Background Immunostimulatory adjuvants used in vaccines to protect against infectious disease have demonstrated efficacy in stimulating anti-cancer immunity. The most commercially advanced ones activate Toll-Like Receptor-4 (TLR4), a transmembrane signaling molecule expressed by macrophages and dendritic cells triggering innate immune responses. Lipopolysaccharide, the first identified TLR4 agonist, induces toxic, unregulated immune activation. Mimetics of monophosphoryl lipid A, the stimulatory component in lipopolysaccharide, reduced immunotoxicity while retaining immunostimulatory properties. Intratumoral injection of formulated TLR4 agonists can stimulate in situ antitumor immune responses by recruitment of immune cells and production of inflammatory cytokines that exert antitumor effects via a variety of mechanisms – including direct cancer cell death and recruitment of effector cells. Human clinical cancer trials have shown efficacy - both locally and through abscopal effects employing this approach. Methods We hypothesized that injection of a novel TLR4 agonist, EmT4™, into a canine soft tissue sarcoma (STS) could alter the tumor microenvironment by attracting and activating immune cells in situ . With the dog owner’s interest and written consent, a 3-cm soft tissue mass on the right forelimb of an 8-year-old female spayed Boston terrier received two intratumoral injections of EmT4™, two weeks apart. There was transient lethargy on the day of the first injection that resolved within hours. The tumor was excised 4 weeks after the second injection. Histopathology, immunohistochemistry, and in situ RNA hybridization were utilized to explore immune cell populations in the tumor microenvironment. Results Histopathology revealed grade 2 STS with large numbers of densely packed perivascular immune cells disseminated within the tumor. Immunohistochemistry for immune cell markers showed heterogeneous positive staining within cell clusters – CD3 (25%), CD20 (57%), FOXP3 (8%), CD204 (5%), and Iba-1 (36%). In situ hybridization performed on serial STS sections with RNAscope™ identified transcripts for CD4 (29%), TNF-α (24%), CD8 (3.2%), and interferon-g (1.3%) in lymphocyte clusters. Conclusions EmT4™ may have elicited an innate immune response that attracted and activated immune effector cells intratumorally. Clinical circumstances prevented acquisition of a pre-EmT4™ biopsy hampering a definitive conclusion although cell infiltrates observed are unusual in canine STS. This case is foundational for continued EmT4™ investigations for canine cancer immunotherapy.

Immunostimulatory adjuvants used in vaccines to protect against infectious disease have demonstrated efficacy in stimulating anti-cancer immunity. The most advanced ones activate Toll-Like Receptor-4 (TLR4), a transmembrane signaling molecule expressed by macrophages and dendritic cells triggering innate immune responses. Lipopolysaccharide, the first identified TLR4 agonist, induces toxic, unregulated immune activation. Mimetics of monophosphoryl lipid A, the stimulatory component in lipopolysaccharide, reduced immunotoxicity while retaining immunostimulatory properties. Intratumoral injection of TLR4 agonists can stimulate in situ antitumor immune responses by recruitment of immune cells and production of inflammatory cytokines mediating antitumor effects via various mechanisms – including direct cancer cell death and recruitment of effector cells. Clinical trials in humans showed intratumoral TLR4 agonist injection augmented cytotoxicity – both locally and through abscopal mechanisms. We considered whether intratumoral administration of TLR4 agonist EmT4™ in a canine soft tissue sarcoma (STS) could improve the surgical outcome by in situ immunostimulation prior to tumor removal. A 3-cm soft tissue mass on the right forelimb of an 8-year-old female spayed Boston terrier was given two intratumoral injections of EmT4™ two weeks apart, authorized by the owner. The tumor was excised 4-weeks after the second injection. Histopathology, immunohistochemistry, and in situ RNA hybridization were utilized to explore immune cell populations in the tumor microenvironment. There was transient lethargy only on the day of the first injection that resolved within hours. Histopathology revealed grade 2 STS with large numbers of densely packed perivascular immune cells disseminated within the tumor. Immunohistochemistry for immune cell markers showed heterogeneous positive staining within cell clusters – CD3 (25

