A Recombinant Protein XBB.1.5 RBD/Alum/CpG Vaccine Elicits High Neutralizing Antibody Titers against Omicron Subvariants of SARS-CoV-2.

(1) Background: We previously reported the development of a recombinant protein SARS-CoV-2 vaccine, consisting of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, adjuvanted with aluminum hydroxide (alum) and CpG oligonucleotides. In mice and non-human primates, our wild-type (WT) RBD vaccine induced high neutralizing antibody titers against the WT isolate of the virus, and, with partners in India and Indonesia, it was later developed into two closely resembling human vaccines, Corbevax and Indovac. Here, we describe the development and characterization of a next-generation vaccine adapted to the recently emerging XBB variants of SARS-CoV-2. (2) Methods: We conducted preclinical studies in mice using a novel yeast-produced SARS-CoV-2 XBB.1.5 RBD subunit vaccine candidate formulated with alum and CpG. We examined the neutralization profile of sera obtained from mice vaccinated twice intramuscularly at a 21-day interval with the XBB.1.5-based RBD vaccine, against WT, Beta, Delta, BA.4, BQ.1.1, BA.2.75.2, XBB.1.16, XBB.1.5, and EG.5.1 SARS-CoV-2 pseudoviruses. (3) Results: The XBB.1.5 RBD/CpG/alum vaccine elicited a robust antibody response in mice. Furthermore, the serum from vaccinated mice demonstrated potent neutralization against the XBB.1.5 pseudovirus as well as several other Omicron pseudoviruses. However, regardless of the high antibody cross-reactivity with ELISA, the anti-XBB.1.5 RBD antigen serum showed low neutralizing titers against the WT and Delta virus variants. (4) Conclusions: Whereas we observed modest cross-neutralization against Omicron subvariants with the sera from mice vaccinated with the WT RBD/CpG/Alum vaccine or with the BA.4/5-based vaccine, the sera raised against the XBB.1.5 RBD showed robust cross-neutralization. These findings underscore the imminent opportunity for an updated vaccine formulation utilizing the XBB.1.5 RBD antigen.

TonB-Dependent Receptor Protein Displayed on Spores of Bacillus subtilis Stimulates Protective Immune Responses against Acinetobacter baumannii.

The emergence of antibiotic-resistant Acinetobacter baumannii strains with limited treatment options has become a significant global health concern. Efforts to develop vaccines against the bacteria have centred on several potential protein targets, including the TonB-dependent receptors (TBDRs). In the present study, TBDRs from A. baumannii were displayed on the surface of Bacillus subtilis spores. The immunogenicity of the recombinant spores was evaluated in orally vaccinated mice. None of the immunized mice demonstrated signs of illness and were observed to be healthy throughout the study. Sera and the intestinal secretions from the recombinant spores-treated mice demonstrated mucosal and humoral antibody responses to the vaccine antigen. In addition, bactericidal activities of the sera against A. baumannii clinical isolates were demonstrated. These observations suggest that the B. subtilis spore-displayed TBDRs should be further explored as much-needed potential oral vaccine candidates against A. baumannii.

From concept to delivery: a yeast-expressed recombinant protein-based COVID-19 vaccine technology suitable for global access.

INTRODUCTION: The development of a yeast-expressed recombinant protein-based vaccine technology co-developed with LMIC vaccine producers and suitable as a COVID-19 vaccine for global access is described. The proof-of-concept for developing a SARS-CoV-2 spike protein receptor-binding domain (RBD) antigen as a yeast-derived recombinant protein vaccine technology is described. AREAS COVERED: Genetic Engineering: The strategy is presented for the design and genetic modification used during cloning and expression in the yeast system. Process and Assay Development: A summary is presented of how a scalable, reproducible, and robust production process for the recombinant protein COVID-19 vaccine antigen was developed. Formulation and Pre-clinical Strategy: We report on the pre-clinical and formulation strategy used for the proof-of-concept evaluation of the SARS-CoV-2 RBD vaccine antigen. Technology Transfer and Partnerships: The process used for the technology transfer and co-development with LMIC vaccine producers is described. Clinical Development and Delivery: The approach used by LMIC developers to establish the industrial process, clinical development, and deployment is described. EXPERT OPINION: Highlighted is an alternative model for developing new vaccines for emerging infectious diseases of pandemic importance starting with an academic institution directly transferring their technology to LMIC vaccine producers without the involvement of multinational pharma companies.

