Epidermal Growth Factor Receptor and Transforming Growth Factor ß Signaling Pathways Cooperate To Mediate Chlamydia Pathogenesis.

Human genital Chlamydia infection is a major public health concern due to the serious reproductive system complications. Chlamydia binds several receptor tyrosine kinases (RTKs) on host cells, including the epidermal growth factor receptor (EGFR), and activates cellular signaling cascades for host invasion, cytoskeletal remodeling, optimal inclusion development, and induction of pathogenic epithelial-mesenchyme transition (EMT). Chlamydia also upregulates transforming growth factor beta (TGF-ß) expression, whose signaling pathway synergizes with the EGFR cascade, but its role in infectivity, inclusions, and EMT induction is unknown. We hypothesized that the EGFR and TGF-ß signaling pathways cooperate during chlamydial infection for optimal inclusion development and stable EMT induction. The results revealed that Chlamydia upregulated TGF-ß expression as early as 6 h postinfection of epithelial cells and stimulated both the EGFR and TGF-ß signaling pathways. Inhibition of either the EGFR or TGF-ßR1 signaling substantially reduced inclusion development; however, the combined inhibition of both EGFR and TGF-ßR1 signaling reduced inclusions by over 90% and prevented EMT induction. Importantly, EGFR inhibition suppressed TGF-ß expression, and an inhibitory thrombospondin-1 (Tsp1)-based peptide inhibited chlamydia-induced EMT, revealing a major source of active TGF-ß during infection. Finally, TGF-ßR signaling inhibition suppressed the expression of transforming acidic coiled-coil protein-3 (TACC3), which stabilizes EGFR signaling, suggesting reciprocal regulation between TGF-ß and EGFR signaling during chlamydial infection. Thus, RTK-mediated host invasion by chlamydia upregulated TGF-ß expression and signaling, which cooperated with other cellular signaling cascades and cytoskeletal remodeling to support optimal inclusion development and EMT induction. This finding may provide new targets for chlamydial disease biomarkers and prevention.

A unique insight into the MiRNA profile during genital chlamydial infection.

BACKGROUND: Genital C. trachomatis infection may cause pelvic inflammatory disease (PID) that can lead to tubal factor infertility (TFI). Understanding the pathogenesis of chlamydial complications including the pathophysiological processes within the female host genital tract is important in preventing adverse pathology. MicroRNAs regulate several pathophysiological processes of infectious and non-infectious etiologies. In this study, we tested the hypothesis that the miRNA profile of single and repeat genital chlamydial infections will be different and that these differences will be time dependent. Thus, we analyzed and compared differentially expressed mice genital tract miRNAs after single and repeat chlamydia infections using a C. muridarum mouse model. Mice were sacrificed and their genital tract tissues were collected at 1, 2, 4, and 8 weeks after a single and repeat chlamydia infections. Histopathology, and miRNA sequencing were performed. RESULTS: Histopathology presentation showed that the oviduct and uterus of reinfected mice were more inflamed, distended and dilated compared to mice infected once. The miRNAs expression profile was different in the reproductive tissues after a reinfection, with a greater number of miRNAs expressed after reinfection. Also, the number of miRNAs expressed each week after chlamydia infection and reinfection varied, with weeks eight and one having the highest number of differentially expressed miRNAs for chlamydia infection and reinfection respectively. Ten miRNAs; mmu-miR-378b, mmu-miR-204-5p, mmu-miR-151-5p, mmu-miR-142-3p, mmu-miR-128-3p, mmu-miR-335-3p, mmu-miR-195a-3p, mmu-miR-142-5p, mmu-miR-106a-5p and mmu-miR-92a-3p were common in both primary chlamydia infection and reinfection. Pathway analysis showed that, amongst other functions, the differentially regulated miRNAs control pathways involved in cellular and tissue development, disease conditions and toxicity. CONCLUSIONS: This study provides insights into the changes in miRNA expression over time after chlamydia infection and reinfection, as well as the pathways they regulate to determine pathological outcomes. The miRNAs networks generated in our study shows that there are differences in the focus molecules involved in significant biological functions in chlamydia infection and reinfection, implying that chlamydial pathogenesis occurs differently for each type of infection and that this could be important when determining treatments regime and disease outcome. The study underscores the crucial role of host factors in chlamydia pathogenesis.

The molecular mechanism of induction of unfolded protein response by Chlamydia.

