Loss of Function Variants in the XPC Causes Severe Xeroderma Pigmentosum in Three Large Consanguineous Families.

BACKGROUND: Xeroderma pigmentosum (XP) is a rare recessively inherited disorder that presents clinical and genetic heterogeneity. Mutations in eight genes, of which seven are involved in nucleotide excision repair (NER) pathway have been reported to cause the XP. METHODS AND RESULTS: Three large consanguineous families of Pakistani origin displaying typical clinical hallmarks of XP were evaluated at clinical and molecular level. Homozygosity mapping using microsatellite markers established linkage of the families to XPC gene on chromosome 3p25.1. Sanger sequencing of the XPC gene identified a novel homozygous single bp deletion [NM_004628.5; c.1934del; p.(Pro645Leufs*5)] and two previously reported mutations that included a nonsense [c.1243 C>T; p.(Arg415*)] and a splice acceptor site (c.2251-1 G>C), all segregating with the disease phenotypes in the families. CONCLUSION: This report has extended the spectrum of mutations in the XPC gene and will also facilitate in diagnosis of XP and counselling of families inheriting it, which is the only inevitable tool for preventing the disease occurrence in future generations.

Fifteen Years of Sm-p80-Based Vaccine Trials in Nonhuman Primates: Antibodies From Vaccinated Baboons Confer Protection in vivo and in vitro From Schistosoma mansoni and Identification of Putative Correlative Markers of Protection.

Recent advances in systems biology have shifted vaccine development from a largely trial-and-error approach to an approach that promote rational design through the search for immune signatures and predictive correlates of protection. These advances will doubtlessly accelerate the development of a vaccine for schistosomiasis, a neglected tropical disease that currently affects over 250 million people. For over 15 years and with contributions of over 120 people, we have endeavored to test and optimize Sm-p80-based vaccines in the non-human primate model of schistosomiasis. Using RNA-sequencing on eight different Sm-p80-based vaccine strategies, we sought to elucidate immune signatures correlated with experimental protective efficacy. Furthermore, we aimed to explore the role of antibodies through in vivo passive transfer of IgG obtained from immunized baboons and in vitro killing of schistosomula using Sm-p80-specific antibodies. We report that passive transfer of IgG from Sm-p80-immunized baboons led to significant worm burden reduction, egg reduction in liver, and reduced egg hatching percentages from tissues in mice compared to controls. In addition, we observed that sera from Sm-p80-immunized baboons were able to kill a significant percent of schistosomula and that this effect was complement-dependent. While we did not find a universal signature of immunity, the large datasets generated by this study will serve as a substantial resource for further efforts to develop vaccine or therapeutics for schistosomiasis.

Chronic whipworm infection exacerbates Schistosoma mansoni egg-induced hepatopathology in non-human primates.

BACKGROUND: Schistosomiasis continues to inflict significant morbidity and mortality in the tropical and subtropical regions of the world. The disease endemicity overlaps with the transmission of other parasitic diseases. Despite the ubiquity of polyparasitism in tropical regions, particularly in rural communities, little is known about the impact of multiple helminth infections on disease progression. In this pilot study, we describe the influence of chronic Trichuris trichiura infection on Schistosoma mansoni egg-induced hepatopathology in infected baboons. METHODS: Baboons with or without underlying whipworm infection were challenged with S. mansoni cercariae to establish schistosomiasis. Adult S. mansoni worms were recovered by perfusion and enumerated, hepatic granulomas were quantified via light microscopy, and transcriptional profiling of tissues were completed using RNA sequencing technologies. RESULTS: Co-infection with both S. mansoni and T. trichiura resulted in higher female schistosome worm burden and significantly larger liver granuloma sizes. Systems biology analyses of peripheral blood mononuclear cells (PBMC) revealed pathways associated with increased liver damage in co-infected baboons. CONCLUSIONS: Underlying chronic whipworm infection intensified schistosome egg-induced liver pathology in infected baboons. RNA-Seq analysis provided insight into pathways associated with increased liver damage, corroborating histological findings.

Sm-p80-based schistosomiasis vaccine: double-blind preclinical trial in baboons demonstrates comprehensive prophylactic and parasite transmission-blocking efficacy.