Vaccine-linked chemotherapy improves cardiac structure and function in a mouse model of chronic Chagas disease.

Introduction: Chagas disease, caused by chronic infection with the protozoan parasite Trypanosoma cruzi, affects 6-7 million people worldwide. The major clinical manifestation of Chagas disease is chronic Chagasic cardiomyopathy (CCC), which encompasses a spectrum of symptoms including arrhythmias, hypertrophy, dilated cardiomyopathy, heart failure, and sudden death. Current treatment is limited to two antiparasitic drugs, benznidazole (BNZ) and nifurtimox, but both have limited efficacy to halt the progression of CCC. We developed a vaccine-linked chemotherapy strategy using our vaccine consisting of recombinant Tc24-C4 protein and a TLR-4 agonist adjuvant in a stable squalene emulsion, in combination with low dose benznidazole treatment. We previously demonstrated in acute infection models that this strategy parasite specific immune responses, and reduced parasite burdens and cardiac pathology. Here, we tested our vaccine-linked chemotherapy strategy in a mouse model of chronic T. cruzi infection to evaluate the effect on cardiac function. Methods: Female BALB/c mice infected with 500 blood form T. cruzi H1 strain trypomastigotes were treated beginning 70 days after infection with a low dose of BNZ and either low or high dose of vaccine, in both sequential and concurrent treatments streams. Control mice were untreated, or administered only one treatment. Cardiac health was monitored throughout the course of treatment by echocardiography and electrocardiograms. Approximately 8 months after infection, endpoint histopathology was performed to measure cardiac fibrosis and cellular infiltration. Results: Vaccine-linked chemotherapy improved cardiac function as evidenced by amelioration of altered left ventricular wall thickness, left ventricular diameter, as well as ejection fraction and fractional shortening by approximately 4 months of infection, corresponding to two months after treatment was initiated. At study endpoint, vaccine-linked chemotherapy reduced cardiac cellular infiltration, and induced significantly increased antigen specific IFN-γ and IL-10 release from splenocytes, as well as a trend toward increased IL-17A. Discussion: These data suggest that vaccine-linked chemotherapy ameliorates changes in cardiac structure and function induced by infection with T. cruzi. Importantly, similar to our acute model, the vaccine-linked chemotherapy strategy induced durable antigen specific immune responses, suggesting the potential for a long lasting protective effect. Future studies will evaluate additional treatments that can further improve cardiac function during chronic infection.

The phytochemical diversity of commercial Cannabis in the United States.

The legal status of Cannabis is changing, fueling an increasing diversity of Cannabis-derived products. Because Cannabis contains dozens of chemical compounds with potential psychoactive or medicinal effects, understanding this phytochemical diversity is crucial. The legal Cannabis industry heavily markets products to consumers based on widely used labeling systems purported to predict the effects of different "strains." We analyzed the cannabinoid and terpene content of commercial Cannabis samples across six US states, finding distinct chemical phenotypes (chemotypes) which are reliably present. By comparing the observed phytochemical diversity to the commercial labels commonly attached to Cannabis-derived product samples, we show that commercial labels do not consistently align with the observed chemical diversity. However, certain labels do show a biased association with specific chemotypes. These results have implications for the classification of commercial Cannabis, design of animal and human research, and regulation of consumer marketing-areas which today are often divorced from the chemical reality of the Cannabis-derived material they wish to represent.

Receptor-binding domain recombinant protein on alum-CpG induces broad protection against SARS-CoV-2 variants of concern.

