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 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.

Chlamydia trachomatis vaccine development - a view on the current challenges and how to move forward.

INTRODUCTION: Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen in the world. A licensed vaccine is not yet available, but the first vaccines have entered clinical trials. AREAS COVERED: We describe the progress that has been made in our understanding of the type of immunity that a protective vaccine should induce, and the challenges that vaccine developers face. We also focus on the clinical development of a chlamydia vaccine. The first chlamydia vaccine candidate has now been tested in a clinical phase I trial, and another phase I trial is currently running. We discuss what it will take to continue this development and what future trial setups could look like. EXPERT OPINION: The chlamydia field is coming of age and the first phase I clinical trial of a C. trachomatis vaccine has been successfully completed. We expect and hope that this will motivate various stakeholders to support further development of chlamydia vaccines in humans.

A Novel Prophylaxis Strategy Using Liposomal Vaccine Adjuvant CAF09b Protects against Influenza Virus Disease.

The SARS-CoV-2 pandemic caused a massive health and societal crisis, although the fast development of effective vaccines reduced some of the impact. To prepare for future respiratory virus pandemics, a pan-viral prophylaxis could be used to control the initial virus outbreak in the period prior to vaccine approval. The liposomal vaccine adjuvant CAF®09b contains the TLR3 agonist polyinosinic:polycytidylic acid, which induces a type I interferon (IFN-I) response and an antiviral state in the affected tissues. When testing CAF09b liposomes as a potential pan-viral prophylaxis, we observed that intranasal administration of CAF09b liposomes to mice resulted in an influx of innate immune cells into the nose and lungs and upregulation of IFN-I-related gene expression. When CAF09b liposomes were administered prior to challenge with mouse-adapted influenza A/Puerto Rico/8/1934 virus, it protected from severe disease, although the virus was still detectable in the lungs. However, when CAF09b liposomes were administered after influenza challenge, the mice had a similar disease course to controls. In conclusion, CAF09b may be a suitable candidate as a pan-viral prophylactic treatment for epidemic viruses, but must be administered prior to virus exposure to be effective.

Th1/Th17 T cell Tissue-Resident Immunity Increases Protection, But Is Not Required in a Vaccine Strategy Against Genital Infection With Chlamydia trachomatis.

The requirement for vaccine-induced tissue-resident immunity for protection against one or repeated infections with Chlamydia trachomatis (C.t.) is still not fully resolved. In this study, our aim was to investigate to which degree tissue-resident Th1/Th17 T cells in the genital tract (GT) could add to the protection mediated by circulating immunity. Out of several mucosal vaccine strategies, a strategy termed SIM (for simultaneous intrauterine and parenteral immunization with CAF01 adjuvanted CTH522), was superior in generating genital tract tissue-resident Th1/Th17 T cell immunity. This led to a faster and stronger local CD4 T cell response post infection, consisting of multifunctional IFNγ/TNFα-producing Th1 T cells and IFNγ/TNFα/IL-17-producing Th17 T cells, and a faster recruitment of innate immune cells. Post infection, SIM animals showed an additional significant reduction in bacterial levels compared to mice having received only a parenteral vaccine. Nevertheless, the parenteral strategy reduced bacterial levels by 75%, and interestingly, post infection, these mice generated their own vaccine-derived genital tract tissue-resident memory Th1/Th17 T cells, which upon a subsequent infection showed as fast an activation in the genital tract, as observed in SIM mice. Furthermore, in contrast to after the first infection, both groups of mice now showed a similar infection-induced boost in local vaginal IgA and IgG titers. Thus, vaccine-induced resident immunity, generated pre-infection, led to an advantage in the response against the first infection, but not the second infection, suggesting that a parenteral vaccine strategy is a suitable vaccine strategy against infections with Chlamydia trachomatis.

Chitin-derived polymer deacetylation regulates mitochondrial reactive oxygen species dependent cGAS-STING and NLRP3 inflammasome activation.

Chitosan is a cationic polysaccharide that has been evaluated as an adjuvant due to its biocompatible and biodegradable nature. The polysaccharide can enhance antibody responses and cell-mediated immunity following vaccination by injection or mucosal routes. However, the optimal polymer characteristics for activation of dendritic cells (DCs) and induction of antigen-specific cellular immune responses have not been resolved. Here, we demonstrate that only chitin-derived polymers with a high degree of deacetylation (DDA) enhance generation of mitochondrial reactive oxygen species (mtROS), leading to cGAS-STING mediated induction of type I IFN. Additionally, the capacity of the polymers to activate the NLRP3 inflammasome was strictly dependent on the degree and pattern of deacetylation and mtROS generation. Polymers with a DDA below 80% are poor adjuvants while a fully deacetylated polyglucosamine polymer is most effective as a vaccine adjuvant. Furthermore, this polyglucosamine polymer enhanced antigen-specific Th1 responses in a NLRP3 and STING-type I IFN-dependent manner. Overall these results indicate that the degree of chitin deacetylation, the acetylation pattern and its regulation of mitochondrial ROS are the key determinants of its immune enhancing effects.

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.