The unfolded protein response (UPR) contributes to chlamydial pathogenesis, as a source of lipids and ATP during replication, and for establishing the initial anti-apoptotic state of host cell that ensures successful inclusion development. The molecular mechanism(s) of UPR induction by Chlamydia is unknown. Chlamydia use type III secretion system (T3SS) effector proteins (e.g, the Translocated Actin-Recruiting Phosphoprotein (Tarp) to stimulate host cell's cytoskeletal reorganization that facilitates invasion and inclusion development. We investigated the hypothesis that T3SS effector-mediated assembly of myosin-II complex produces activated non-muscle myosin heavy chain II (NMMHC-II), which then binds the UPR master regulator (BiP) and/or transducers to induce UPR. Our results revealed the interaction of the chlamydial effector proteins (CT228 and Tarp) with components of the myosin II complex and UPR regulator and transducer during infection. These interactions caused the activation and binding of NMMHC-II to BiP and IRE1α leading to UPR induction. In addition, specific inhibitors of myosin light chain kinase, Tarp oligomerization and myosin ATPase significantly reduced UPR activation and Chlamydia replication. Thus, Chlamydia induce UPR through T3SS effector-mediated activation of NMMHC-II components of the myosin complex to facilitate infectivity. The finding provides greater insights into chlamydial pathogenesis with the potential to identify therapeutic targets and formulations.

Molecular Pathogenesis of Chlamydia Disease Complications: Epithelial-Mesenchymal Transition and Fibrosis.

The reproductive system complications of genital chlamydial infection include fallopian tube fibrosis and tubal factor infertility. However, the molecular pathogenesis of these complications remains poorly understood. The induction of pathogenic epithelial-mesenchymal transition (EMT) through microRNA (miRNA) dysregulation was recently proposed as the pathogenic basis of chlamydial complications. Focusing on fibrogenesis, we investigated the hypothesis that chlamydia-induced fibrosis is caused by EMT-driven generation of myofibroblasts, the effector cells of fibrosis that produce excessive extracellular matrix (ECM) proteins. The results revealed that the targets of a major category of altered miRNAs during chlamydial infection are key components of the pathophysiological process of fibrogenesis; these target molecules include collagen types I, III, and IV, transforming growth factor ß (TGF-ß), TGF-ß receptor 1 (TGF-ßR1), connective tissue growth factor (CTGF), E-cadherin, SRY-box 7 (SOX7), and NFAT (nuclear factor of activated T cells) kinase dual-specificity tyrosine (Y) phosphorylation-regulated kinase 1a (Dyrk1a). Chlamydial induction of EMT resulted in the generation of α-smooth muscle actin (α-SMA)-positive myofibroblasts that produced ECM proteins, including collagen types I and III and fibronectin. Furthermore, the inhibition of EMT prevented the generation of myofibroblasts and production of ECM proteins during chlamydial infection. These findings may provide useful avenues for targeting EMT or specific components of the EMT pathways as a therapeutic intervention strategy to prevent chlamydia-related complications.

The Roles of Unfolded Protein Response Pathways in Chlamydia Pathogenesis.

Chlamydia is an obligate intracellular bacterium that relies on host cells for essential nutrients and adenosine triphosphate (ATP) for a productive infection. Although the unfolded protein response (UPR) plays a major role in certain microbial infectivity, its role in chlamydial pathogenesis is unknown. We hypothesized that Chlamydia induces UPR and exploits it to upregulate host cell uptake and metabolism of glucose, production of ATP, phospholipids, and other molecules required for its replicative development and host survival. Using a combination of biochemical and pathway inhibition assays, we showed that the 3 UPR pathway transducers-protein kinase RNA-activated (PKR)-like ER kinase (PERK), inositol-requiring enzyme-1α (IRE1α), and activating transcription factor-6α (ATF6α)-were activated during Chlamydia infection. The kinase activity of PERK and ribonuclease (RNase) of IRE1α mediated the upregulation of hexokinase II and production of ATP via substrate-level phosphorylation. In addition, the activation of PERK and IRE1α promoted autophagy formation and apoptosis resistance for host survival. Moreover, the activation of IRE1α resulted in the generation of spliced X-box binding protein 1 (sXBP1) and upregulation of lipid production. The vital role of UPR pathways in Chlamydia development and pathogenesis could lead to the identification of potential molecular targets for therapeutics against Chlamydia.

The immunoregulatory role of alpha enolase in dendritic cell function during Chlamydia infection.

