Abstract We report the generation and propagation of not only the first heritable transgenic schistosome line but also a line that secretes a functional therapeutic protein in vivo. Using multiplexed CRISPR/Cas-mediated homology-directed knock-in targeted to a predicted genomic safe-harbor, we inserted a VHH–IgG1 Fc (termed C5-Fc) transgene into Schistosoma mansoni eggs. Single-miracidium infections of Biomphalaria glabrata yielded parental P0 lines; serial passage through snail and mouse hosts produced an F2 cohort in which all parasites carried the C5-Fc transgene and secreted C5-Fc into the murine venous circulation. Molecular assays confirmed chromosomal insertion, germline transmission and systemic secretion. Sera from mice harboring C5-Fc transgenic worms neutralized SARS-CoV-2 in vitro with potent activity consistent with the expected ACE2-binding blockade by the C5 variable domain of heavy-chain-only antibody (VHH). These results demonstrate (i) stable, heritable transgenesis of a platyhelminth, (ii) delivery of a biologically active antibody fragment by a live helminth in a mammalian host, and (iii) feasibility of using transgenic schistosomes as sustained, single-dose protein delivery platforms. This technology and delivery system enable new experimental approaches for schistosome biology and motivate exploration of living-foundry therapeutics.

BACKGROUND:Necator americanus glutathione S-transferase-1 (Na-GST-1) performs a crucial enzymatic step in hookworm blood feeding and is a leading target for vaccine development. Phase 1 trials in Brazil, Gabon, and the USA of recombinant Na-GST-1 adsorbed on Alhydrogel (Na-GST-1/Al), co-administered with or without AP 10-701 (Toll-like receptor [TLR] 4 agonist) or CpG 10104 (TLR9 agonist), showed the vaccine was well tolerated, safe, and immunogenic. A controlled human hookworm infection (CHHI) model was previously developed to evaluate the effect of candidate hookworm vaccines on infection. We aimed to assess the effect of vaccination with three different Na-GST-1 vaccine formulations on subsequent CHHI challenge with infectious N americanus larvae (NaL3), compared with placebo, and to evaluate vaccine safety. METHODS:In this double-blind, randomised, placebo-controlled phase 2 trial, we enrolled non-pregnant, hookworm-naive adults aged 18-49 years who were deemed healthy by means of screening procedures at The George Washington University Medical Faculty Associates (Washington, DC, USA) and The George Washington University School of Medicine and Health Sciences (Washington, DC, USA). Participants were randomly assigned (1:1:1:1) in blocks of four to receive three intramuscular injections at 2-month intervals of Na-GST-1/Al, Na-GST-1/Al in combination with CpG 10104 (Na-GST-1/Al-CpG), Na-GST-1/Al in combination with AP 10-701 (Na-GST-1/Al-AP), or placebo. Participants, vaccinators, investigators conducting safety assessments, and laboratory personnel were all blinded to assignment. Only pharmacy personnel were unblinded. 4 weeks after the final vaccination, participants underwent CHHI challenge with 50 infectious NaL3. Faecal samples were collected every second week until treatment with three consecutive daily oral doses of 400 mg albendazole starting on day 280. After albendazole administration, clearance of infection was documented by three negative post-treatment faecal examinations, collected once per week. Primary outcomes were efficacy and safety. The primary efficacy outcome was infection incidence at any timepoint up to day 380 detected by faecal microscopy in the per-protocol efficacy population. Safety was assessed in the vaccine safety analysis population (ie, all participants who received at least one dose of a vaccine or placebo) and CHHI safety analysis population (ie, all participants who underwent CHHI challenge) from day 0 to day 380. Secondary endpoints were infection intensity and vaccine immunogenicity. The trial is registered at ClinicalTrials.gov (NCT03172975) and is complete. FINDINGS:Between May 24, 2018, and Dec 9, 2020, 57 volunteers were assessed for eligibility, 39 of whom were enrolled (mean age 26·7 years [SD 5·4], 22 [56%] self-reported as female, 17 [44%] self-reported as male). Ten (26%) participants were randomly assigned to Na-GST-1/Al, nine (23%) to Na-GST-1/Al-CpG, ten (26%) to Na-GST-1/Al-AP, and ten (26%) to placebo. All 39 participants received at least one dose and were included in the vaccine safety analysis; 33 received CHHI, of whom 31 were included in the per-protocol efficacy analysis. Participants in the Na-GST-1/Al-CpG group had lower incidence of infection (two [40%] of five) compared to participants in the placebo (four [57%] of seven), Na-GST-1/Al (five [56%] of nine), and Na-GST-1/Al-AP (six [60%] of ten) groups, although differences were not significant. Maximum faecal hookworm egg counts were significantly lower in the Na-GST-1/Al-CpG (median 0·0 eggs per g) group compared with the placebo group (median 66·7 eggs per g; rate ratio 0·00, 95% credible interval 0·00-0·00). Maximum eosinophil counts were significantly lower in the Na-GST-1/Al-CpG group (median 0·6 × 103 cells per μL) compared with the placebo group (median 3·1 × 103 cells per μL; geometric mean ratio 0·33, 95% CI 0·13-0·88; p=0·027). Vaccine-induced anti-Na-GST-1 IgG responses were highest in the Na-GST-1/Al-CpG group. No related serious adverse events were observed and most adverse events were mild. INTERPRETATION:Based on the protection observed against infection, Na-GST-1/Al-CpG has been selected for further clinical testing. The higher levels of anti-Na-GST-1 IgG seen in association with protection against challenge infection suggests that humoral immune responses might correlate with protection. FUNDING:US National Institute of Allergy and Infectious Diseases.