This study was conducted to develop a robust, scalable manufacturing process for the candidate vaccine rBmHAXT for human lymphatic filariasis (LF). During scale-up production, rBmHAXT showed significant antigen aggregation leading to a loss of purified vaccine antigens. This project aims to create an improved formulation suitable for industrial-scale production while maintaining robust protection. We generated three variants: (1) ∆Cys, in which all cysteinyl residues were mutated to serinyl. (2) GS, which has a flexible glycine-serine linker inserted between each of the component antigens, and (3) GS/∆Cys, a third variant with a combination of both the cysteine deletion and the addition of linkers. We then evaluated the immunogenicity and efficacy of each variant in a mouse model. We demonstrated that the ΔCys mutant retained immunogenicity and vaccine efficacy of the parent tag-free rBmHAXT protein. We also performed an accelerated stability study. All preparations remained stable at 4°C, and the ΔCys variant remained stable even at 25°C throughout the study (6 weeks). The ∆Cys protein was stable with equivalent potency in mice. Therefore, ∆Cys is an optimal candidate for progression to cGMP (Current Good Manufacturing Practices) manufacturing and human clinical trials as a vaccine for lymphatic filariasis.

Schistosomiasis (bilharzia) is a neglected tropical disease caused by Schistosoma spp. Clinical manifestation of chronic schistosomiasis include but not restricted to anemia, growth stunting, hepatosplenomegaly, cognitive impairment in children and male/female genital schistosomiasis. Praziquantel (PZQ) remains the principal standard treatment for schistosomiasis, but concerns about its reduced effectiveness against larval stages, reinfections, and emerging drug resistance reinforces the urgent need for a vaccine. SchistoShield®, composed of Sm-p80 antigen with GLA-SE adjuvant, is a promising vaccine candidate that has successfully completed phase 1 and 1b clinical trials in the USA and two countries in Africa (Madagascar and Burkina Faso). In this study, SchistoShield® vaccine specific total IgG antibody titers were measured from serum samples collected at multiple time points from both the USA and Africa trials. Results demonstrate that total IgG titers increased at week 5 after the first booster and peaked at weeks 9 and 12. Furthermore, in vitro schistosomula killing assays and heterologous passive transfer of purified total IgG in mice were performed to evaluate the role of vaccine-induced antibodies against schistosomes. Sera collected from individuals enrolled in the USA, Burkina Faso, and Madagascar trials, exhibited 69.2%, 55.1%, and 34.3% in vitro schistosomula killing, respectively, indicative of potent anti-worm antibody responses. Passive transfer of human total IgG from vaccinated individuals in mice revealed notable reductions in worm burden, egg counts, and egg-hatching ability compared to groups given pre-vaccination sera across all trials. Overall, these findings support that SchistoShield® induces generation of functional antibodies that may play a crucial role in antibody-mediated protection against schistosomiasis.

Helminth parasites of the genus Schistosoma cause 290,000 deaths annually, mostly in tropical and subtropical regions. An estimated 250 million people are currently chronically infected with Schistosoma parasites, imposing a risk of new and recurrent infections in an additional 800 million people. SchistoShield® (Sm-p80 + GLA-SE) is a leading vaccine candidate for schistosomiasis that has successfully completed Phase 1 (USA) and Phase 1b (Africa) safety and immunogenicity clinical trials. Using Peripheral Blood Mononuclear Cells (PBMCs) obtained from intercontinental Phase 1 and Phase 1b trial participants, adaptive immune effector and memory responses to SchistoShield® were investigated. Functional recall responses were measured in vitro using Sm-p80 vaccine antigen. Results clearly demonstrate that the vaccine induced pronounced effector and memory T-cell responses. Upon recall with Sm-p80 antigen, cytokines including IFN-γ, TNF-α, IL-17A, IL-9, and granzyme B were produced, indicating the generation of functionally heterogeneous CD4 T-helper and cytotoxic lymphocyte responses. Consistent with T-helper responses that promote humoral immunity, Sm-p80 antigen-specific antibody-secreting plasmablasts were detected in vaccinated volunteers who were tracked longitudinally. Taken together, the SchistoShield® vaccine induced robust cell-mediated effector and memory responses, hallmarks of a potentially efficacious vaccine against schistosome/helminth parasites.