BACKGROUND: We have previously reported that interleukin-10 (IL-10) deficient dendritic cells (DCs) are potent antigen presenting cells that induced elevated protective immunity against Chlamydia. To further investigate the molecular and biochemical mechanism underlying the superior immunostimulatory property of IL-10 deficient DCs we performed proteomic analysis on protein profiles from Chlamydia-pulsed wild-type (WT) and IL-10-/- DCs to identify differentially expressed proteins with immunomodulatory properties. RESULTS: The results showed that alpha enolase (ENO1), a metabolic enzyme involved in the last step of glycolysis was significantly upregulated in Chlamydia-pulsed IL-10-/- DCs compared to WT DCs. We further studied the immunoregulatory role of ENO1 in DC function by generating ENO1 knockdown DCs, using lentiviral siRNA technology. We analyzed the effect of the ENO1 knockdown on DC functions after pulsing with Chlamydia. Pyruvate assay, transmission electron microscopy, flow cytometry, confocal microscopy, cytokine, T-cell activation and adoptive transfer assays were also used to study DC function. The results showed that ENO1 knockdown DCs had impaired maturation and activation, with significant decrease in intracellular pyruvate concentration as compared with the Chlamydia-pulsed WT DCs. Adoptive transfer of Chlamydia-pulsed ENO1 knockdown DCs were poorly immunogenic in vitro and in vivo, especially the ability to induce protective immunity against genital chlamydia infection. The marked remodeling of the mitochondrial morphology of Chlamydia-pulsed ENO1 knockdown DCs compared to the Chlamydia-pulsed WT DCs was associated with the dysregulation of translocase of the outer membrane (TOM) 20 and adenine nucleotide translocator (ANT) 1/2/3/4 that regulate mitochondrial permeability. The results suggest that an enhanced glycolysis is required for efficient antigen processing and presentation by DCs to induce a robust immune response. CONCLUSIONS: The upregulation of ENO1 contributes to the superior immunostimulatory function of IL-10 deficient DCs. Our studies indicated that ENO1 deficiency causes the reduced production of pyruvate, which then contributes to a dysfunction in mitochondrial homeostasis that may affect DC survival, maturation and antigen presenting properties. Modulation of ENO1 thus provides a potentially effective strategy to boost DC function and promote immunity against infectious and non-infectious diseases.

Interleukin-10 modulates antigen presentation by dendritic cells through regulation of NLRP3 inflammasome assembly during Chlamydia infection.

Interleukin-10 (IL-10) has been implicated in susceptibility to genital chlamydial infection and the development of tubal pathologies. IL-10 limitation also resulted in the rapid elicitation of immune responses against Chlamydia, and decreased levels of IL-10 correlated with protective anti-Chlamydia immunity. To investigate the molecular basis for these effects, we compared the reproductive pathologies and fertility rates in Chlamydia-infected wild-type (WT) and IL-10-knockout (IL-10(-/-)) mice; we also analyzed the expression of the Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) superfamily, IL-1ß production, NLRP3 inflammasome assembly and activation, and the immunostimulatory capacity and apoptotic predilection of Chlamydia-exposed dendritic cells (DCs) from WT and IL-10(-/-) mice. Our results revealed that, in addition to the rapid clearance of infection, genitally infected IL-10(-/-) mice were protected from tubal pathologies and infertility, whereas WT (IL-10(+/+)) mice were not. Chlamydia-pulsed IL-10(-/-) DCs expressed larger numbers of TLR4/IL-1R molecules and had enhanced IL-1ß production. In addition, NLRP3 inflammasome assembly was suppressed in IL-10(-/-) DCs through the inhibition of the P2X purinoceptor 7 (P2X7) receptor (P2X7R), an ATP-gated ion channel, and a decrease in intracellular Ca(2+) levels, which inhibited DC apoptosis. Thus, the potent immunostimulatory capacity of IL-10-deficient DCs is due, at least in part, to the suppression of the intracellular inflammasome assembly, which prevents DC apoptosis, allowing efficient antigen presentation. The results indicate that IL-10 deficiency enables efficient antigen presentation by DCs for rapid and enhanced immune activation against Chlamydia, which results in rapid microbial clearance, which prevents tubal pathologies during infection. Our finding has important implications for the induction of protective immunity against Chlamydia and other infectious and noninfectious diseases by vaccines.

Genetic diversity of Plasmodium falciparum parasite by microsatellite markers after scale-up of insecticide-treated bed nets in western Kenya.