Ascaris infection-ascariasis-is the most common human parasitic worm infection worldwide and induces damage and cellular changes within the stomach. Helicobacter pylori is a bacterium of the stomach that also causes gastric pathologies globally. Importantly, Ascaris, H. pylori, and gastric diseases all disproportionately affect individuals in resource-limited settings. However, the associations between ascariasis, H. pylori infection, and gastric diseases remain relatively unexplored. We synthesized the existing research associating Ascaris infection with H. pylori infection and/or gastric pathologies. Records were identified in OVID Medline, Embase and Web of Science using search terms for ascariasis, H. pylori infection, and gastric abnormalities and additional citation searching. Blinded screening and extraction were performed by two independent reviewers. We found 410 unique citations, of which 86 (21.0%) were included. A total of 24 articles (27.9%) associated ascariasis with H. pylori infection, 68 (79.1%) associated ascariasis with gastric abnormalities, and 6 (7.0%) associated ascariasis with both H. pylori infection and gastric abnormalities. Although predominantly comprised of case reports and observational studies, these data supported significant co-endemicity of ascariasis and H. pylori infection, co-infections with both pathogens, and concurrency of ascariasis with gastric pathologies in human and non-human host organisms. The available literature thus supports associations between ascariasis, H. pylori infections, and gastric pathology, warranting further epidemiologic and mechanistic studies.

Biologics, protein- and peptide-based drugs derived from living organisms or cell lines have emerged as effective therapies across a broad range of indications. However, parenteral administration and the need for frequent dosing increase costs and limit compliance, creating an urgent need for innovative platforms capable of continuously, safely, and efficiently delivering sustained biologics in situ within the host. Here, we describe a bioengineered hookworm platform to manufacture and deliver biologic therapeutics in vivo. As proof of concept, we engineer the Ancylostoma ceylanicum secretome by inserting a human single-chain variable fragment antibody (s16-HuScFv) into its genome. Transgene expression does not perturb surrounding gene expression, and heritable transgenesis is confirmed. The s16-HuScFv transgene product is secreted into host circulation and partially neutralizes tetrodotoxin. Given the availability of controlled human infections, our disease-agnostic bioengineered hookworm platform offers a next-generation approach to address a suite of chronic human diseases, and with a single-dose administration, could potentially produce and deliver biologic medicines within the human host for years.