Schistosomiasis is of public health importance to an estimated one billion people in 79 countries. A vaccine is urgently needed. Here, we report the results of four independent, double-blind studies of an Sm-p80-based vaccine in baboons. The vaccine exhibited potent prophylactic efficacy against transmission of Schistosoma mansoni infection and was associated with significantly less egg-induced pathology, compared with unvaccinated control animals. Specifically, the vaccine resulted in a 93.45% reduction of pathology-producing female worms and significantly resolved the major clinical manifestations of hepatic/intestinal schistosomiasis by reducing the tissue egg-load by 89.95%. A 35-fold decrease in fecal egg excretion in vaccinated animals, combined with an 81.51% reduction in hatching of eggs into the snail-infective stage (miracidia), demonstrates the parasite transmission-blocking potential of the vaccine. Substantially higher Sm-p80 expression in female worms and Sm-p80-specific antibodies in vaccinated baboons appear to play an important role in vaccine-mediated protection. Preliminary analyses of RNA sequencing revealed distinct molecular signatures of vaccine-induced effects in baboon immune effector cells. This study provides comprehensive evidence for the effectiveness of an Sm-p80-based vaccine for schistosomiasis.

Schistosoma mansoni antigen Sm-p80: prophylactic efficacy using TLR4 agonist vaccine adjuvant glucopyranosyl lipid A-Alum in murine and non-human primate models.

Sm-p80, the large subunit of Schistosoma mansoni calpain, is a leading candidate for a schistosomiasis vaccine. The prophylactic and antifecundity efficacy of Sm-p80 has been tested in three animal models (mouse, hamster and baboon) using a multitude of vaccine formulations and approaches. In our continual effort to enhance the vaccine efficacy, in this study, we have utilized the adjuvant, synthetic hexa-acylated lipid A derivative, glucopyranosyl lipid A (GLA) formulated in aluminum (GLA-Alum) with recombinant Sm-p80. The rSm-p80+GLA-Alum immunization regimen provided 33.33%-53.13% reduction in worm burden in the mouse model and 38% worm burden reduction in vaccinated baboons. Robust Sm-p80-specific immunoglobulin (Ig)G, IgG1, IgG2a and IgM responses were observed in all immunized animals. The rSm-p80+GLA-Alum coadministration induced a mix of T-helper (Th) cells (Th1, Th2 and Th17) responses as determined via the release of interleukin (IL)-2, IL-4, IL-18, IL-21, IL-22 and interferon-γ.

Sm-p80-based vaccine trial in baboons: efficacy when mimicking natural conditions of chronic disease, praziquantel therapy, immunization, and Schistosoma mansoni re-encounter.

Sm-p80-based vaccine efficacy for Schistosoma mansoni was evaluated in a baboon model of infection and disease. The study was designed to replicate a human vaccine implementation scenario for endemic regions in which vaccine would be administered following drug treatment of infected individuals. In our study, the Sm-p80-based vaccine reduced principal pathology producing hepatic egg burdens by 38.0% and egg load in small and large intestines by 72.2% and 49.4%, respectively, in baboons. Notably, hatching rates of eggs recovered from liver and small and large intestine of vaccinated animals were significantly reduced, by 60.4%, 48.6%, and 82.3%, respectively. Observed reduction in egg maturation/hatching rates was supported by immunofluorescence and confocal microscopy showing unique differences in Sm-p80 expression in worms of both sexes and matured eggs. Vaccinated baboons had a 64.5% reduction in urine schistosome circulating anodic antigen, a parameter that reflects worm numbers/health status in infected hosts. Preliminary analyses of RNA sequencing revealed unique genes and canonical pathways associated with establishment of chronic disease, praziquantel-mediated parasite killing, and Sm-p80-mediated protection in vaccinated baboons. Overall, our study demonstrated efficacy of the Sm-p80 vaccine and provides insight into some of the epistatic interactions associated with protection.

Schistosoma egg-induced liver pathology resolution by Sm-p80-based schistosomiasis vaccine in baboons.

