Rare Variable M. tuberculosis Antigens induce predominant Th17 responses in human infection.

CD4 T cells are essential for immunity to M. tuberculosis (Mtb), and emerging evidence indicates that IL-17-producing Th17 cells contribute to immunity to Mtb. While identifying protective T cell effector functions is important for TB vaccine design, T cell antigen specificity is also likely to be important. To identify antigens that induce protective immunity, we reasoned that as in other pathogens, effective immune recognition drives sequence diversity in individual Mtb antigens. We previously identified Mtb genes under evolutionary diversifying selection pressure whose products we term Rare Variable Mtb Antigens (RVMA). Here, in two distinct human cohorts with recent exposure to TB, we found that RVMA preferentially induce CD4 T cells that express RoRγt and produce IL-17, in contrast to 'classical' Mtb antigens that induce T cells that produce IFNγ. Our results suggest that RVMA can be valuable antigens in vaccines for those already infected with Mtb to amplify existing antigen-specific Th17 responses to prevent TB disease.

Rifampin-resistant Tuberculosis in the United States, 1998-2014.

BACKGROUND: Monoresistance to rifamycins necessitates longer and more toxic regimens for tuberculosis (TB). We examined characteristics and mortality associated with rifampin-monoresistant (RMR) TB in the United States. METHODS: We analyzed Mycobacterium tuberculosis culture-positive cases reported to the National TB Surveillance System (excluding California) between 1998 and 2014. We defined RMR TB found on initial drug susceptibility testing and possible acquired rifampin-resistant (ARR) TB. We assessed temporal trends in RMR TB. For both classifications of rifampin resistance, we calculated adjusted risk ratios (adjRRs) and 95% confidence intervals (CIs) for characteristics associated with mortality when compared with drug-susceptible TB in multivariable models using backward selection. RESULTS: Of 180 329 TB cases, 126 431 (70%) were eligible for analysis, with 359 (0.28%) of eligible cases reported as RMR. The percentage of RMR TB cases with HIV declined 4% annually between 1998 and 2014. Persons with HIV and prior TB were more likely to have RMR TB (adjRR, 25.9; 95% CI, 17.6-38.1), as were persons with HIV and no prior TB (adjRR, 3.1; 95% CI, 2.4-4.1) vs those without either characteristic, controlling for other statistically significant variables. RMR cases had greater mortality (adjRR, 1.4; 95% CI, 1.04-1.8), controlling for HIV and other variables. Persons with HIV had greater risk of ARR than persons without HIV (adjRR, 9.6; 95% CI, 6.9-13.3), and ARR was also associated with increased mortality, controlling for HIV and other variables. CONCLUSIONS: All forms of rifampin resistance were positively associated with HIV infection and increased mortality.

The Impact of a Fogarty International Center-Supported Tuberculosis Research Training Program in the Country of Georgia.

In 2004, there existed limited tuberculosis (TB) research capacity in the country of Georgia. In response, a collaborative research training program (RTP) supported by a National Institutes of Health Fogarty International Center Global Infectious Diseases grant was formed between a U.S. academic institution and the National Center for Tuberculosis and Lung Disease (NCTLD) and other institutions in Georgia. We sought to assess outcomes of this RTP. The TB RTP combined didactic and mentored research training for Georgian trainees. Long-term trainees were supported for a 2-year period and with posttrainee career development mentoring. Metrics used to measure program performance included publications, grants received, and career advancement. From 2004 to 2015, 20 trainees participated in the program with 15 (75%) authoring a total of 65 publications in PubMed-listed journals. The median number of publications per trainee was six (interquartile range 2-14). A total of 16 (80%) trainees remain working in the area of TB; nine were promoted to leadership positions and three to lead research units at Georgian institutions. Ten (50%) trainees were the principal investigator (PI) of a peer-reviewed external grant after Fogarty-supported training, and 40% served as research mentors. Annual TB-related research funding at the NCTLD increased from $5,000 in 2005 to ∼$1.5 million in 2017. A Georgian Fogarty trainee was either PI, site PI, or coinvestigator on > 90% of all research funding. We believe that the NIH Fogarty-funded TB research training grant has made critical contributions to increasing the TB-related research infrastructure and capacity in Georgia, particularly at the NCTLD.

Knowledge, attitudes, and practices regarding cervical cancer and screening among Ethiopian health care workers.