BACKGROUND: An initial study of genetic diversity of Plasmodium falciparum in Asembo, western Kenya showed that the parasite maintained overall genetic stability 5 years after insecticide-treated bed net (ITN) introduction in 1997. This study investigates further the genetic diversity of P. falciparum 10 years after initial ITN introduction in the same study area and compares this with two other neighbouring areas, where ITNs were introduced in 1998 (Gem) and 2004 (Karemo). METHODS: From a cross-sectional survey conducted in 2007, 235 smear-positive blood samples collected from children ≤15-year-old in the original study area and two comparison areas were genotyped employing eight neutral microsatellites. Differences in multiple infections, allele frequency, parasite genetic diversity and parasite population structure between the three areas were assessed. Further, molecular data reported previously (1996 and 2001) were compared to the 2007 results in the original study area Asembo. RESULTS: Overall proportion of multiple infections (MA) declined with time in the original study area Asembo (from 95.9 %-2001 to 87.7 %-2007). In the neighbouring areas, MA was lower in the site where ITNs were introduced in 1998 (Gem 83.7 %) compared to where they were introduced in 2004 (Karemo 96.7 %) in 2007. Overall mean allele count (MAC ~ 2.65) and overall unbiased heterozygosity (H e  ~ 0.77) remained unchanged in 1996, 2001 and 2007 in Asembo and was the same level across the two neighbouring areas in 2007. Overall parasite population differentiation remained low over time and in the three areas at FST < 0.04. Both pairwise and multilocus linkage disequilibrium showed limited to no significant association between alleles in Asembo (1996, 2001 and 2007) and between three areas. CONCLUSIONS: This study showed the P. falciparum high genetic diversity and parasite population resilience on samples collected 10 years apart and in different areas in western Kenya. The results highlight the need for long-term molecular monitoring after implementation and use of combined and intensive prevention and intervention measures in the region.

Assessment of molecular markers for anti-malarial drug resistance after the introduction and scale-up of malaria control interventions in western Kenya.

BACKGROUND: Although it is well known that drug pressure selects for drug-resistant parasites, the role of transmission reduction by insecticide-treated bed nets (ITNs) on drug resistance remains unclear. In this study, the drug resistance profile of current and previous first-line anti-malarials in Kenya was assessed within the context of drug policy change and scale-up of ITNs. National first-line treatment changed from chloroquine (CQ) to sulphadoxine-pyrimethamine (SP) in 1998 and to artemether-lumefantrine (AL) in 2004. ITN use was scaled-up in the Asembo, Gem and Karemo areas of western Kenya in 1997, 1999 and 2006, respectively. METHODS: Smear-positive samples (N = 253) collected from a 2007 cross-sectional survey among children in Asembo, Gem and Karemo were genotyped for mutations in pfcrt and pfmdr1 (CQ), dhfr and dhps (SP), and at pfmdr-N86 and the gene copy number in pfmdr1 (lumefantrine). Results were compared among the three geographic areas in 2007 and to retrospective molecular data from children in Asembo in 2001. RESULTS: In 2007, 69 and 85% of samples harboured the pfmdr1-86Y mutation and dhfr/dhps quintuple mutant, respectively, with no significant differences by study area. However, the prevalence of the pfcrt-76T mutation differed significantly among areas (p <0.02), between 76 and 94%, with the highest prevalence in Asembo. Several 2007 samples carried mutations at dhfr-164L, dhps-436A, or dhps-613T. From 2001 to 2007, there were significant increases in the pfcrt-76T mutation from 82 to 94% (p <0.03), dhfr/dhps quintuple mutant from 62 to 82% (p <0.03), and an increase in the septuple CQ and SP combined mutant haplotype, K 76 Y 86 I 51 R 59 N 108 G 437 E 540 , from 28 to 39%. The prevalence of the pfmdr1-86Y mutation remained unchanged. All samples were single copy for pfmdr1. CONCLUSIONS: Molecular markers associated with lumefantrine resistance were not detected in 2007. More recent samples will be needed to detect any selective effects by AL. The prevalence of CQ and SP resistance markers increased from 2001 to 2007 in the absence of changes in transmission intensity. In 2007, only the prevalence of pfcrt-76T mutation differed among study areas of varying transmission intensity. Resistant parasites were most likely selected by sustained drug pressure from the continued use of CQ, SP, and mechanistically similar drugs, such as amodiaquine and cotrimoxazole. There was no clear evidence that differences in transmission intensity, as a result of ITN scale-up, influenced the prevalence of drug resistance molecular markers.

Prevention of Chlamydia-induced infertility by inhibition of local caspase activity.