We conducted preclinical studies in mice using a yeast-produced SARS-CoV-2 RBD subunit vaccine candidate formulated with aluminum hydroxide (alum) and CpG deoxynucleotides. This formulation is equivalent to the CorbevaxTM vaccine that recently received emergency use authorization by the Drugs Controller General ofIndia. We compared the immune response of mice vaccinated with RBD/alum to mice vaccinated with RBD/alum + CpG. We also evaluated mice immunized with RBD/alum + CpG and boosted with RBD/alum. Mice were immunized twice intramuscularly at a 21-day interval. Compared to two doses of the /alum formulation, the RBD/alum + CpG vaccine induced a stronger and more balanced Th1/Th2 cellular immune response, with high levels of neutralizing antibodies against the original Wuhan isolate of SARS-CoV-2 as well as the B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 and (Delta) variants. Neutralizing antibody titers against the B.1.1.529 (BA.1, Omicron) variant exceeded those in human convalescent plasma after Wuhan infection but were lower than against the other variants. Interestingly, the second dose did not benefit from the addition of CpG, possibly allowing dose-sparing of the adjuvant in the future. The data reported here reinforces that the RBD/alum + CpG vaccine formulation is suitable for inducing broadly neutralizing antibodies against SARS-CoV-2, including variants of concern.

Mucosal Vaccination With Recombinant Tm-WAP49 Protein Induces Protective Humoral and Cellular Immunity Against Experimental Trichuriasis in AKR Mice.

Trichuriasis is one of the most common neglected tropical diseases of the world's poorest people. A recombinant vaccine composed of Tm-WAP49, an immunodominant antigen secreted by adult Trichuris stichocytes into the mucosa of the cecum to which the parasite attaches, is under development. The prototype is being evaluated in a mouse model of Trichuris muris infection, with the ultimate goal of producing a mucosal vaccine through intranasal delivery. Intranasal immunization of mice with Tm-WAP49 formulated with the adjuvant OCH, a truncated analog of alpha-GalCer with adjuvanticity to stimulate natural killer T cells (NKT) and mucosal immunity, induced significantly high levels of IgG and its subclasses (IgG1 and IgG2a) in immunized mice. This also resulted in a significant reduction of worm burden after challenge with T. muris-infective eggs. The addition of QS-21 adjuvant to this vaccine formulation further reduced worm counts. The improved protection from the dual-adjuvanted vaccine correlated with higher serum antibody responses (IgG, IgG1, IgG2a, IgA) as well as with the induction of antigen-specific IgA in the nasal mucosa. It was also associated with the robust cellular responses including functional subsets of CD4 T cells producing IL-4, and cytotoxic CD8 T cells expressing granzyme B. The worm reduction achieved by mucosal immunization was higher than that induced by subcutaneous immunization. Intranasal immunization also induced a significantly higher nasal mucosa-secreted antigen-specific IgA response, as well as higher functional cellular responses including CD4+IL4+ (Th1) and CD8+GnzB+ (Th2) T cells, and antigen-specific INFγ-producing T cells in both spleen and MLNs and antibody-producing B cells (CD19+B220+/B220+GL7+). Mucosal immunization further induced long-term T lymphocyte memory with increased central (CD62L+CD44+) and effector (CD62L-CD44+) memory subsets of both CD4 and CD8 T cells at 60 days after the last immunization. In summary, intranasal immunization with recombinant Tm-WAP49 protein induced strong protection versus murine trichuriasis. It represents a promising vaccination approach against intestinal nematodes.

Yeast-expressed recombinant SARS-CoV-2 receptor binding domain RBD203-N1 as a COVID-19 protein vaccine candidate.

SARS-CoV-2 protein subunit vaccines are currently being evaluated by multiple manufacturers to address the global vaccine equity gap, and need for low-cost, easy to scale, safe, and effective COVID-19 vaccines. In this paper, we report on the generation of the receptor-binding domain RBD203-N1 yeast expression construct, which produces a recombinant protein capable of eliciting a robust immune response and protection in mice against SARS-CoV-2 challenge infections. The RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity was evaluated in mice. Sera were evaluated for their efficacy using a SARS-CoV-2 pseudovirus assay. The RBD203-N1 protein was expressed with a yield of 492.9 ± 3.0 mg/L of fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 ± 3% (total yield of purified protein: 270.5 ± 13.2 mg/L fermentation supernatant). The protein was characterized to be a homogeneous monomer that showed a well-defined secondary structure, was thermally stable, antigenic, and when adjuvanted on Alhydrogel in the presence of CpG it was immunogenic and induced high levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. The characteristics of the RBD203-N1 protein-based vaccine show that this candidate is another well suited RBD-based construct for technology transfer to manufacturing entities and feasibility of transition into the clinic to evaluate its immunogenicity and safety in humans.