BACKGROUND: Though cervical cancer incidence has dramatically decreased in resource rich regions due to the implementation of universal screening programs, it remains one of the most common cancers affecting women worldwide and has one of the highest mortality rates. The vast majority of cervical cancer-related deaths are among women that have never been screened. Prior to implementation of a screening program in Addis Ababa University-affiliated hospitals in Ethiopia, a survey was conducted to assess knowledge of cervical cancer etiology, risk factors, and screening, as well as attitudes and practices regarding cervical cancer screening among women's health care providers. METHODS: Between February and March 2012 an anonymous, self-administered survey to assess knowledge, attitudes, and practices related to cervical cancer and its prevention was distributed to 334 health care providers at three government hospitals in Addis Ababa, Ethiopia and three Family Guidance Association clinics in Awassa, Adama, and Bahir Dar. Data were analyzed using SPSS software and chi-square test was used to test differences in knowledge, attitudes, and practices across provider type. RESULTS: Overall knowledge surrounding cervical cancer was high, although awareness of etiology and risk factors was low among nurses and midwives. Providers had no experience performing cervical cancer screening on a routine basis with <40% having performed any type of cervical cancer screening. Reported barriers to performing screening were lack of training (52%) and resources (53%); however the majority (97%) of providers indicated cervical cancer screening is an essential part of women's health care. CONCLUSION: There is a clear need among women's health care providers for education regarding cervical cancer etiology, risk factors and for training in low-tech, low-cost screening methods. Meeting these needs and improving the infrastructure necessary to implement appropriate screening programs is essential to reduce the burden of cervical cancer in Ethiopia.

Selective and potent urea inhibitors of cryptosporidium parvum inosine 5'-monophosphate dehydrogenase.

Cryptosporidium parvum and related species are zoonotic intracellular parasites of the intestine. Cryptosporidium is a leading cause of diarrhea in small children around the world. Infection can cause severe pathology in children and immunocompromised patients. This waterborne parasite is resistant to common methods of water treatment and therefore a prominent threat to drinking and recreation water even in countries with strong water safety systems. The drugs currently used to combat these organisms are ineffective. Genomic analysis revealed that the parasite relies solely on inosine-5'-monophosphate dehydrogenase (IMPDH) for the biosynthesis of guanine nucleotides. Herein, we report a selective urea-based inhibitor of C. parvum IMPDH (CpIMPDH) identified by high-throughput screening. We performed a SAR study of these inhibitors with some analogues exhibiting high potency (IC(50) < 2 nM) against CpIMPDH, excellent selectivity >1000-fold versus human IMPDH type 2 and good stability in mouse liver microsomes. A subset of inhibitors also displayed potent antiparasitic activity in a Toxoplasma gondii model.

Structure-activity relationship study of selective benzimidazole-based inhibitors of Cryptosporidium parvum IMPDH.

Cryptosporidium parasites are important waterborne pathogens of both humans and animals. The Cryptosporidium parvum and Cryptosporidium hominis genomes indicate that the only route to guanine nucleotides is via inosine 5'-monophosphate dehydrogenase (IMPDH). Thus the inhibition of the parasite IMPDH presents a potential strategy for treating Cryptosporidium infections. A selective benzimidazole-based inhibitor of C. parvum IMPDH (CpIMPDH) was previously identified in a high throughput screen. Here we report a structure-activity relationship study of benzimidazole-based compounds that resulted in potent and selective inhibitors of CpIMPDH. Several compounds display potent antiparasitic activity in vitro.

A screening pipeline for antiparasitic agents targeting cryptosporidium inosine monophosphate dehydrogenase.

BACKGROUND: The protozoan parasite Cryptosporidium parvum is responsible for significant disease burden among children in developing countries. In addition Cryptosporidiosis can result in chronic and life-threatening enteritis in AIDS patients, and the currently available drugs lack efficacy in treating these severe conditions. The discovery and development of novel anti-cryptosporidial therapeutics has been hampered by the poor experimental tractability of this pathogen. While the genome sequencing effort has identified several intriguing new targets including a unique inosine monophosphate dehydrogenase (IMPDH), pursuing these targets and testing inhibitors has been frustratingly difficult. METHODOLOGY AND PRINCIPAL FINDINGS: Here we have developed a pipeline of tools to accelerate the in vivo screening of inhibitors of C. parvum IMPDH. We have genetically engineered the related parasite Toxoplasma gondii to serve as a model of C. parvum infection as the first screen. This assay provides crucial target validation and a large signal window that is currently not possible in assays involving C. parvum. To further develop compounds that pass this first filter, we established a fluorescence-based assay of host cell proliferation, and a C. parvum growth assay that utilizes automated high-content imaging analysis for enhanced throughput. CONCLUSIONS AND SIGNIFICANCE: We have used these assays to evaluate C. parvum IMPDH inhibitors emerging from our ongoing medicinal chemistry effort and have identified a subset of 1,2,3-triazole ethers that exhibit excellent in vivo selectivity in the T. gondii model and improved anti-cryptosporidial activity.