Chagas disease is a poverty-related neglected tropical disease caused by the protozoan Trypanosoma cruzi affecting approximately 6.3 million people, predominantly in the Americas. Approximately 30% of T. cruzi infections progress to chronic cardiomyopathy and 10% of these cases end in death from cardiac failure. The first-line drug Benznidazole (BNZ) require a prolonged treatment regimen and can be highly toxic. Here, we evaluate a therapeutic vaccine in T. cruzi-infected mice, based on the recombinant Trypomastigote Surface Antigen 1 (TSA-1.C4) protein and the emulsified adjuvant E6020, and in combination with a suboptimal dose of BNZ. We observed a reduced burden parasite in blood in mice receiving either with TSA-1.C4 vaccine alone or in combination with a low dose of BNZ. TSA-1.C4-specific IgG and isotype levels were increased in all experimental groups receiving TSA-1.C4 protein treatment, confirming its immunogenicity. Mice treated with TSA-1.C4 vaccine in combination with BNZ exhibit a reduced cardiac inflammation as well as an antigen-specific IFN-γ CD4+ T cells with an IL-2 and IL-4 cytokine production. Even though TSA-1.C4 vaccine alone induced a cytokine response by IFN-γ and IL-10 production, only the TSA-1.C4 vaccine plus BNZ reduced cardiac inflammatory infiltrate compared to infected untreated mice. In conclusion the therapeutic vaccine with a low dose of BNZ prevent cardiac inflammation and provide a balanced Th1/Th2 cytokine immune response in a murine model of acute Chagas disease.

Rational adjuvant selection is a systematic approach based on adjuvant-mediated immunomodulation to identify safe vaccine regimens that enhance protective immunity. Transmitted through ticks and caused by the bacterium Borrelia burgdorferi (Bb), Lyme disease (LD) is the most common vector-borne disease in the Northern hemisphere. There are no approved human vaccines, making it suitable for testing the concept of rational adjuvant selection. Here, we formulated our previously developed and effective LD vaccine antigen, CspZ-YAC187S, with different adjuvants suitable for human use; we analyzed the immune response by transcriptomics and tested the vaccine efficacy after Bb infection. We identified Alum-CpG and Alum-αGal to elicit the highest titers of CspZ-YAC187S-dependent protective antibodies and robust levels of protection, but through distinct mechanisms of immunomodulation. We demonstrated that immunization with Alum-CpG formulated CspZ-YAC187S provided up to nine months of protective bactericidal antibody titers, as well as recall-memory response to prevent LD after natural infection. Immunity was linked to elevated levels of IgG1 memory cells in the vaccine-triggered immune responses. This work thus identified a durable, memory immunity-driven LD vaccine, ultimately paving the road to understand the mechanisms of rationale adjuvant selection for vaccine development.

Background The antigen Na-GST-1, expressed by the hookworm Necator americanus, plays crucial biochemical roles in parasite survival. This study explores the development of mRNA vaccine candidates based on Na-GST-1, building on the success of recombinant Na-GST-1 (rNa-GST-1) protein, currently assessed as a subunit vaccine candidate, which has shown promise in preclinical and clinical studies. Methodology/findings By leveraging the flexible design of RNA vaccines and protein intracellular trafficking signal sequences, we developed three variants of Na-GST-1 as native (cytosolic), secretory, and plasma membrane-anchored (PM) antigens. After one immunization in mice, mRNA vaccines induced an earlier onset of antigen-specific antibodies compared to rNa-GST-1. Following two immunizations, mRNA vaccines induced similar or superior levels of antigen-specific antibodies compared to rNa-GST-1. Secretory Na-GST-1 was comparable to rNa-GST1 in producing neutralizing antibodies against Na-GST-1’s thiol transferase activity, while native Na-GST-1 induced a more robust CD8+ T cell response due to its intracellular accumulation. Although PM Na-GST-1 elicited one of highest titers of antigen-specific antibody and a diverse set of memory T-cell populations, it resulted in a lower ratio of neutralizing antibodies after IgG purification compared to the other vaccine candidates. Conclusions/significance These findings emphasize the importance of antigen localization in tailoring immune responses and suggest that extracellular antigens are more effective for inducing humoral responses, whereas cytosolic antigen accumulation enhances MHC-1 peptide presentation. Future studies will determine if these in vitro and immunogenicity findings translate to in vivo efficacy. Altogether, mRNA vaccines offer numerous possibilities in the development of multivalent vaccines with single or multiple antigens.