Receptor-binding domain recombinant protein on alum-CpG induces broad protection against SARS-CoV-2 variants of concern.

We conducted preclinical studies in mice using a yeast-produced SARS-CoV-2 RBD subunit vaccine candidate formulated with aluminum hydroxide (alum) and CpG deoxynucleotides. This formulation is equivalent to the CorbevaxTM vaccine that recently received emergency use authorization by the Drugs Controller General of India. We compared the immune response of mice vaccinated with RBD/alum to mice vaccinated with RBD/alum+CpG. We also evaluated mice immunized with RBD/alum+CpG and boosted with RBD/alum. Mice were immunized twice intramuscularly at a 21-day interval. Compared to two doses of the /alum formulation, the RBD/alum+CpG vaccine induced a stronger and more balanced Th1/Th2 cellular immune response, with high levels of neutralizing antibodies against the original Wuhan isolate of SARS-CoV-2 as well as the B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 and (Delta) variants. Neutralizing antibody titers against the B.1.1.529 (BA.1, Omicron) variant exceeded those in human convalescent plasma after Wuhan infection but were lower than against the other variants. Interestingly, the second dose did not benefit from the addition of CpG, possibly allowing dose-sparing of the adjuvant in the future. The data reported here reinforces that the RBD/alum+CpG vaccine formulation is suitable for inducing broadly neutralizing antibodies against SARS-CoV-2 including variants of concern.

Potency testing for a recombinant protein vaccine early in clinical development: Lessons from the Schistosoma mansoni Tetraspanin 2 vaccine.

INTRODUCTION: As a primary stability-indicating parameter, potency should be strategically evaluated during each phase of vaccine development. Herein, we present potency testing during the early clinical development of the Schistosoma mansoni (Sm) Tetraspanin-2 vaccine formulated on Alhydrogel (Sm-TSP-2/Al). As Sm-TSP-2/Al does not induce sterilizing immunity against its target pathogen (Sm) in animal models, potency is measured by "serological substitution", a method that can add significant variation to the potency metric, especially when used in a compliance (or 'single data point') approach. METHODS: Potency data were analyzed using the compliance approach to determine if two clinical lots of Sm-TSP-2/Al retained potency over 84 and 36 months post-release, respectively. These same data were also analyzed by: i) least-squares regression with a joinpoint regression analysis; ii) control charting of stability slopes; and iii) bootstrap modeling. Nested-regression and bootstrapping were used to compare the potency of the first (#11-69F-003) and second (#1975) clinical lots of Sm-TSP-2/Al. RESULTS: Despite significant variability in the immune assay, both clinical lots of Sm-TSP-2/Al remained potent for 84 and 36 months, respectively, in all four statistical approaches. The first lot of Sm-TSP-2/Al showed a gain in potency starting at 36 months post-release as captured by joinpoint regression. The two clinical lots of Sm-TSP-2/Al had comparable long-term potency. CONCLUSION: While a compliance approach can monitor the long-term stability of Sm-TSP-2/Al, it risks putting this critical stability-indicating parameter out of specification with each time point tested due to statistical multiplicity. Alternative statistical methods, such as joinpoint regression or bootstrapping, do not have this limitation and offer even more precise estimations of potency, with the added benefit of also providing predictive analytics. Nested regression and bootstrapping were shown to be a viable alternatives for lot-to-lot comparisons of the stability of Sm-TSP-2/Al. Instructions for implementing both these potency testing approaches are provided.

SARS­CoV-2 RBD219-N1C1: A yeast-expressed SARS-CoV-2 recombinant receptor-binding domain candidate vaccine stimulates virus neutralizing antibodies and T-cell immunity in mice.