Schistosomiasis remains a serious chronic debilitating hepato-intestinal disease. Current control measures based on mass drug administration are inadequate due to sustained re-infection rates, low treatment coverage and emergence of drug resistance. Hence, there is an urgent need for a schistosomiasis vaccine for disease control. In this study, we assessed the anti-pathology efficacy of Schistosoma mansoni large subunit of calpain (Sm-p80)-based vaccine against schistosomiasis caused by infections with Schistosoma mansoni in baboons. We also evaluated the disease transmission-blocking potential of Sm-p80 vaccine. Immunisations with Sm-p80-based vaccine resulted in significant reduction of hepatic egg load in vaccinated baboons (67.7% reduction, p = 0.0032) when compared to the control animals, indicative of reduction in pathology. There was also a significant reduction in sizes of egg-induced granulomas in baboons immunised with Sm-p80 vaccine compared to their control counterparts. Egg hatching rate analysis revealed an overall 85.6% reduction (p = 0.0018) in vaccinated animals compared to the controls, highlighting the potential role of Sm-p80 vaccine in disease transmission. The findings on anti-pathology efficacy and transmission-blocking potential presented in this study have formed the basis for a large-scale double-blinded baboon experiment that is currently underway.

The self-curing phenomenon of schistosome infection in rhesus macaques: insight from in vitro studies.

A reduction in the burden of schistosomiasis is potentially achievable by integrating a schistosomiasis vaccine with current control measures. Here, we determine parasite-specific in vitro responses of B, T, and NK cells from naive uninfected rhesus macaques to Schistosoma mansoni (Sm) egg (SmEA) and worm antigen (SmWA) preparations isolated from infected baboons. Pronounced B cell responses to SmEA and NK cell responses to both SmEA and SmWA were observed. High levels of IL-2 and IL-21 responses against Sm antigens were observed in T and non-T cells of lymph nodes (LNs) and gut lamina propria-derived lymphocytes (LPLs). Data analysis showed multifunctionality of LN-derived CD4+ , CD8+ , and CD4+ CD8+ double positive T cells against either SmWA or SmWA+SmEA antigen preparations. Distinct SmEA-specific multifunctional responses were observed in gut LPLs, suggesting simultaneous responses against egg antigens. These data provide insight into the immune effectors involved in schistosome responses by rhesus macaques.

Gastrointestinal helminths of Coyotes (Canis latrans) from Southeast Nebraska and Shenandoah area of Iowa.

AIM: This survey was carried out on the carcasses of 29 coyotes from Southeastern Nebraska and Shenandoah area of Iowa to document the helminths present in the intestinal track of these carnivorous animals. MATERIALS AND METHODS: A total of 29 adult coyote carcasses were generously donated in the autumn and winter (November-February) of 2014-2015 by trappers, fur buyers and hunters of Southeast Nebraska and Shenandoah area of Iowa. The intestine of individual animals were examined for the recovery of helminth parasites as per the established procedures. RESULTS: We found that as many as 93.10% of the investigated coyotes were infected with one or more helminth infections. A total of 10 different species of helminth parasites were recovered from the intestines of coyotes under investigation. Among the 10 species of helminths, 5 were identified as cestodes while the remaining 5 were nematodes. A total of 82.75% of the animals were infected with one or more species of nematodes, while 75.86% of them were colonized with one or more species of cestode parasites. The most abundant species in coyotes were Toxascaris leonina (68.95%) closely followed by Taenia hydatigena (58.62%). The prevalence of Ancylostoma caninum and Taenia pisiformis were recorded at 31.03%, followed by those of Toxocara canis and Echinococcus spp. at 24.13%, respectively. Three animals were infected with Trichuris vulpis while three other coyotes each were found to be harboring Uncinaria stenocephala, Dipylidium caninum, or Hymenolepis diminuta. The presence of H. diminuta might have been the result of the ingestion of a rodent by the respective coyotes. CONCLUSION: From the overall analysis of the present data and comparing it with the previous reports of various scientists over several decades, we can conclude that intestinal helminths are still very much prevalent among the coyote population in the Southeast Nebraska and Iowa area. The relatively high prevalence of the zoonotic parasite species further warrants a more comprehensive investigation with larger numbers of wild predators from the region to ascertain the possible contribution of coyotes to the disease cycle as these animals are more frequently spotted in and around the densely populated urban areas.

Longevity of Sm-p80-specific antibody responses following vaccination with Sm-p80 vaccine in mice and baboons and transplacental transfer of Sm-p80-specific antibodies in a baboon.