Tubal factor infertility (TFI) represents 36% of female infertility and genital infection by Chlamydia trachomatis (C. trachomatis) is a major cause. Although TFI is associated with host inflammatory responses to bacterial components, the molecular pathogenesis of Chlamydia-induced infertility remains poorly understood. We investigated the hypothesis that activation of specific cysteine proteases, the caspases, during C. trachomatis genital infection causes the disruption of key fertility-promoting molecules required for embryo development and implantation. We analyzed the effect of caspase inhibition on infertility and the integrity of Dicer, a caspase-sensitive, fertility-promoting ribonuclease III enzyme, and key micro-RNAs in the reproductive system. Genital infection with the inflammation- and caspase-inducing, wild-type C. trachomatis serovar L2 led to infertility, but the noninflammation-inducing, plasmid-free strain did not. We confirmed that caspase-mediated apoptotic tissue destruction may contribute to chlamydial pathogenesis. Caspase-1 or -3 deficiency, or local administration of the pan caspase inhibitor, Z-VAD-FMK into normal mice protected against Chlamydia-induced infertility. Finally, the oviducts of infected infertile mice showed evidence of caspase-mediated cleavage inactivation of Dicer and alteration in critical miRNAs that regulate growth, differentiation, and development, including mir-21. These results provide new insight into the molecular pathogenesis of TFI with significant implications for new strategies for treatment and prevention of chlamydial complications.

In silico design and analysis of a multiepitope vaccine against Chlamydia.

Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterial infection worldwide, potentially leading to severe pathologies including pelvic inflammatory disease, ectopic pregnancy, and tubal infertility if left untreated. Current strategies, including screening and antibiotics, have limited effectiveness due to high rates of asymptomatic cases and logistical challenges. A multiepitope prophylactic vaccine could afford long-term protection against infection. Immunoinformatic analyses were employed to design a multiepitope Chlamydia vaccine antigen. B- and T-cell epitopes from five highly conserved and immunogenic Ct antigens were predicted and selected for the vaccine design. The final construct, adjuvanted with cholera toxin A1 subunit (CTA1), was further screened for immunogenicity. CTA1-MECA (multiepitope Chlamydia trachomatis antigen) was identified as antigenic and nonallergenic. A tertiary structure was predicted, refined, and validated as a good quality model. Molecular docking exhibited strong interactions between the vaccine and toll-like receptor 4 (TLR4). Additionally, immune responses consistent with protection including IFN-γ, IgG + IgM antibodies, and T- and B-cell responses were predicted following vaccination in an immune simulation. Expression of the construct in an Escherichia coli expression vector proved efficient. To further validate the vaccine efficacy, we assessed its immunogenicity in mice. Immunization with CTA1-MECA elicited high levels of Chlamydia-specific antibodies in mucosal and systemic compartments.

The COVID-19 pandemic in sub-Saharan Africa: The significance of presumed immune sufficiency.

A novel coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in China in 2019 and later ignited a global pandemic. Contrary to expectations, the effect of the pandemic was not as devastating to Africa and its young population compared to the rest of the world. To provide insight into the possible reasons for the presumed immune sufficiency to coronavirus disease 2019 (COVID-19) in Africa, this review critically examines literature published from 2020 onwards on the dynamics of COVID-19 infection and immunity and how other prevalent infectious diseases in Africa might have influenced the outcome of COVID-19. Studies characterising the immune response in patients with COVID-19 show that the correlates of protection in infected individuals are T-cell responses against the SARS-CoV-2 spike protein and neutralising titres of immunoglobin G and immunoglobin A antibodies. In some other studies, substantial pre-existing T-cell reactivity to SARS-CoV-2 was detected in many people from diverse geographical locations without a history of exposure. Certain studies also suggest that innate immune memory, which offers protection against reinfection with the same or another pathogen, might influence the severity of COVID-19. In addition, an initial analysis of epidemiological data showed that COVID­19 cases were not severe in some countries that implemented universal Bacillus Calmette-Guerin (BCG) vaccination policies, thus supporting the potential of BCG vaccination to boost innate immunity. The high burden of infectious diseases and the extensive vaccination campaigns previously conducted in Africa could have induced specific and non-specific protective immunity to infectious pathogens in Africans.

Genital tract microbiome dynamics are associated with time of Chlamydia infection in mice.

We have previously shown that the time of Chlamydia infection was crucial in determining the chlamydial infectivity and pathogenesis. This study aims to determine whether the time of Chlamydia infection affects the genital tract microbiome. This study analyzed mice vaginal, uterine, and ovary/oviduct microbiome with and without Chlamydia infection. The mice were infected with Chlamydia at either 10:00 am (ZT3) or 10:00 pm (ZT15). The results showed that mice infected at ZT3 had higher Chlamydia infectivity than those infected at ZT15. There was more variation in the compositional complexity of the vaginal microbiome (alpha diversity) of mice infected at ZT3 than those mice infected at ZT15 throughout the infection within each treatment group, with both Shannon and Simpson diversity index values decreased over time. The analysis of samples collected four weeks post-infection showed that there were significant taxonomical differences (beta diversity) between different parts of the genital tract-vagina, uterus, and ovary/oviduct-and this difference was associated with the time of infection. Firmicutes and Proteobacteria were the most abundant phyla within the microbiome in all three genital tract regions for all the samples collected during this experiment. Additionally, Firmicutes was the dominant phylum in the uterine microbiome of ZT3 Chlamydia infected mice. The results show that the time of infection is associated with the microbial dynamics in the genital tract. And this association is more robust in the upper genital tract than in the vagina. This result implies that more emphasis should be placed on understanding the changes in the microbial dynamics of the upper genital tract over the course of infection.