There is an urgent need for an accessible and low-cost COVID-19 vaccine suitable for low- and middle-income countries. Here, we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed at high levels in yeast (Pichia pastoris), as a suitable vaccine candidate against COVID-19. After introducing two modifications into the wild-type RBD gene to reduce yeast-derived hyperglycosylation and improve stability during protein expression, we show that the recombinant protein, RBD219-N1C1, is equivalent to the wild-type RBD recombinant protein (RBD219-WT) in an in vitro ACE-2 binding assay. Immunogenicity studies of RBD219-N1C1 and RBD219-WT proteins formulated with Alhydrogel® were conducted in mice, and, after two doses, both the RBD219-WT and RBD219-N1C1 vaccines induced high levels of binding IgG antibodies. Using a SARS-CoV-2 pseudovirus, we further showed that sera obtained after a two-dose immunization schedule of the vaccines were sufficient to elicit strong neutralizing antibody titers in the 1:1,000 to 1:10,000 range, for both antigens tested. The vaccines induced IFN-γ IL-6, and IL-10 secretion, among other cytokines. Overall, these data suggest that the RBD219-N1C1 recombinant protein, produced in yeast, is suitable for further evaluation as a human COVID-19 vaccine, in particular, in an Alhydrogel® containing formulation and possibly in combination with other immunostimulants.

Genetic modification to design a stable yeast-expressed recombinant SARS-CoV-2 receptor binding domain as a COVID-19 vaccine candidate.

BACKGROUND: Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has now spread worldwide to infect over 110 million people, with approximately 2.5 million reported deaths. A safe and effective vaccine remains urgently needed. METHOD: We constructed three variants of the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (residues 331-549) in yeast as follows: (1) a "wild type" RBD (RBD219-WT), (2) a deglycosylated form (RBD219-N1) by deleting the first N-glycosylation site, and (3) a combined deglycosylated and cysteine-mutagenized form (C538A-mutated variant (RBD219-N1C1)). We compared the expression yields, biophysical characteristics, and functionality of the proteins produced from these constructs. RESULTS AND CONCLUSIONS: These three recombinant RBDs showed similar secondary and tertiary structure thermal stability and had the same affinity to their receptor, angiotensin-converting enzyme 2 (ACE-2), suggesting that the selected deletion or mutations did not cause any significant structural changes or alteration of function. However, RBD219-N1C1 had a higher fermentation yield, was easier to purify, was not hyperglycosylated, and had a lower tendency to form oligomers, and thus was selected for further vaccine development and evaluation. GENERAL SIGNIFICANCE: By genetic modification, we were able to design a better-controlled and more stable vaccine candidate, which is an essential and important criterion for any process and manufacturing of biologics or drugs for human use.

Vaccine-linked chemotherapy induces IL-17 production and reduces cardiac pathology during acute Trypanosoma cruzi infection.

Chagas disease resulting from Trypanosoma cruzi infection leads to a silent, long-lasting chronic neglected tropical disease affecting the poorest and underserved populations around the world. Antiparasitic treatment with benznidazole does not prevent disease progression or death in patients with established cardiac disease. Our consortium is developing a therapeutic vaccine based on the T. cruzi flagellar-derived antigen Tc24-C4 formulated with a Toll-like receptor 4 agonist adjuvant, to complement existing chemotherapy and improve treatment efficacy. Here we demonstrate that therapeutic treatment of acutely infected mice with a reduced dose of benznidazole concurrently with vaccine treatment - also known as "vaccine-linked chemotherapy"-induced a TH17 like immune response, with significantly increased production of antigen specific IL-17A, IL-23 and IL-22, and CD8 + T lymphocytes, as well as significantly increased T. cruzi specific IFNγ-producing CD4 + T lymphocytes. Significantly reduced cardiac inflammation, fibrosis, and parasite burdens and improved survival were achieved by vaccine-linked chemotherapy and individual treatments. Importantly, low dose treatments were comparably efficacious to high dose treatments, demonstrating potential dose sparing effects. We conclude that through induction of TH17 immune responses vaccine-linked chemotherapeutic strategies could bridge the tolerability and efficacy gaps of current drug treatment in Chagasic patients.

SARS-CoV-2 RBD219-N1C1: A Yeast-Expressed SARS-CoV-2 Recombinant Receptor-Binding Domain Candidate Vaccine Stimulates Virus Neutralizing Antibodies and T-cell Immunity in Mice.