Soil-transmitted helminth (STH) infections rank among the most prevalent communicable diseases of humans, yet detection of these parasites is mostly restricted to identifying active infection through fecal examinations. Currently, there are no commercial diagnostic tools to identify a prior whipworm or hookworm exposure, and the few serological assays for roundworm infection have not been well validated for crossreactivity or infections in humans. Such diagnostic restrictions limit the range of scientific and clinical questions that surround STH exposures and their implicated relationship to chronic diseases, such as autoimmunity, allergy, and cancer. The goal of this investigation was to evaluate the diagnostic potential of 13 STH recombinant proteins. As there are no gold standard tests to verify positive STH antisera, we used sera from active STH-infected individuals in Honduras (measured by quantitative real-time polymerase chain reaction of helminth DNA in stool) and compared antibody recognition by both ELISA and western blot with nonendemic control sera from age-matched individuals in the United States split into screening and validation cohorts. One recombinant protein, rTm-WAP49, shows potential as a whipworm diagnostic tool by receiver-operator characteristic analysis (area under the curve = 0.997, P <0.001) and indirect ELISA with sensitivity of 100% and specificity of 91% as defined by mean plus two SDs from the nonendemic screening cohort. We found discrepancies in serological recognition of previously tested STH antigens, highlighting the need to consider different technologies before down selection of a promising diagnostic candidate and screen multiple endemic populations before widely accepting an STH serological assay.

Introduction:Chagas disease (CD), caused by the parasite Trypanosoma cruzi, affects millions globally. Despite treatment options in the acute phase, most infections progress to a chronic indeterminate form or develop severe cardiac/gastrointestinal complications. Understanding the immune response is crucial for the development of vaccines and more efficient drugs for the disease control. Methods:This work investigates the immune response to T. cruzi H1 K68 strain infection in female BALB/c and C57BL/6 mice to characterize differences in Tfh and B cell responses that may be involved in the poor parasite-specific antibody production during acute infection. For this, mice were euthanized 14, 28, and 49 days after infection, and splenic T and B cell populations were evaluated by flow cytometry. Results:BALB/c mice exhibited a strong Th2-biased response with a massive expansion of classic Tfh cells and GC B cells, potentially linked with polyclonal B cell activation and hypergammaglobulinemia, but not with efficient parasite clearance. C57BL/6 mice displayed a Th1-skewed response with a population of "Th1-like Tfh" cells expressing IFN-γ and CXCR5 associated with lower parasite burden and more focused antibody response, including parasitespecific IgG2c during early acute infection. Discussion:These findings suggest that these mouse models develop different immune responses mediated by Tfh cells, which are crucial for B cell activation and antibody production. The massive expansion of Tfh cells in BALB/c mice might lead to unspecific antibody production due to excessive B cell activation. Conversely, C57BL/6 mice exhibit a "Th1-like Tfh" response lacking classic Tfh cells, potentially explaining their weak parasite-specific antibody production throughout the acute infection. Overall, this study provides for the first time insights into the complex interplay between Tfh cells and antibody production during T. cruzi infection, suggesting potential targets for therapeutic intervention in CD.