A novel cold-chain free VCG-based subunit vaccine protects against Chlamydia abortus-induced neonatal mortality in a pregnant mouse model.

Chlamydia abortus (Cab) causes spontaneous abortion and neonatal mortality in infected ruminants and pregnant women. Most Cab infections are asymptomatic, although they can be treated with antibiotics, signifying that control of these infections may require alternative strategies, including the use of effective vaccines. However, the limitations imposed by live attenuated and inactivated vaccines further suggest that employment of subunit vaccines may need to be considered. The efficacy of a newly generated Vibrio cholerae ghost (rVCG)-based subunit vaccine harboring the N-terminal portion of the Cab Pmp18D protein (rVCG-Pmp18.3) in preventing Cab-induced abortion or neonatal mortality was evaluated in pregnant mice. Mice were intranasally (IN) immunized and boosted twice, 2 weeks apart with the vaccine, and immunized and unimmunized mice were caged with males 4 weeks postimmunization. The mice were then infected either IN or transcervically (TC) 10 days after pregnancy, and the fertility rate was determined 7 days postpartum. Eight days after delivery, the mice were sacrificed, and Cab infectivity in the lungs and spleens was evaluated by culturing tissue homogenates in tissue culture. Our results demonstrated that the vaccine induced immune effectors that mediated complete clearance of infection in the lungs and significantly reduced Cab infectivity in the spleen following IN immunization. Vaccine immunization also afforded protection against Cab-induced upper genital tract pathology (uterine dilation). Furthermore, while there was no incidence of abortion in both immunized and unimmunized mice, immunized mice were completely protected against neonatal mortality compared to unimmunized infected controls, which lost a significant percentage of their litter 7 days postpartum. Our results establish the capability of the rVCG-Pmp18.3 vaccine to prevent infection in the lungs (mucosal) and spleen (systemic) and protect mice from Cab-induced tubal pathologies and neonatal mortality, a hallmark of Cab infection in ruminants. To advance the commercial potential of this vaccine, future studies will optimize the antigen dose and the number of vaccine doses required for protection of ruminants.

Temporal trends of sulphadoxine-pyrimethamine (SP) drug-resistance molecular markers in Plasmodium falciparum parasites from pregnant women in western Kenya.

BACKGROUND: Resistance to sulphadoxine-pyrimethamine (SP) in Plasmodium falciparum parasites is associated with mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes and has spread worldwide. SP remains the recommended drug for intermittent preventive treatment for malaria in pregnancy (IPTp) and information on population prevalence of the SP resistance molecular markers in pregnant women is limited. METHODS: Temporal trends of SP resistance molecular markers were investigated in 489 parasite samples collected from pregnant women at delivery from three different observational studies between 1996 and 2009 in Kenya, where SP was adopted for both IPTp and case treatment policies in 1998. Using real-time polymerase chain reaction, pyrosequencing and direct sequencing, 10 single-nucleotide polymorphisms (SNPs) of SP resistance molecular markers were assayed. RESULTS: The prevalence of quintuple mutant (dhfr N51I/C59R/S108N and dhps A437G/K540E combined genotype) increased from 7% in the first study (1996-2000) to 88% in the third study (2008-2009). When further stratified by sample collection year and adoption of IPTp policy, the prevalence of the quintuple mutant increased from 2.4% in 1998 to 44.4% three years after IPTp policy adoption, seemingly in parallel with the increase in percentage of SP use in pregnancy. However, in the 1996-2000 study, more mutations in the combined dhfr/dhps genotype were associated with SP use during pregnancy only in univariable analysis and no associations were detected in the 2002-2008 and 2008-2009 studies. In addition, in the 2008-2009 study, 5.3% of the parasite samples carried the dhps triple mutant (A437G/K540E/A581G). There were no differences in the prevalence of SP mutant genotypes between the parasite samples from HIV + and HIV- women over time and between paired peripheral and placental samples. CONCLUSIONS: There was a significant increase in dhfr/dhps quintuple mutant and the emergence of new genotype containing dhps 581 in the parasites from pregnant women in western Kenya over 13 years. IPTp adoption and SP use in pregnancy only played a minor role in the increased drug-resistant parasites in the pregnant women over time. Most likely, other major factors, such as the high prevalence of resistant parasites selected by the use of SP for case management in large non-pregnant population, might have contributed to the temporally increased prevalence of SP resistant parasites in pregnant women. Further investigations are needed to determine the linkage between SP drug resistance markers and efficacy of IPTp-SP.