BackgroundSchistosomiasis is caused by parasitic blood flukes of the genus Schistosoma. Despite ongoing mass drug administration efforts, the disease remains a major public health burden in endemic regions. A better understanding of early host responses to schistosomiasis is critical for developing effective vaccines and therapeutics.MethodsWe conducted a longitudinal transcriptomic study of peripheral blood samples from 30 Schistosoma-naïve volunteers participating in two controlled human infection trials with male- or female-only S. mansoni cercariae. Blood was collected at six time points over 20 weeks post-infection. Whole-transcriptome RNA sequencing and integrative analyses, including differential gene expression, gene set enrichment, protein interaction networks, co-expression clustering, and immune module profiling, were employed to characterize temporal modulation of genes related to immune responses.ResultsRobust and highly time-dependent transcriptional responses were observed, peaking at Week 4 post-infection. Differential gene expression and pathway analyses revealed activation of immune responses, including type I and II interferon signaling, chemokine-mediated pathways, and antigen presentation. Notably, both Th1 and Th2 signatures were evident at Week 4. Key immune hubs included IFNG, TNF, and IL1B, along with transcriptional regulators such as STAT1 and IRF7. Blood transcription module analysis further highlighted transient activation of interferon and plasma cell-related responses.ConclusionsThis study provides a comprehensive transcriptional map of early host responses to S. mansoni infection in humans. The findings underscore the central role of interferon pathways, early mixed Th1/Th2 polarization, and inflammation-associated gene signatures in shaping host response to S. mansoni infection. These insights may inform the rational design of vaccines and biomarkers for schistosomiasis.

Onchocerciasis remains a significant cause of morbidity and economic loss in sub-Saharan Africa. Despite the existence of effective therapeutics, a prophylactic vaccine targeting the etiologic agent, Onchocerca volvulus, is needed to control ongoing disease and transmission. Mice were vaccinated against O. volvulus with a fusion of the recombinant antigens Ov-103 and Ov-RAL-2 (Ov-FUS-1) with Advax-CpG adjuvant. Immunized mice developed protective immunity with the killing of third-stage larvae (L3) within 36 h of challenge infection. IgG from immunized mice passively transferred protective immunity to naïve mice, indicating that antigen-specific IgG mediated parasite elimination. Neutrophils were the most abundant subset of immune cells recruited to the parasite microenvironment in vivo, and treating mice with a granulocyte-depleting antibody resulted in the total loss of immune-mediated larval killing. Analysis of neutrophil gene expression revealed that both vaccination and the presence of O. volvulus larvae were capable of modulating neutrophil transcriptional activity. The mechanism by which antigen-specific IgG and neutrophils collaborated to kill L3 was independent of Fcγ receptors. However, the elimination of complement component C3 prevented vaccine-induced protection, which suggests these components may interact through the complement system. This study describes a vaccine-induced mechanism of protective immunity against O. volvulus L3 dependent on IgG, neutrophils, and complement, highlighting an effective collaboration between the innate and adaptive arms of the immune system to control O. volvulus infection.

Schistosomiasis is a chronic disease that inflicts high morbidity and significant mortality with an estimated 90% of cases occurring in sub-Saharan Africa. Development of a vaccine against schistosomiasis is a major global health goal. The SchistoShield (Sm-p80 + GLA-SE) vaccine has consistently exhibited robust efficacy in non-human primate pre-clinical trials, has recently completed Phase 1 clinical trials in the US and Phase 1b trials are ongoing in Africa. To delineate a potential clinical/parasitological endpoint to measure the efficacy of schistosomiasis vaccine during human clinical trials, we have designed an in vitro schistosomula killing assay using sera collected from SchistoShield-vaccinated baboons and human volunteers from the Phase 1 clinical trial. Sm-p80-specific antibodies produced by immunized non-human primates and humans induced statistically significant larval schistosomula killing ex vivo. This reproducible functional assay has the potential to serve as one of the biomarkers for the effectiveness of the SchistoShield vaccine in human trials in Africa.

Schistosomiasis is a neglected tropical disease with the greatest burden in sub-Saharan Africa. An efficacious and safe vaccine would have a major global public health impact. The investigational SchistoShield® (Sm-p80 [antigen] + GLA-SE [adjuvant]) vaccine targets the Sm-p80 surface membrane antigen of Schistosoma mansoni and in nonhuman primate challenge studies was shown to be highly effective in killing pathogenic female worms and reducing host organ pathology and egg excretion. In this Phase 1 first-in-human, dose-escalation trial with sequential assignment, we evaluated the safety and immunogenicity of the vaccine in healthy adults in the United States. The vaccine formulations, given as a three dose intramuscular series, were well tolerated and adjuvanted formulations induced robust IgG ELISA responses against the Sm-p80 antigen. The vaccine has been advanced to a Phase 1b trial among adults in endemic areas of Africa. Clinicaltrials.gov registration: NCT05292391 https://Clinicaltrials.gov/study/NCT05292391 .