There is an urgent need for an accessible and low-cost COVID-19 vaccine suitable for low- and middle-income countries. Here we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed at high levels in yeast ( Pichia pastoris ), as a suitable vaccine candidate against COVID-19. After introducing two modifications into the wild-type RBD gene to reduce yeast-derived hyperglycosylation and improve stability during protein expression, we show that the recombinant protein, RBD219-N1C1, is equivalent to the wild-type RBD recombinant protein (RBD219-WT) in an in vitro ACE-2 binding assay. Immunogenicity studies of RBD219-N1C1 and RBD219-WT proteins formulated with Alhydrogel ® were conducted in mice, and, after two doses, both the RBD219-WT and RBD219-N1C1 vaccines induced high levels of binding IgG antibodies. Using a SARS-CoV-2 pseudovirus, we further showed that sera obtained after a two-dose immunization schedule of the vaccines were sufficient to elicit strong neutralizing antibody titers in the 1:1,000 to 1:10,000 range, for both antigens tested. The vaccines induced IFN-γ, IL-6, and IL-10 secretion, among other cytokines. Overall, these data suggest that the RBD219-N1C1 recombinant protein, produced in yeast, is suitable for further evaluation as a human COVID-19 vaccine, in particular, in an Alhydrogel ® containing formulation and possibly in combination with other immunostimulants.

Modeling cannabinoids from a large-scale sample of Cannabis sativa chemotypes.

The widespread legalization of Cannabis has opened the industry to using contemporary analytical techniques for chemotype analysis. Chemotypic data has been collected on a large variety of oil profiles inherent to the cultivars that are commercially available. The unknown gene regulation and pharmacokinetics of dozens of cannabinoids offer opportunities of high interest in pharmacology research. Retailers in many medical and recreational jurisdictions are typically required to report chemical concentrations of at least some cannabinoids. Commercial cannabis laboratories have collected large chemotype datasets of diverse Cannabis cultivars. In this work a data set of 17,600 cultivars tested by Steep Hill Inc., is examined using machine learning techniques to interpolate missing chemotype observations and cluster cultivars into groups based on chemotype similarity. The results indicate cultivars cluster based on their chemotypes, and that some imputation methods work better than others at grouping these cultivars based on chemotypic identity. Due to the missing data and to the low signal to noise ratio for some less common cannabinoids, their behavior could not be accurately predicted. These findings have implications for characterizing complex interactions in cannabinoid biosynthesis and improving phenotypical classification of Cannabis cultivars.

TLR4 agonist protects against Trypanosoma cruzi acute lethal infection by decreasing cardiac parasite burdens.

E6020 is a synthetic agonist of Toll-like receptor-4 (TLR4). The purpose of this study was to evaluate the effect of different doses of E6020-SE on Trypanosoma cruzi-specific immune responses and its ability to confer protection against acute lethal infection in mice. Forty female BALB/c were infected with 500 trypomastigotes of T cruzi H1 strain, divided into four groups (n = 10) and treated at 7- and 14-day post-infection (dpi) with different doses of E6020-SE or PBS (control). Survival was followed for 51 days, mice were euthanized and hearts were collected to evaluate parasite burden, inflammation and fibrosis. We found significantly higher survival and lower parasite burdens in mice injected with E6020-SE at all doses compared to the control group. However, E6020-SE treatment did not significantly reduce cardiac inflammation or fibrosis. On the other hand, E6020-SE modulated Th1 and Th2 cytokines, decreasing IFN-γ and IL-4 in a dose-dependent manner after stimulation with parasite antigens. We conclude that E6020-SE alone increased survival by decreasing cardiac parasite burdens in BALB/c mice acutely infected with T cruzi but failed to prevent cardiac damage. Our results suggest that for optimal protection, a vaccine antigen is necessary to balance and orient a protective immune response.

Protective immunity elicited by the nematode-conserved As37 recombinant protein against Ascaris suum infection.