Based on data obtained using vaccine efficacy studies in mice, hamsters, and baboons, the credentials of Sm-p80 as a first tier vaccine candidate for schistosomiasis have been well established. Sm-p80-based vaccine formulation(s) have consistently exhibited potent prophylactic efficacy in reducing adult worm burden following cercarial challenge and induce killing of established adult worms in chronic infection. This vaccine is protective against both intestinal and urinary schistosomiasis. In this study, the longevity of Sm-p80-specific antibody responses was studied in mice and in baboons. Robust antibody titers were detected in mice for up to 60 weeks following vaccination with Sm-p80 recombinant vaccine (Sm-p80 + GLA-SE). In the follow-up experiments to our published studies, Sm-p80-specific IgG was also detected in baboons 5-8 years following the initial vaccination with an Sm-p80 DNA vaccine. In one baboon, transfer of Sm-p80-specific antibody was detected in umbilical cord blood and in the baby. These long-lasting humoral immune response data coupled with the vaccine efficacy data in rodents and nonhuman primates further strengthens the case for Sm-p80 to be moved forward through development leading to human clinical trials.

Cross-species protection: Schistosoma mansoni Sm-p80 vaccine confers protection against Schistosoma haematobium in hamsters and baboons.

The ability of the Schistosoma mansoni antigen, Sm-p80, to provide cross-species protection against Schistosoma haematobium challenge was evaluated in hamster and baboon models. Pronounced reduction in worm burden (48%) and in tissue egg load (64%) was observed in hamsters vaccinated with recombinant Sm-p80 admixed with glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE). Similarly, in baboons, the Sm-p80/GLA-SE vaccine produced a 25% reduction in S. haematobium adult worms and decreased the egg load in the urinary bladder by 64%. A 40% and 53% reduction in fecal and urine egg output, respectively, was observed in vaccinated baboons. A balanced pro-inflammatory (Th17 and Th1) and Th2 type of response was generated after vaccination and appears indicative of augmented prophylactic efficacy. These data on cross-species protection coupled with the prophylactic, therapeutic and antifecundity efficacy against the homologous parasite, S. mansoni, reinforces Sm-p80 as a promising vaccine candidate. It is currently being prepared for GMP-compliant manufacture and for further pre-clinical development leading to human clinical trials. These results solidify the expectation that the Sm-p80 vaccine will provide relief for both the intestinal and the urinary schistosomiasis and thus will be greatly beneficial in reducing the overall burden of schistosomiasis.

Simultaneous priming with DNA encoding Sm-p80 and boosting with Sm-p80 protein confers protection against challenge infection with Schistosoma mansoni in mice.

Prophylactic efficacy of Sm-p80 was tested in the mouse model using DNA priming and boosting with protein approach. However, the novelty of the approach utilized in this study is that both the DNA priming and protein boosting was performed on a single day and no further vaccine inoculations were given to mice; the animals were challenged 1 month after the initial vaccine administration. Using this approach, significant reduction in worm burden (33 to 57 %) and marked decrease in egg retention in tissues (34 to 66%) was observed. Robust antibody titers and upregulation of cytokines (IL-1α/ß, IL-12α, and IFN-γ) appears to correlate with the protection. This approach of administering vaccine on a single day could be greatly helpful in the field setting because it will eliminate the compliance issues that may arise with multiple boosters that may be required for optimal efficacy for some vaccines.

Use of an Sm-p80-based therapeutic vaccine to kill established adult schistosome parasites in chronically infected baboons.

No vaccines are available for human use for any parasitic infections, including the helminthic disease schistosomiasis. Sm-p80, the large subunit of Schistosoma mansoni calpain, is a leading antigen candidate for a schistosomiasis vaccine. Prophylactic and antifecundity efficacies of Sm-p80 have been tested using a variety of vaccine approaches in both rodent and nonhuman primate models. However, the therapeutic efficacy of a Sm-p80-based vaccine had not been determined. In this study, we evaluated the therapeutic efficacy of Sm-p80 by using 2 different strategies and 3 Sm-p80-based vaccine formulations in baboons. Vaccine formulations were able to decrease established adult worms by 10%-36%, reduce retention of eggs in tissues by 10%-57%, and decrease egg excretion in feces by 13%-33%, compared with control formulations. Marked differences were observed in B and T cell immune correlates between vaccinated and control animals. This is the first report of killing of established adult schistosome worms by a vaccine. In addition to distinct prophylactic efficacy of Sm-p80, this study adds to the evidence that Sm-p80 is a potentially important antigen with both substantial prophylactic and therapeutic efficacies. These data reinforce that Sm-p80 should be moved forward along the path toward human clinical trials.

