also reduced protection after vaccination. In contrast, infection with an MPT70-overexpressing Mtb strain promoted highly differentiated KLRG1+CX3CR1+ CD4 T cells with limited lung-homing capacity. Importantly, this differentiated phenotype could be prevented by vaccination, and against the overexpressing strain, vaccination with MPT70 conferred protection similar to vaccination with ESAT-6. Together, our data indicate that high in vivo antigen expression drives T cells toward terminal differentiation and that targeted vaccination with adjuvanted protein can counteract this phenomenon by maintaining T cells in a protective less differentiated state. These observations shed new light on host-pathogen interactions and provide guidance on how future Mtb vaccines can be designed to tip the immune balance in favor of the host.IMPORTANCE Tuberculosis, caused by Mtb, constitutes a global health crisis of massive proportions and the impact of the current coronavirus disease 2019 (COVID-19) pandemic is expected to cause a rise in tuberculosis-related deaths. Improved vaccines are therefore needed more than ever, but a lack of knowledge on protective immunity hampers their development. The present study shows that constitutively expressed antigens with high availability drive highly differentiated CD4 T cells with diminished protective capacity, which could be a survival strategy by Mtb to evade T cell immunity against key antigens. We demonstrate that immunization with such antigens can counteract this phenomenon by maintaining antigen-specific T cells in a state of low differentiation. Future vaccine strategies should therefore explore combinations of multiple highly expressed antigens and we suggest that T cell differentiation could be used as a readily measurable parameter to identify these in both preclinical and clinical studies.

In vivo antigen expression regulates CD4 T cell differentiation and vaccine efficacy against Mycobacterium tuberculosis infection.

New vaccines are urgently needed against Mycobacterium tuberculosis (Mtb), which kills more than 1.4 million people each year. CD4 T cell differentiation is a key determinant of protective immunity against Mtb, but it is not fully understood how host-pathogen interactions shape individual antigen-specific T cell populations and their protective capacity. Here, we investigated the immunodominant Mtb antigen, MPT70, which is upregulated in response to IFN-γ or nutrient/oxygen deprivation of in vitro infected macrophages. Using a murine aerosol infection model, we compared the in vivo expression kinetics of MPT70 to a constitutively expressed antigen, ESAT-6, and analysed their corresponding CD4 T cell phenotype and vaccine-protection. For wild-type Mtb, we found that in vivo expression of MPT70 was delayed compared to ESAT-6. This delayed expression was associated with induction of less differentiated MPT70-specific CD4 T cells but, compared to ESAT-6, also reduced protection after vaccination. In contrast, infection with an MPT70-overexpressing Mtb strain promoted highly differentiated KLRG1+CX3CR1+ CD4 T cells with limited lung-homing capacity. Importantly, this differentiated phenotype could be prevented by vaccination and, against the overexpressing strain, vaccination with MPT70 conferred similar protection as ESAT-6. Together our data indicate that high in vivo antigen expression drives T cells towards terminal differentiation and that targeted vaccination with adjuvanted protein can counteract this phenomenon by maintaining T cells in a protective less-differentiated state. These observations shed new light on host-pathogen interactions and provide guidance on how future Mtb vaccines can be designed to tip the immune-balance in favor of the host.

Rescuing ESAT-6 Specific CD4 T Cells From Terminal Differentiation Is Critical for Long-Term Control of Murine Mtb Infection.