BACKGROUND: Ascaris lumbricoides is one of the three major soil-transmitted gastrointestinal helminths (STHs) that infect more than 440 million people in the world, ranking this neglected tropical disease among the most common afflictions of people living in poverty. Children infected with this roundworm suffer from malnutrition, growth stunting as well as cognitive and intellectual deficits. An effective vaccine is urgently needed to complement anthelmintic deworming as a better approach to control helminth infections. As37 is an immunodominant antigen of Ascaris suum, a pig roundworm closely related to the human A. lumbricoides parasite, recognized by protective immune sera from A. suum infected mice. In this study, the immunogenicity and vaccine efficacy of recombinant As37 were evaluated in a mouse model. METHODOLOGY/PRINCIPAL FINDINGS: As37 was cloned and expressed as a soluble recombinant protein (rAs37) in Escherichia coli. The expressed rAs37 was highly recognized by protective immune sera from A. suum egg-infected mice. Balb/c mice immunized with 25 µg rAs37 formulated with AddaVax™ adjuvant showed significant larval worm reduction after challenge with A. suum infective eggs when compared with a PBS (49.7%) or adjuvant control (48.7%). Protection was associated with mixed Th1/2-type immune responses characterized by high titers of serological IgG1 and IgG2a and stimulation of the production of cytokines IL-4, IL-5, IL-10 and IL-13. In this experiment, the AddaVax™ adjuvant induced better protection than the Th1-type adjuvant MPLA (38.9%) and the Th2-type adjuvant Alhydrogel (40.7%). Sequence analysis revealed that As37 is a member of the immunoglobulin superfamily (IgSF) and highly conserved in other human STHs. Anti-As37 antibodies strongly recognized homologs in hookworms (Necator americanus, Ancylostoma ceylanicum, A. caninum) and in the whipworm Trichuris muris, but there was no cross-reaction with human spleen tissue extracts. These results suggest that the nematode-conserved As37 could serve as a pan-helminth vaccine antigen to prevent all STH infections without cross-reaction with human IgSF molecules. CONCLUSIONS/SIGNIFICANCE: As37 is an A. suum expressed immunodominant antigen that elicited significant protective immunity in mice when formulated with AddaVax™. As37 is highly conserved in other STHs, but not in humans, suggesting it could be further developed as a pan-helminth vaccine against STH co-infections.

Reproductive history and progressive multiple sclerosis risk in women.

Being a woman is one of the strongest risk factors for multiple sclerosis. The natural reproductive period from menarche to natural menopause corresponds to the active inflammatory disease period in multiple sclerosis. The fifth decade marks both the peri-menopausal transition in the reproductive aging and a transition from the relapsing-remitting to the progressive phase in multiple sclerosis. A short reproductive period with premature/early menopause and/or low number of pregnancies may be associated with an earlier onset of the progressive multiple sclerosis phase. A cross-sectional study of survey-based reproductive history in a multiple sclerosis clinical series enriched for patients with progressive disease, and a case-control study of multiple sclerosis and age/sex matched controls from a population-based cohort were conducted. Menarche age, number of complete/incomplete pregnancies, menopause type and menopause age were compared between 137 cases and 396 control females. Onset of relapsing-remitting phase of multiple sclerosis, progressive disease onset and reaching severe disability (expanded disability status scale 6) were studied as multiple sclerosis-related outcomes (n = 233). Menarche age was similar between multiple sclerosis and control females (P = 0.306). Females with multiple sclerosis had fewer full-term pregnancies than the controls (P < 0.001). Non-natural menopause was more common in multiple sclerosis (40.7%) than in controls (30.1%) (P = 0.030). Age at natural menopause was similar between multiple sclerosis (median, interquartile range: 50 years, 48-52) and controls (median, interquartile range: 51 years, 49-53) (P = 0.476). Nulliparous females had earlier age at progressive multiple sclerosis onset (mean ± standard deviation: 41.9 ± 12.5 years) than females with ≥1 full-term pregnancies (mean ± standard deviation: 47.1 ± 9.7 years) (P = 0.069) with a pregnancy-dose effect [para 0 (mean ± standard deviation: 41.9 ± 12.5 years), para 1-3 (mean ± standard deviation: 46.4 ± 9.2 years), para ≥4 (mean ± standard deviation: 52.6 ± 12.9 years) (P = 0.005)]. Menopause age was associated with progressive multiple sclerosis onset age (R 2 = 0.359, P < 0.001). Duration from onset of relapses to onset of progressive multiple sclerosis was shorter for females with premature/early menopause (n = 26; mean ± standard deviation: 12.9 ± 9.0 years) than for females with normal menopause age (n = 39; mean ± standard deviation: 17.8 ± 10.3 years) but was longer than for males (mean ±standard deviation: 10.0 ± 9.4 years) (P = 0.005). There was a pregnancy-dose effect of age at expanded disability status scale 6 (para 0: 43.0 ± 13.2 years, para 1-3: 51.7 ± 11.3 years, para ≥4: 53.5 ± 4.9 years) (P = 0.013). Age at menopause was associated with age at expanded disability status scale 6 (R 2 = 0.229, P < 0.003). Premature/early menopause or nulliparity was associated with earlier onset of progressive multiple sclerosis with a 'dose effect' of pregnancies on delaying progressive multiple sclerosis and severe disability. Although causality remains uncertain, our results suggest a beneficial impact of oestrogen in delaying progressive multiple sclerosis. If confirmed in prospective studies, our findings have implications for counselling women with multiple sclerosis about pregnancy, surgical menopause and menopausal hormone therapy.