Complement plays a minimal role in Sm-p80-mediated protection against Schistosoma mansoni.

Sm-p80, the large subunit of Schistosoma masoni calpain, is a leading antigen candidate for a schistosome vaccine. Prophylactic and antifecundity efficacy of Sm-p80 has been tested using a variety of vaccine approaches. However, the mechanism of Sm-p80-mediated killing is still unknown. In this study, potential role of complement in Sm-p80-mediated protection was studied using both in vitro (cobra venom factor inhibition) and in vivo using mice deficient in C3 (C3 -/-; B6.129S4-C3tm1Crr/J). In the absence of C3, Sm-p80-based vaccine was able to provide significant reduction in adult worm burden following challenge with schistosome cercariae in mice suggesting the effector functions of complement may be limited in this vaccine-induced protection.

Immunodetection of coproantigens for the diagnosis of amphistomosis in naturally infected Indian Water Buffalo, Bubalus bubalis.

The infection of gastrointestinal helminths in livestock is routinely diagnosed by microscopical examination of faecal samples for the presence of ova/eggs but this approach becomes ineffective for the seasonally egg producing trematodes. Therefore, an alternative approach to detect the coproantigens of liver and rumen amphistomes, Gigantocotyle explanatum and Gastrothylax crumenifer respectively, infecting Indian water buffalo Bubalus bubalis, was undertaken using ELISA, immunodot and countercurrent immunoelectrophoresis (CCIEP). The hyperimmune polyclonal antisera were separately raised in rabbits against excretory/secretory (ES) antigens of both the flukes under study. An overall 70% buffalo faecal samples were tested positive for G. crumenifer and 75% for G. explanatum in Aligarh region. The ELISA results reflected higher infection intensity among individual buffaloes that was also observed at necropsy. Using the respective homologous hyperimmune antiserum, 55% buffaloes tested positive for G. crumenifer and 65% positive for G. explanatum in immunodot assay. Further, the faecal samples with high absorbance values in ELISA and strong immunodot reaction tested positive in CCIEP. The analysis of CCIEP result revealed two and one precipitin bands in G. crumenifer and G. explanatum respectively, indicating prominent antigenic differences in the coproantigens of these two parasites. Taken together, it is suggested that polyclonal antibodies could be conveniently used for the detection of coproantigens by ELISA and immunodot methods, particularly during the non-egg producing phase of the seasonally regulated reproductive cycle of the rumen amphistome G. crumenifer. It is concluded that the coproantigen detection is a good alternative over conventional method for the diagnosis of amphistomosis in livestock; however, further studies are required on a larger sample size of field buffaloes to augment the reproducibility of the present results.

Role of antibody dependent cell mediated cytotoxicity (ADCC) in Sm-p80-mediated protection against Schistosoma mansoni.

Schistosomiasis is a major health problem in the developing world and for international travelers to the endemic countries. Existing strategies to control schistosomiasis have had limited successes so far. The addition of an effective vaccine in existing control measures would be greatly beneficial in reducing the impact of the disease. In this regard, Sm-p80 mediated protection against intestinal schistosomiasis caused by Schistosoma mansoni has been observed to be promising in two animal models of infection and disease. In this study, the role of antibody dependent cell mediated cytotoxicity (ADCC) was deciphered in Sm-p80-mediated protection especially in the elimination of lung stage schistosomula. This was achieved using lung lavage cells and lung cells that were isolated from mice immunized with and without Sm-p80 formulated in a recombinant vaccine formulation. Significant differences were observed in cytotoxicity assays using immune sera with the lung lavage cells which showed 51% more killing of schistosomula and elevated levels of nitric oxide in the supernatants were detected compared to controls.