Parenteral vaccination protects against transcervical infection with Chlamydia trachomatis and generate tissue-resident T cells post-challenge.

The optimal protective immunity against Chlamydia trachomatis (C.t.) is still not fully resolved. One of the unresolved issues concerns the importance of resident immunity, since a recent study showed that optimal protection against a transcervical (TC) infection required genital tissue-resident memory T cells. An important question in the Chlamydia field is therefore if a parenteral vaccine strategy, inducing only circulating immunity primed at a nonmucosal site, should be pursued by Chlamydia vaccine developers. To address this question we studied the protective efficacy of a parenteral Chlamydia vaccine, formulated in the Th1/Th17 T cell-inducing adjuvant CAF01. We found that a parenteral vaccination induced significant protection against a TC infection and against development of chronic pathology. Protection correlated with rapid recruitment of Th1/Th17 T cells to the genital tract (GT), which efficiently prevented infection-driven generation of low quality Th1 or Th17 T cells, and instead maintained a pool of high quality multifunctional Th1/Th17 T cells in the GT throughout the infection. After clearance of the infection, a pool of these cells settled in the GT as tissue-resident Th1 and Th17 cells expressing CD69 but not CD103, CD49d, or CCR7, where they responded rapidly to a reinfection. These results show that a nonmucosal parenteral strategy inducing Th1 and Th17 T cells mediates protection against both infection with C.t. as well as development of chronic pathology, and lead to post-challenge protective tissue-resident memory immunity in the genital tract.

Type I IFN signalling is required for cationic adjuvant formulation (CAF)01-induced cellular immunity and mucosal priming.

Despite being in the midst of a global pandemic of infections caused by the pathogen Chlamydia trachomatis, a vaccine capable of inducing protective immunity remains elusive. Given the C. trachomatis mucosal port of entry, a formulation compatible with mucosal administration and capable of eliciting potent genital tract immunity is highly desirable. While subunit vaccines are considered safer and better tolerated, these are typically poorly immunogenic and require co-formulation with immune-potentiating adjuvants. However, of the adjuvants licensed for use in humans, very few drive robust cellular responses, a pre-requisite for protection against C. trachomatis infection. Recently, the cationic adjuvant formulations (CAF) have been shown to induce robust humoral and cellular immunity in pre-clinical models of chlamydia, malaria and tuberculosis (TB). Here, we demonstrate that CAF01 induces potent immune responses when combined with the major outer membrane protein (MOMP) of C. trachomatis following parenteral immunisation and also as part of a heterologous prime/boost regime. We show that a subcutaneous prime with CAF01-adjuvanted recombinant MOMP licenses antigen-specific immunity at distant mucosal sites which can be activated following oral antigen re-encounter in the absence of concomitant adjuvant stimulation. Finally, we shed light on the mechanism(s) through which CAF01 elicits robust antigen-specific immunity to co-formulated MOMP via type I interferon (IFN) signalling.

Safety and immunogenicity of the chlamydia vaccine candidate CTH522 adjuvanted with CAF01 liposomes or aluminium hydroxide: a first-in-human, randomised, double-blind, placebo-controlled, phase 1 trial.

BACKGROUND: Chlamydia is the most common sexually transmitted bacterial infection worldwide. National screening programmes and antibiotic treatment have failed to decrease incidence, and to date no vaccines against genital chlamydia have been tested in clinical trials. We aimed to assess the safety and immunogenicity, in humans, of a novel chlamydia vaccine based on a recombinant protein subunit (CTH522) in a prime-boost immunisation schedule. METHODS: This phase 1, first-in-human, double-blind, parallel, randomised, placebo-controlled trial was done at Hammersmith Hospital in London, UK, in healthy women aged 19-45 years. Participants were randomly assigned (3:3:1) to three groups: CTH522 adjuvanted with CAF01 liposomes (CTH522:CAF01), CTH522 adjuvanted with aluminium hydroxide (CTH522:AH), or placebo (saline). Participants received three intramuscular injections of 85 µg vaccine (with adjuvant) or placebo to the deltoid region of the arm at 0, 1, and 4 months, followed by two intranasal administrations of 30 µg unadjuvanted vaccine or placebo (one in each nostril) at months 4·5 and 5·0. The primary outcome was safety and the secondary outcome was humoral immunogenicity (anti-CTH522 IgG seroconversion). This study is registered with Clinicaltrials.gov, number NCT02787109. FINDINGS: Between Aug 15, 2016, and Feb 13, 2017, 35 women were randomly assigned (15 to CTH522:CAF01, 15 to CTH522:AH, and five to placebo). 32 (91%) received all five vaccinations and all participants were included in the intention-to-treat analyses. No related serious adverse reactions were reported, and the most frequent adverse events were mild local injection-site reactions, which were reported in all (15 [100%] of 15) participants in the two vaccine groups and in three (60%) of five participants in the placebo group (p=0·0526 for both comparisons). Intranasal vaccination was not associated with a higher frequency of related local reactions (reported in seven [47%] of 15 participants in the active treatment groups vs three [60%] of five in the placebo group; p=1·000). Both CTH522:CAF01 and CTH522:AH induced anti-CTH522 IgG seroconversion in 15 (100%) of 15 participants after five immunisations, whereas no participants in the placebo group seroconverted. CTH522:CAF01 showed accelerated seroconversion, increased IgG titres, an enhanced mucosal antibody profile, and a more consistent cell-mediated immune response profile compared with CTH522:AH. INTERPRETATION: CTH522 adjuvanted with either CAF01 or aluminium hydroxide appears to be safe and well tolerated. Both vaccines were immunogenic, although CTH522:CAF01 had a better immunogenicity profile, holding promise for further clinical development. FUNDING: European Commission and The Innovation Fund Denmark.