Energy Efficient Hybrid Routing Protocol Based on the Artificial Fish Swarm Algorithm and Ant Colony Optimisation for WSNs.

Wireless Sensor Networks (WSNs) are a particular type of distributed self-managed network with limited energy supply and communication ability. The most significant challenge of a routing protocol is the energy consumption and the extension of the network lifetime. Many energy-efficient routing algorithms were inspired by the development of Ant Colony Optimisation (ACO). However, due to the inborn defects, ACO-based routing algorithms have a slow convergence behaviour and are prone to premature, stagnation phenomenon, which hinders further route discovery, especially in a large-scale network. This paper proposes a hybrid routing algorithm by combining the Artificial Fish Swarm Algorithm (AFSA) and ACO to address these issues. We utilise AFSA to perform the initial route discovery in order to find feasible routes quickly. In the route discovery algorithm, we present a hybrid algorithm by combining the crowd factor in AFSA and the pseudo-random route select strategy in ACO. Furthermore, this paper presents an improved pheromone update method by considering energy levels and path length. Simulation results demonstrate that the proposed algorithm avoids the routing algorithm falling into local optimisation and stagnation, whilst speeding up the routing convergence, which is more prominent in a large-scale network. Furthermore, simulation evaluation reports that the proposed algorithm exhibits a significant improvement in terms of network lifetime.

Trichuris muris whey acidic protein induces type 2 protective immunity against whipworm.

Human whipworm (Trichuris trichiura) infects approximately 1 in 15 people worldwide, representing the leading infectious cause of colitis and subsequent, inflammatory bowel disease (IBD). Current control measures focused on mass deworming have had limited success due to low drug efficacies. Vaccination would be an ideal, cost-effective strategy to induce protective immunity, leading to control of infection and transmission. Here we report the identification of whey acidic protein, a whipworm secretory protein, as a strong immunogen for inducing protective efficacy in a surrogate mouse T. muris infection model. The recombinant WAP protein (rTm-WAP49), as well as a single, highly conserved repeat within WAP (fragment 8) expressed as an Na-GST-1 fusion protein (rTm-WAP-F8+Na-GST-1), generate a strong T helper type 2 (Th2) immune response when delivered as subcutaneous vaccines formulated with Montanide ISA 720. Oral challenge with T. muris infective eggs following vaccination led to a significant reduction in worm burden of 48% by rTm-WAP49 and 33% by rTm-WAP-F8+Na-GST-1. The cellular immune correlates of protection included significant antigen-specific production of Th2 cytokines IL-4, IL-9, and IL-13 by cells isolated from the vaccine-draining inguinal lymph nodes, parasite-draining mesenteric lymph nodes, and spleen in mice vaccinated with either rTm-WAP49 or rTm-WAP-F8+Na-GST-1. The humoral immune correlates included a high antigen-specific ratio of IgG1 to IgG2a, without eliciting an IgE-mediated allergic response. Immunofluorescent staining of adult T. muris with WAP antisera identified the worm's pathogenic stichosome organ as the site of secretion of native Tm-WAP protein into the colonic mucosa. Given the high sequence conservation for the WAP proteins from T. muris and T. trichiura, the results presented here support the WAP protein to be further evaluated as a potential human whipworm vaccine candidate.