Cellular Basis for the Enhanced Efficacy of the Fms-Like Tyrosine Kinase 3 Ligand (FL) Adjuvanted VCG-Based Chlamydia abortus Vaccine.

Efficacious vaccines are needed to control genital chlamydial diseases in humans and the veterinary industry. We previously reported a C. abortus (Cab) vaccine comprising recombinant Vibrio cholerae ghosts (rVCG) expressing the conserved and immunogenic N-terminal region of the Cab polymorphic membrane protein D (rVCG-Pmp18.1) protein that protected mice against intravaginal challenge. In this study, we investigated the immunomodulatory effect of the hematopoietic progenitor activator cytokine, Fms-like tyrosine kinase 3-ligand (FL) when co-administered with the rVCG-Pmp18.1 vaccine as a strategy to enhance the protective efficacy and the potential mechanism of immunomodulation. Groups of female C57BL/6J mice were immunized and boosted twice intranasally (IN) with rVCG-PmpD18.1 with and without FL or purified rPmp18.1 or rVCG-gD2 (antigen control) or PBS (medium) per mouse. The results revealed that co-administration of the vaccine with FL enhanced antigen-specific cellular and humoral immune responses and protected against live Cab genital infection. Comparative analysis of immune cell phenotypes infiltrating mucosal and systemic immune inductive tissue sites following immunization revealed that co-administration of rVCG-Pmp18.1 with FL significantly enhanced the number of macrophages, dendritic and NK cells, γδ and NK T cells in the spleen (systemic) and iliac lymph nodes (ILN) draining the genital tract (mucosal) tissues compared to rVCG-Pmp18.1 alone. Furthermore, FL enhanced monocyte infiltration in the ILN, while CD19+ B cells and CD4+ T cells were enhanced in the spleen. These results indicate that the immunomodulatory effect of FL is associated with its ability to mobilize innate immune cells and subsequent activation of robust antigen-specific immune effectors in mucosal and systemic lymphoid tissues.

MiR-378b Modulates Chlamydia-Induced Upper Genital Tract Pathology.

Genital Chlamydia trachomatis infection causes severe reproductive pathologies such as salpingitis and pelvic inflammatory disease that can lead to tubal factor infertility. MicroRNAs (miRNAs) are evolutionarily conserved regulators of mammalian gene expression in development, immunity and pathophysiologic processes during inflammation and infection, including Chlamydia infection. Among the miRNAs involved in regulating host responses and pathologic outcome of Chlamydia infection, we have shown that miR-378b was significantly differentially expressed during primary infection and reinfection. In this study, we tested the hypothesis that miR-378b is involved in the pathological outcome of Chlamydia infection. We developed miR-378b knockout mice (miR-378b-/-) using Crispr/Cas and infected them along with their wild-type (WT) control with Chlamydia to compare the infectivity and reproductive pathologies. The results showed that miR-378b-/- mice were unable to clear the infection compared to WT mice; also, miR-378b-/- mice exhibited a relatively higher Chlamydia burden throughout the duration of infection. However, gross pathology results showed that miR-378b-/- mice had significantly reduced uterine dilatations and pathologic lesions after two infections compared to WT mice. In addition, the pregnancy and fertility rates for infected miR-378b-/- mice showed protection from Chlamydia-induced infertility with fertility rate that was comparable to uninfected WT mice. These results are intriguing as they suggest that miR-378b is important in regulating host immune responses that control Chlamydial replication and drive the inflammation that causes complications such as infertility. The finding has important implications for biomarkers of Chlamydial complications and targets for prevention of disease.

Differential miRNA Profiles Correlate With Disparate Immunity Outcomes Associated With Vaccine Immunization and Chlamydial Infection.