In most cases, Mycobacterium tuberculosis (Mtb) causes life-long chronic infections, which poses unique challenges for the immune system. Most of the current tuberculosis (TB) subunit vaccines incorporate immunodominant antigens and at this point, it is poorly understood how the CD4 T cell subsets recognizing these antigens are affected during long-term infection. Very little is known about the requirements for sustainable vaccine protection against TB. To explore this, we screened 62 human-recognized Mtb antigens during chronic murine Mtb infection and identified the four most immunodominant antigens in this setting (MPT70, Rv3020c, and Rv3019c and ESAT-6). Combined into a subunit vaccine, this fusion protein induced robust protection both in a standard short-term model and in a long-term infection model where immunity from BCG waned. Importantly, replacement of ESAT-6 with another ESAT-6-family antigen, Rv1198, led to similar short-term protection but a complete loss of bacterial control during chronic infection. This observation was further underscored, as the ESAT-6 containing vaccine mediated sustainable protection in a model of post-exposure vaccination, where the ESAT-6-replacement vaccine did not. An individual comparison of the CD4 T cell responses during Mtb infection revealed that ESAT-6-specific T cells were more terminally differentiated than the other immunodominant antigens and immunization with the ESAT-6 containing vaccine led to substantially greater reduction in the overall T cell differentiation status. Our data therefore associates long-term bacterial control with the ability of a vaccine to rescue infection-driven CD4T cell differentiation and future TB antigen discovery programs should focus on identifying antigens with the highest accompanying T cell differentiation, like ESAT-6. This also highlights the importance of long-term readouts in both preclinical and clinical studies with TB vaccines.

Immunization with Mycobacterium tuberculosis-Specific Antigens Bypasses T Cell Differentiation from Prior Bacillus Calmette-Guérin Vaccination and Improves Protection in Mice.

Despite the fact that the majority of people in tuberculosis (TB)-endemic areas are vaccinated with the Bacillus Calmette-Guérin (BCG) vaccine, TB remains the leading infectious cause of death. Data from both animal models and humans show that BCG and subunit vaccines induce T cells of different phenotypes, and little is known about how BCG priming influences subsequent booster vaccines. To test this, we designed a novel Mycobacterium tuberculosis-specific (or "non-BCG") subunit vaccine with protective efficacy in both mice and guinea pigs and compared it to a known BCG boosting vaccine. In naive mice, this M. tuberculosis-specific vaccine induced similar protection compared with the BCG boosting vaccine. However, in BCG-primed animals, only the M. tuberculosis-specific vaccine added significantly to the BCG-induced protection. This correlated with the priming of T cells with a lower degree of differentiation and improved lung-homing capacity. These results have implications for TB vaccine design.

The increased protection and pathology in Mycobacterium tuberculosis-infected IL-27R-alpha-deficient mice is supported by IL-17A and is associated with the IL-17A-induced expansion of multifunctional T cells.

During Mycobacterium tuberculosis (Mtb) infection, mice lacking the IL-27R exhibit lower bacterial burdens but develop an immunopathological sequelae in comparison to wild-type mice. We here show that this phenotype correlates with an enhanced recruitment of antigen-specific CCR6+ CD4+ T cells and an increased frequency of IL-17A-producing CD4+ T cells. By comparing the outcome of Mtb infection in C57BL/6, IL-27R-deficient and IL-27R/IL-17A-double deficient mice, we observed that both the increased protection and elevated immunopathology are supported by IL-17A. Whereas IL-17A neither impacts the development of Tr1 cells nor the expression of PD1 and KLRG1 on T cells in IL-27R-deficient mice during infection, it regulates the presence of multifunctional T-cells in the lungs, co-expressing IFN-γ, IL-2 and TNF. Eventually, IL-17A supports Cxcl9, Cxcl10 and Cxcl13 expression and the granulomatous response in the lungs of infected IL-27R-deficient mice. Taken together, IL-17A contributes to protection in Mtb-infected IL-27R-deficient mice probably through a chemokine-mediated recruitment and strategic positioning of multifunctional T cells in granulomas. As IL-27 limits optimal antimycobacterial protection by inhibiting IL-17A production, blocking of IL-27R-mediated signaling may represent a strategy for improving vaccination and host-directed therapy in tuberculosis. However, because IL-27 also prevents IL-17A-mediated immunopathology, such intervention has to be tightly controlled.

A flow cytometry-based assay to determine the phagocytic activity of both clinical and nonclinical antibody samples against Chlamydia trachomatis.