Effects of cationic adjuvant formulation particle type, fluidity and immunomodulators on delivery and immunogenicity of saRNA.

Self-amplifying RNA (saRNA) is well suited as a vaccine platform against chlamydia, as it is relatively affordable and scalable, has been shown to induce immunity against multivalent antigens, and can result in protein expression for up to 60 days. Cationic adjuvant formulations (CAFs) have been previously investigated as an adjuvant for protein subunit vaccines; here we optimize the CAFs for delivery of saRNA in vivo and observe the immunogenicity profile in the context of both cellular and humoral immunity against the major outer membrane protein (MOMP) of Chlamydia trachomatis. We tested both liposomal and emulsion based CAFs with solid and fluid phase lipids, with or without the TLR agonists R848 and 3M-052, for in vitro transfection efficiency and cytotoxicity. We then optimized the RNA/delivery system ratio for in vivo delivery using saRNA coding for firefly luciferase (fLuc) as a reporter protein in vivo. We observed that while the fluid phase liposome formulations showed the highest in vitro transfection efficiency, the fluid and solid phase liposomes had equivalent luciferase expression in vivo. Incorporation of R848 or 3M-052 into the formulation was not observed to affect the delivery efficiency of saRNA either in vitro or in vivo. MOMP-encoding saRNA complexed with CAFs resulted in both MOMP-specific cellular and humoral immunity, and while there was a slight enhancement of IFN-γ+ T-cell responses when R848 was incorporated into the formulation, the self-adjuvanting effects of RNA appeared to dominate the immune response. These studies establish that CAFs are efficient delivery vehicles for saRNA both for in vitro transfections and in vivo immunogenicity and generate cellular and humoral responses that are proportionate to protein expression.

Genital Infiltrations of CD4+ and CD8+ T Lymphocytes, IgA+ and IgG+ Plasma Cells and Intra-Mucosal Lymphoid Follicles Associate With Protection Against Genital Chlamydia trachomatis Infection in Minipigs Intramuscularly Immunized With UV-Inactivated Bacteria Adjuvanted With CAF01.

The development of a vaccine against genital chlamydia in women is advancing, and the evaluation of in situ immune responses following vaccination and challenge infections is crucial for development of a safe and protective vaccine. This study employs the sexually mature minipig model to characterize the genital in situ immune response to Chlamydia trachomatis infection in pigs previously immunized intramuscularly with UV-inactivated C. trachomatis serovar D (UV-SvD) adjuvanted/formulated with CAF01 adjuvant compared to a CAF01-alone control group. Pigs immunized with UV-SvD were significantly protected against vaginal challenge with C. trachomatis on day 3 post inoculation and showed significantly higher cervical infiltrations of approximately equal numbers of CD4+ and CD8+ T-cells, and IgG+ and IgA+ plasma cells compared to adjuvant-alone immunized controls. These immunological signatures correspond to findings in mice and are similar to those described in female chlamydia patients. This proves important potential for the pig model in elucidating immunological in situ signatures in future translational research in chlamydia vaccinology.

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.

Unusual Self-Assembly of the Recombinant Chlamydia trachomatis Major Outer Membrane Protein-Based Fusion Antigen CTH522 Into Protein Nanoparticles.

Sexually transmitted Chlamydia trachomatis infects more than 100 million people annually, and untreated chlamydia infections can cause severe complications. Therefore, there is an urgent need for a chlamydia vaccine. The Ctrachomatis major outer membrane protein (MOMP) is highly immunogenic but is a challenging vaccine candidate by being an integral membrane protein, and the immunogenicity depends on a correctly folded structure. We investigated the biophysical properties of the recombinant MOMP-based fusion antigen CTH522, which is tested in early human clinical trials. It consists of a truncated and cysteine-free version of MOMP fused to 4 variable domains from serovars D-G. In the native state, CTH522 did not exist as a monomer but showed an unusual self-assembly into nanoparticles with a negative zeta potential. In contrast to the ß-barrel structure of MOMP, native CTH522 contained no well-defined secondary structural elements, and no thermal transitions were measurable. Chemical unfolding resulted in monomers that upon removal of the denaturant self-assembled into higher order structures, comparable to the structure of the native protein. The conformation of CTH522 in nanoparticles is thus not entirely random and contains structural elements stabilized via denaturant-disruptable hydrophobic interactions. In conclusion, CTH522 has an unusual quaternary structure of supramolecular self-assemblies.