Prodrug activation by Cryptosporidium thymidine kinase.

Cryptosporidium spp. cause acute gastrointestinal disease that can be fatal for immunocompromised individuals. These protozoan parasites are resistant to conventional antiparasitic chemotherapies and the currently available drugs to treat these infections are largely ineffective. Genomic studies suggest that, unlike other protozoan parasites, Cryptosporidium is incapable of de novo pyrimidine biosynthesis. Curiously, these parasites possess redundant pathways to produce dTMP, one involving thymidine kinase (TK) and the second via thymidylate synthase-dihydrofolate reductase. Here we report the expression and characterization of TK from C. parvum. Unlike other TKs, CpTK is a stable trimer in the presence and absence of substrates and the activator dCTP. Whereas the values of k(cat) = 0.28 s(-1) and K(m)(,ATP) = 140 microm are similar to those of human TK1, the value of K(m)(thymidine) = 48 microm is 100-fold greater, reflecting the abundance of thymidine in the gastrointestinal tract. Surprisingly, the antiparasitic nucleosides AraT, AraC, and IDC are not substrates for CpTK, indicating that Cryptosporidium possesses another deoxynucleoside kinase. Trifluoromethyl thymidine and 5-fluorodeoxyuridine are good substrates for CpTK, and both compounds inhibit parasite growth in an in vitro model of C. parvum infection. Trifluorothymidine is also effective in a mouse model of acute disease. These observations suggest that CpTK-activated pro-drugs may be an effective strategy for treating cryptosporidiosis.

Targeting a prokaryotic protein in a eukaryotic pathogen: identification of lead compounds against cryptosporidiosis.

Cryptosporidium parvum is an important human pathogen and potential bioterrorism agent. No vaccines exist against C. parvum, the drugs currently approved to treat cryptosporidiosis are ineffective, and drug discovery is challenging because the parasite cannot be maintained continuously in cell culture. Mining the sequence of the C. parvum genome has revealed that the only route to guanine nucleotides is via inosine-5'-monophosphate dehydrogenase (IMPDH). Moreover, phylogenetic analysis suggests that the IMPDH gene was obtained from bacteria by lateral gene transfer. Here we exploit the unexpected evolutionary divergence of parasite and host enzymes by designing a high-throughput screen to target the most diverged portion of the IMPDH active site. We have identified four parasite-selective IMPDH inhibitors that display antiparasitic activity with greater potency than paromomycin, the current gold standard for anticryptosporidial activity.

Rapid and specific biotin labelling of the erythrocyte surface antigens of both cultured and ex-vivo Plasmodium parasites.

BACKGROUND: Sensitive detection of parasite surface antigens expressed on erythrocyte membranes is necessary to further analyse the molecular pathology of malaria. This study describes a modified biotin labelling/osmotic lysis method which rapidly produces membrane extracts enriched for labelled surface antigens and also improves the efficiency of antigen recovery compared with traditional detergent extraction and surface radio-iodination. The method can also be used with ex-vivo parasites. METHODS: After surface labelling with biotin in the presence of the inhibitor furosemide, detergent extraction and osmotic lysis methods of enriching for the membrane fractions were compared to determine the efficiency of purification and recovery. Biotin-labelled proteins were identified on silver-stained SDS-polyacrylamide gels. RESULTS: Detergent extraction and osmotic lysis were compared for their capacity to purify biotin-labelled Plasmodium falciparum and Plasmodium chabaudi erythrocyte surface antigens. The pellet fraction formed after osmotic lysis of P. falciparum-infected erythrocytes is notably enriched in surface antigens, including PfEMP1, when compared to detergent extraction. There is also reduced co-extraction of host proteins such as spectrin and Band 3. CONCLUSION: Biotinylation and osmotic lysis provides an improved method to label and purify parasitised erythrocyte surface antigen extracts from both in vitro and ex vivo Plasmodium parasite preparations.