Globally, an estimated 131 million new cases of chlamydial infection occur annually. Chlamydia trachomatis infection can cause permanent damage to the fallopian tubes in woman, resulting in infertility and a risk of ectopic pregnancy. There is a great need for a vaccine against Chlamydia trachomatis and as a result there is a need for assays to evaluate functional immune responses for use in future clinical trials and epidemiological studies. Antibodies play a crucial role in the defense against infection and can be protective by several functions, including phagocytosis and neutralization. Vaccine development could greatly benefit from a method to measure functional C. trachomatis-specific antibodies in a large number of samples. In the current in vitro antibody protection assays, which measure the capacity of antibodies to facilitate phagocytic uptake of C. trachomatis, the phagocytosed bacteria have to be counted manually. This is both labor demanding, time consuming, and it prevents high-throughput usage of this method. In this study, we, therefore, developed a simple and rapid flow cytometry based assay to measure the capacity of antibodies to mediate Fc-receptor dependent phagocytosis. This method is highly reproducible and suitable to analyze large numbers of clinical and nonclinical samples. © 2018 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC.

Africa-wide evaluation of host biomarkers in QuantiFERON supernatants for the diagnosis of pulmonary tuberculosis.

We investigated host-derived biomarkers that were previously identified in QuantiFERON supernatants, in a large pan-African study. We recruited individuals presenting with symptoms of pulmonary TB at seven peripheral healthcare facilities in six African countries, prior to assessment for TB disease. We then evaluated the concentrations of 12 biomarkers in stored QuantiFERON supernatants using the Luminex platform. Based on laboratory, clinical and radiological findings and a pre-established algorithm, participants were classified as TB disease or other respiratory diseases(ORD). Of the 514 individuals included in the study, 179(34.8%) had TB disease, 274(51.5%) had ORD and 61(11.5%) had an uncertain diagnosis. A biosignature comprising unstimulated IFN-γ, MIP-1ß, TGF-α and antigen-specific levels of TGF-α and VEGF, identified on a training sample set (n = 311), validated by diagnosing TB disease in the test set (n = 134) with an AUC of 0.81(95% CI, 0.76-0.86), corresponding to a sensitivity of 64.2%(95% CI, 49.7-76.5%) and specificity of 82.7%(95% CI, 72.4-89.9%). Host biomarkers detected in QuantiFERON supernatants can contribute to the diagnosis of active TB disease amongst people presenting with symptoms requiring investigation for TB disease, regardless of HIV status or ethnicity in Africa.

The dynamics of immune responses to Mycobacterium tuberculosis during different stages of natural infection: A longitudinal study among Greenlanders.

OBJECTIVE: Understanding human immunity to Mycobacterium tuberculosis (Mtb) during different stages of infection is important for development of an effective tuberculosis (TB) vaccine. We aimed to evaluate immunity to Mtb infection by measuring immune responses to selected Mtb antigens expressed during different stages of infection over time and to observe sustainability of immunity. METHODS: In a cohort study comprising East Greenlanders aged 17-22 years (2012 to 2014) who had either; undetectable Mtb infection, ongoing or prior Mtb infection at enrolment, we measured immunity to 15 antigens over a one-year period. Quantiferon-TB Gold testing (QFT) defined Mtb infection status (undetected/detected). The eligible study population of East Greenlanders aged 17-22 years was identified from the entire population using the Civil Registration System. From the source population 65 participants were selected by stratified random sampling according to information on Mtb infection stage. Retrospective and prospective information on notified TB (including treatment) was obtained through the mandatory TB notification system and was used to characterise Mtb infection stage (ongoing/prior). Immunity to 15 antigens including two QFT antigens, PPD and 12 non-QFT antigens (representing early, constitutive and latent Mtb infection) was assessed by measuring immune responses using whole-blood antigen stimulation and interferon gamma measurement. RESULTS: Of 65 participants, 54 were considered Mtb-infected. Immunity to Mtb infection fluctuated with high annual risk of conversion (range: 6-69%) and reversion (range: 5-95%). During follow-up, five (8%) participants were notified with TB; neither conversion nor reversion was associated with an increased risk of progressing to TB. CONCLUSIONS: Our findings suggest that human immunity to natural Mtb infection over time is versatile with fluctuations, resulting in high levels of conversion and reversion of immunity, thus human immunity to Mtb is much more dynamic than anticipated. The study findings suggest future use of longitudinal assessment of immune responses when searching for TB vaccine candidate antigens.