More than 470 million people globally are infected with the hookworms Ancylostoma ceylanicum and Necator americanus, resulting in an annual loss of 2.1 to 4 million disability-adjusted-life-years. Current infection management approaches are limited by modest drug efficacy, the costs associated with frequent mass drug administration campaigns, and the risk of reinfection and burgeoning drug resistance. Subunit vaccines based on proteins excreted and secreted (ES) by hookworms that reduce worm numbers and associated disease burden are a promising management strategy to overcome these limitations. However, studies on the ES proteomes of hookworms have mainly described proteins from the adult life stage which may preclude the opportunity to target the infective larva. Here, we employed high resolution mass spectrometry to identify 103 and 57 ES proteins from the infective third larvae stage (L3) as well as 106 and 512 ES proteins from the adult N. americanus and A. ceylanicum respectively. Comparisons between these developmental stages identified 91 and 41 proteins uniquely expressed in the L3 ES products of N. americanus and A. ceylanicum, respectively. We characterized these proteins based on functional annotation, KEGG pathway analysis, InterProScan signature and gene ontology. We also performed reciprocal BLAST analysis to identify orthologs across species for both the L3 and adult stages and identified five orthologous proteins in both life stages and 15 proteins that could be detected only in the L3 stage of both species. Last, we performed a three-way reciprocal BLAST on the L3 proteomes from both hookworm species together with a previously reported L3 proteome from the rodent hookworm Nippostrongylus brasiliensis, and identified eight L3 proteins that could be readily deployed for testing using well established rodent models. This novel characterization of L3 proteins and taxonomic conservation across hookworm species provides a raft of potential candidates for vaccine discovery for prevention of hookworm infection and disease.

BACKGROUND:Long coronavirus disease (COVID), which affects an estimated 44.69-48.04 million people in the United States, is an ongoing public health concern that will persist as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread. METHODS:We developed a computational simulation model representing the clinical course, health effects, and associated costs of a person with long COVID. RESULTS:Simulations show that the average total cost of a long COVID case can range from $5084-$11 646 (assuming symptoms only last 1 year) with 92.5%-95.2% of these costs being productivity losses. Therefore, the current number of long COVID cases could cost society at least $2.01-$6.56 billion, employers at least $1.99-$6.49 billion in productivity losses, and third-party payers $21.0-$68.5 million annually (6%-20% probability of developing long COVID). These cases would accrue 35 808-121 259 quality-adjusted life-years (QALYs) lost and 13 484-45 468 disability-adjusted life-years (DALYs) and would rise as COVID-19 incidence increases. CONCLUSIONS:The current health and economic burden of long COVID may already exceed that of a number of other chronic diseases and will continue to grow each year as COVID-19 cases increase. This could be a significant drain on businesses, third-party payers, the healthcare system, and society.

Despite the efficacy of approved severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines in preventing severe disease and death, breakthrough infections continue to occur in vaccinated individuals, contributing to further viral mutation and spread. These limitations may be attributable to the poor induction of mucosal immunity by parenteral vaccination. Mucosal adjuvants, such as T-vant, can enhance vaccine-induced immune responses through the generation of antigen-specific antibodies and T cells in the respiratory tract. In this study, we evaluated the protective efficacy of adjuvanted SARS-CoV-2 receptor binding domain (RBD) subunit vaccines administered by homologous and heterologous routes. Immunized mice were challenged with SARS-CoV-2-XBB.1.5 and monitored for weight loss and survival. Lung and nasopharynx tissues were collected at pre-scheduled timepoints to assess viral loads and histopathology. Additionally, vaccine-induced humoral and cell-mediated immune responses were evaluated in the mucosal and systemic compartments. A prime-pull vaccination strategy - comprising an intramuscular prime immunization with aluminum hydroxide (alum) and CpG-adjuvanted RBD followed by an intranasal boost with T-vant-adjuvanted RBD - conferred protection against mortality and lung pathology and cleared virus from the nasopharynx by three days post infection. The prime-pull vaccine regimen elicited superior anti-RBD IgA in the bronchoalveolar lavage fluid and nasal washes, when compared to other vaccine groups. Given that much of the global population has already received parenteral SARS-CoV-2 vaccination or has been naturally exposed, a prime-pull approach could leverage pre-existing systemic immunity using a single mucosal boost.