Plasmodium cysteine repeat modular proteins 1-4: complex proteins with roles throughout the malaria parasite life cycle.

The Cysteine Repeat Modular Proteins (PCRMP1-4) of Plasmodium, are encoded by a small gene family that is conserved in malaria and other Apicomplexan parasites. They are very large, predicted surface proteins with multipass transmembrane domains containing motifs that are conserved within families of cysteine-rich, predicted surface proteins in a range of unicellular eukaryotes, and a unique combination of protein-binding motifs, including a >100 kDa cysteine-rich modular region, an epidermal growth factor-like domain and a Kringle domain. PCRMP1 and 2 are expressed in life cycle stages in both the mosquito and vertebrate. They colocalize with PfEMP1 (P. falciparum Erythrocyte Membrane Antigen-1) during its export from P. falciparum blood-stage parasites and are exposed on the surface of haemolymph- and salivary gland-sporozoites in the mosquito, consistent with a role in host tissue targeting and invasion. Gene disruption of pcrmp1 and 2 in the rodent malaria model, P. berghei, demonstrated that both are essential for transmission of the parasite from the mosquito to the mouse and has established their discrete and important roles in sporozoite targeting to the mosquito salivary gland. The unprecedented expression pattern and structural features of the PCRMPs thus suggest a variety of roles mediating host-parasite interactions throughout the parasite life cycle.

Antibodies from malaria-exposed pregnant women recognize trypsin resistant epitopes on the surface of Plasmodium falciparum-infected erythrocytes selected for adhesion to chondroitin sulphate A.

BACKGROUND: The ability of Plasmodium falciparum-infected erythrocytes to adhere to the microvasculature endothelium is thought to play a causal role in malaria pathogenesis. Cytoadhesion to endothelial receptors is generally found to be highly sensitive to trypsinization of the infected erythrocyte surface. However, several studies have found that parasite adhesion to placental receptors can be markedly less sensitive to trypsin. This study investigates whether chondroitin sulphate A (CSA) binding parasites express trypsin-resistant variant surface antigens (VSA) that bind female-specific antibodies induced as a result of pregnancy associated malaria (PAM). METHODS: Fluorescence activated cell sorting (FACS) was used to measure the levels of adult Scottish and Ghanaian male, and Ghanaian pregnant female plasma immunoglobulin G (IgG) that bind to the surface of infected erythrocytes. P. falciparum clone FCR3 cultures were used to assay surface IgG binding before and after selection of the parasite for adhesion to CSA. The effect of proteolytic digestion of parasite erythrocyte surface antigens on surface IgG binding and adhesion to CSA and hyaluronic acid (HA) was also studied. RESULTS: P. falciparum infected erythrocytes selected for adhesion to CSA were found to express trypsin-resistant VSA that are the target of naturally acquired antibodies from pregnant women living in a malaria endemic region of Ghana. However in vitro adhesion to CSA and HA was relatively trypsin sensitive. An improved labelling technique for the detection of VSA expressed by CSA binding isolates has also been described. CONCLUSION: The VSA expressed by CSA binding P. falciparum isolates are currently considered potential targets for a vaccine against PAM. This study identifies discordance between the trypsin sensitivity of CSA binding and surface recognition of CSA selected parasites by serum IgG from malaria exposed pregnant women. Thus, the complete molecular definition of an antigenic P. falciparum erythrocyte surface protein that can be used as a malaria in pregnancy vaccine has not yet been achieved.

High throughput immuno-screening of cDNA expression libraries produced by in vitro recombination; exploring the Plasmodium falciparum proteome.

Improved Plasmodium falciparum cDNA expression libraries were constructed by combining mRNA oligo-capping with in vitro recombination and directional cloning of cDNA inserts into a plasmid vector that expresses sequences as thioredoxin fusion proteins. A novel procedure has also been developed for the rapid identification of seropositive clones on high-density filters, using direct labelling of P. falciparum immune immunoglobulin with fluorescein isothiocynate (FITC). This approach combines the advantages of recombination-assisted cDNA cloning with high throughput, non-radioactive serological screening of expression libraries. Production of replicate colony matrices allows the identification of antigens recognised by different pools with different specificities from residents of a malaria endemic region. Analyses of DNA sequences derived from sero-reactive colonies indicate that this is an effective method for producing recombinant proteins that react with antibodies from malaria-exposed individuals. This approach permits the systematic construction of a database of antigenic proteins recognised by sera from malaria-exposed individuals.