Preclinical prophylactic efficacy testing of Sm-p80-based vaccine in a nonhuman primate model of Schistosoma mansoni infection and immunoglobulin G and E responses to Sm-p80 in human serum samples from an area where schistosomiasis is endemic.

The prophylactic efficacy of a schistosome antigen (Sm-p80) was tested in a nonhuman primate model, the baboon. Using a total of 28 baboons, different vaccination strategies were used including recombinant Sm-p80 protein formulated in Toll-like receptor 7 and Toll-like receptor 9 agonists, and DNA priming followed by boosting with protein plus adjuvants. Recombinant protein approaches provided levels of prophylactic efficacy of 52%-58%, whereas prime-boost approaches conferred 38%-47% protection in baboons. An appropriately balanced pro-inflammatory (T-helper 17 [Th17] and Th1) and anti-inflammatory (Th2) type of response was generated; the Th1 and Th17 types of immune responses appear to be indicative of increased prophylactic efficacy. Production and expression of several cytokines (interleukin 2 [IL-2], interferon γ, IL-12α, IL-1ß, IL-6, and IL-22) were up-regulated in vaccinated animals. Human correlate studies revealed Sm-p80 reactivity with immunoglobulin G in human serum samples from schistosome-infected individuals. In addition, a complete lack of prevailing Sm-p80-specific immunoglobulin E in a high-risk or infected population was observed, thus minimizing the risk of hypersensitivity reaction following vaccination with Sm-p80 in humans. This study provided the proof of concept to move Sm-p80 forward into further preclinical development leading to human clinical trials.

Isolation and partial characterization of excretory/secretory antigens of Gastrothylax crumenifer.

The present study was carried out to identify the excretory/secretory (E/S) antigens of the rumen infecting digenetic trematode Gastrothylax crumenifer that may be useful for the immunodiagnosis of rumen amphistomosis particularly during the pre-monsoon season during which this rumen parasite stops shedding eggs. The in vitro released E/S proteins were purified on a Sephadex G-200 column. The gel filtration profile revealed three distinct fractions F1-F3 where F1 and F3 appeared as sharp peaks while the F2 fraction was dispersed. The antibody titre against each of the purified E/S fractions was determined by ELISA using anti-whole E/S polyclonal antibodies raised in rabbit. Among the three fractions, the antibody titre against F1 was highest (1:12,800) whereas IgG titre was very low (1:50) for fraction F2 and F3 (1:100). Of the total polypeptides resolved on gradient SDS-PAGE, only a few antigenic polypeptides were detected in each fraction with hyperimmune anti-serum as revealed by Western Blot analysis. However, a 33 kDa antigen detected in each fraction appeared to be immunodominant which could be exploited for the diagnosis of the pouched amphistome.

Role of antibodies in Sm-p80-mediated protection against Schistosoma mansoni challenge infection in murine and nonhuman primate models.

Schistosomiasis is an important public health concern in more than 76 developing countries. Advent of an anti-schistosome vaccine would undoubtedly add to the existing control measures and may eventually help in the elimination of this disease. In the present study we have attempted to dissect the role(s) of antibodies in Sm-p80 mediated protection by intravenously transferring pooled sera from mice immunized with Sm-p80-pcDNA3 or purified IgG from baboons immunized with Sm-p80-pcDNA3, into naïve C57BL/6 mice, respectively, prior to challenge with cercariae. The passive transfer of antibodies from protected mice (homologous transfers) as well as transfer of total IgG from baboons (heterologous transfers), into naïve mice showed statistically significant reductions in worm burden and in the number of eggs in the tissues. Immunizations of antibody knockout mice (µMt-/-; B10.129S2 (B6)-Igh-6(tm1Cgn)/J) with recombinant Sm-p80 in the presence of CpG-motif oligodeoxynucleotides as an adjuvant, resulted in substantial reduction of Sm-p80-mediated protection, compared to C57BL/6 (normal) control group of mice. Down regulation of cytokines that have important effects on B cell proliferation as well as the recovery of higher number of parasites in antibody knockout indicated a significant role(s) of antibodies in Sm-p80-mediated protection against Schistosoma mansoni in mice. In toto, these studies appear to suggest that antibodies play a significant role in Sm-p80 mediated protection.