Vaccine-induced immune responses following immunization with promising Chlamydia vaccines protected experimental animals from Chlamydia-induced upper genital tract pathologies and infertility. In contrast, primary genital infection with live Chlamydia does not protect against these pathologies. We hypothesized that differential miRNA profiles induced in the upper genital tracts (UGT) of mice correlate with the disparate immunity vs. pathologic outcomes associated with vaccine immunization and chlamydial infection. Thus, miRNA expression profiles in the UGT of mice after Chlamydia infection (Live EB) and immunization with dendritic cell (DC)-based vaccine (DC vaccine) or VCG-based vaccine (VCG vaccine) were compared using the NanoString nCounter Mouse miRNA assay. Of the 602 miRNAs differentially expressed (DE) in the UGT of immunized and infected mice, we selected 58 with counts >100 and p-values < 0.05 for further analysis. Interestingly, vaccine immunization and Chlamydia infection induced the expression of distinct miRNA profiles with a higher proportion in vaccine-immunized compared to Chlamydia infected mice; DC vaccine (41), VCG vaccine (23), and Live EB (15). Hierarchical clustering analysis showed notable differences in the uniquely DE miRNAs for each experimental group, with DC vaccine showing the highest number (21 up-regulated, five down-regulated), VCG vaccine (two up-regulated, five down-regulated), and live EB (two up-regulated, four down-regulated). The DC vaccine-immunized group showed the highest number (21 up-regulated and five down-regulated compared to two up-regulated and four down-regulated in the live Chlamydia infected group). Pathway analysis showed that the DE miRNAs target genes that regulate several biological processes and functions associated with immune response and inflammation. These results suggest that the induction of differential miRNA expression plays a significant role in the disparate immunity outcomes associated with Chlamydia infection and vaccination.

Shift work influences the outcomes of Chlamydia infection and pathogenesis.

Shift work, performed by approximately 21 million Americans, is irregular or unusual work schedule hours occurring after 6:00 pm. Shift work has been shown to disrupt circadian rhythms and is associated with several adverse health outcomes and chronic diseases such as cancer, gastrointestinal and psychiatric diseases and disorders. It is unclear if shift work influences the complications associated with certain infectious agents, such as pelvic inflammatory disease, ectopic pregnancy and tubal factor infertility resulting from genital chlamydial infection. We used an Environmental circadian disruption (ECD) model mimicking circadian disruption occurring during shift work, where mice had a 6-h advance in the normal light/dark cycle (LD) every week for a month. Control group mice were housed under normal 12/12 LD cycle. Our hypothesis was that compared to controls, mice that had their circadian rhythms disrupted in this ECD model will have a higher Chlamydia load, more pathology and decreased fertility rate following Chlamydia infection. Results showed that, compared to controls, mice that had their circadian rhythms disrupted (ECD) had higher Chlamydia loads, more tissue alterations or lesions, and lower fertility rate associated with chlamydial infection. Also, infected ECD mice elicited higher proinflammatory cytokines compared to mice under normal 12/12 LD cycle. These results imply that there might be an association between shift work and the increased likelihood of developing more severe disease from Chlamydia infection.

Cellular responses to modified Plasmodium falciparum MSP119 antigens in individuals previously exposed to natural malaria infection.

BACKGROUND: MSP1 processing-inhibitory antibodies bind to epitopes on the 19 kDa C-terminal region of the Plasmodium falciparum merozoite surface protein 1 (MSP1(19)), inhibiting erythrocyte invasion. Blocking antibodies also bind to this antigen but prevent inhibitory antibodies binding, allowing invasion to proceed. Recombinant MSP1(19) had been modified previously to allow inhibitory but not blocking antibodies to continue to bind. Immunization with these modified proteins, therefore, has the potential to induce more effective protective antibodies. However, it was unclear whether the modification of MSP1(19) would affect critical T-cell responses to epitopes in this antigen. METHODS: The cellular responses to wild-type MSP1(19) and a panel of modified MSP1(19) antigens were measured using an in-vitro assay for two groups of individuals: the first were malaria-naïve and the second had been naturally exposed to Plasmodium falciparum infection. The cellular responses to the modified proteins were examined using cells from malaria-exposed infants and adults. RESULTS: Interestingly, stimulation indices (SI) for responses induced by some of the modified proteins were at least two-fold higher than those elicited by the wild-type MSP1(19). A protein with four amino acid substitutions (Glu27-->Tyr, Leu31-->Arg, Tyr34-->Ser and Glu43-->Leu) had the highest stimulation index (SI up to 360) and induced large responses in 64% of the samples that had significant cellular responses to the modified proteins. CONCLUSION: This study suggests that specific MSP1(19) variants that have been engineered to improve their antigenicity for inhibitory antibodies, retain T-cell epitopes and the ability to induce cellular responses. These proteins are candidates for the development of MSP1-based malaria vaccines.