Borrelia burgdorferi ( Bb ) causes Lyme disease (LD), one of the most common vector-borne diseases in the Northern Hemisphere. Here, we solve the crystal structure of a mutated Bb vaccine antigen, CspZ-YA that lacks the ability to bind to host complement factor H (FH). We generate point mutants of CspZ-YA and identify CspZ-YA I183Y and CspZ-YA C187S to trigger more robust bactericidal responses. Compared to CspZ-YA, these CspZ-YA mutants require a lower immunization frequency to protect mice from LD-associated inflammation and bacterial colonization. Antigenicity of wild-type and mutant CspZ-YA proteins are similar, as measured using sera from infected people or immunized female mice. Structural comparison of CspZ-YA with CspZ-YA I183Y and CspZ-YA C187S shows enhanced interactions of two helices adjacent to the FH-binding sites in the mutants, consistent with their elevated thermostability. In line with these findings, protective CspZ-YA monoclonal antibodies show increased binding to CspZ-YA at a physiological temperature (37 °C). In summary, this proof-of-concept study applies structural vaccinology to enhance intramolecular interactions for the long-term stability of a Bb antigen while maintaining its protective epitopes, thus promoting LD vaccine development.

Chagas disease (CD) is caused by the parasite Trypanosoma cruzi. This work investigates the immune response to T. cruzi H1 K68 strain infection in female BALB/c and C57BL/6 mice to characterize differences in Tfh and B cell responses that may be involved in the poor parasite-specific antibody production during acute infection. For this, mice were euthanized 14, 28, and 49 days after infection, and splenic T and B cell populations were evaluated by flow cytometry. BALB/c mice exhibited a strong Th2-biased response with a massive expansion of classic Tfh cells and GC B cells, potentially linked with polyclonal B cell activation and hypergammaglobulinemia, but not with efficient parasite clearance. C57BL/6 mice displayed a Th1-skewed response with a population of “Th1-like Tfh” cells expressing IFN-γ and CXCR5 associated with lower parasite burden and more focused antibody response, including parasite-specific IgG2c during early acute infection. The massive expansion of Tfh cells in BALB/c mice might lead to unspecific antibody production due to excessive B cell activation. Conversely, C57BL/6 mice exhibit a “Th1-like Tfh” response lacking classic Tfh cells, potentially explaining their weak parasite-specific antibody production throughout the acute infection. Overall, this study provides for the first time insights into the complex interplay between Tfh cells and antibody production during T. cruzi infection, suggesting potential targets for therapeutic intervention in CD. This work was funded by the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation (PJH) and R01AI168038 (KMJ). This project was supported by the Cytometry and Cell Sorting Core at Baylor College of Medicine with funding from the NIH (NIAID P30AI036211, NCI P30CA125123, and NCRR S10RR024574) and the assistance of Joel M. Sederstrom. Microbial, Parasitic, and Fungal Immunology (MPF)

This study explored the development and evaluation of mRNA vaccine candidates targeting Schistosoma mansoni tetraspanin-2 (Sm-TSP-2). We designed constructs encoding either full-length Sm-TSP-2, or its large extracellular loop (EC2) domain in secretory, membrane-anchored, or cytosolic forms. In a murine model, the secreted and membrane-anchored versions of Sm-TSP-2-EC2 induced the highest antigen-specific antibody titers. These two construct designs, along with full-length Sm-TSP-2 mRNA, also significantly reduced adult worm and egg burden compared to controls. The membrane-anchored Sm-TSP-2-EC2 mRNA was the most effective, lowering worm and egg burdens by 66.7% and 66.9%, respectively. Protective responses induced by the mRNA vaccines were comparable to those elicited by the Sm-TSP-2-EC2 protein formulated with Alum. Histopathological analysis revealed smaller hepatic granulomas surrounding worm eggs, supporting the immunopathological benefit of vaccination. Using a systematic mRNA-based approach, we optimized the presentation of the Sm-TSP-2-EC2 and demonstrated that extracellular exposure of EC2 is essential for eliciting a protective response.