Background/Objectives: mRNA vaccines introduced during the COVID-19 pandemic were a significant step forward in the rapid development and deployment of vaccines in a global pandemic context. These vaccines showed good protective efficacy, but—due to limited breadth of the immune response—they required frequent boosters with manufactured spike sequences that often lagged behind the circulating strains. In order to enhance the breadth, durability, and magnitude of immune responses, we studied the effect of combining priming with an RNA vaccine technology with boosting with protein/adjuvant using a TLR4-agonist based adjuvant. Methods: Specifically, four proprietary adjuvants (EmT4TM, LiT4QTM, MiT4TM, and AlT4TM) were investigated in combination with multiple modes of SARS-CoV-2 vaccination (protein, peptide, RNA) for their effectiveness in boosting antibody responses to SARS-CoV-2 spike protein in murine models. Results: Results showed significant improvement in immune response strength and breadth—especially against more distant SARS-CoV-2 variants such as Omicron—when adjuvants were used in combination with boosters following an RNA vaccine prime. Conclusions: The use of novel TLR4 adjuvants in combination with protein or RNA vaccinations presents a promising strategy for improving the efficacy of vaccines in the event of future pandemics, by leveraging rapid response using an RNA vaccine prime and following up with protein/adjuvant-based vaccines to enhance the breadth of immunity.

Tuberculosis (TB) is again the deadliest infectious disease globally, and more efficacious vaccines are needed to reduce this mortality. Successful subunit TB vaccines need antigens and adjuvants that are immunogenic, inexpensive, and accessible. Here we evaluated the potential of synthetically produced Monophosphoryl lipid A (SyMLP), a TLR4-agonist, formulated in an oil-in-water emulsion (EmT4™) in combination with selected fusion proteins, to drive an effective vaccine-mediated immunogenic response in C57BL/6 mice against Mycobacterium tuberculosis (M.tb) HN878 and H37Rv challenge. We first observed that EmT4™ enhances activation of C57BL/6 bone-marrow derived macrophages and dendritic cells measured by CD40, CD86, and MHCII expression by flow cytometry. EmT4™ did not induce safety signals in a scaled tolerability study. In immunogenicity studies, mice immunized 3 times 3 weeks apart with ID93 antigen + EmT4™ produced a significantly higher magnitude of circulating proinflammatory cytokines and ID93-specific immunoglobulin G (IgG) antibodies pre- and post-challenge with M.tb than saline control animals. Ex vivo ID93 restimulated splenocytes and lung cells elicited significant polyfunctional CD4+ T-helper 1 responses. Importantly, ID93 + EmT4™ immunizations significantly reduced bacterial burden in C57BL/6 mice 4 weeks post-challenge. Interestingly, EmT4™ paired with a next generation protein fusion ID91 also afforded prophylactic protection against M.tb HN878 challenge in both young (6 to 8 wk) and aged (20 mo) immunocompromised Beige mice. These protection and immunogenicity findings suggest that synthetically derived EmT4™ adjuvant is not only suitable to help backfill the preclinical TB vaccine candidate pipeline but is also suitable for the needs of the global community.

An estimated 200 million people are currently infected with schistosomiasis and an additional 800 million reside in high transmission-risk areas in 78 endemic countries. In this report we describe a functional enzymatic assay based on the core calpain antigen (Sm-p80) of the schistosomiasis vaccine, SchistoShield®. A 44 kDa soluble variant of the core Sm-p80 antigen (B7), was assessed for its enzymatic activity using a fluorescent synthetic substrate. Inhibition of the B7 enzymatic activity by Sm-p80-specific antibodies obtained from pre-clinical trials in rodents, non-human primates as well as from participants of the human clinical trials was measured. The B7 enzyme activity followed a Michaelis-Menten-like kinetic behavior. Statistically significant inhibition of the B7 activity was observed by Sm-p80-specific antibodies produced by immunized mice, non-human primates and humans. This quantitative serological assay could be of value in assessing the effectiveness of the SchistoShield® vaccine in human trials in Africa.

IntroductionSchistosomiasis is a neglected tropical disease that puts over 200 million people at risk, and prevention options are sparse with no approved vaccine. Our vaccine candidate, SchistoShield®, is based on an approximately 87 kDa large subunit of calcium activated neutral protease - termed Sm-p80 - combined with a potent TLR4 agonist-based adjuvant. SchistoShield® has been shown to prevent disease throughout the parasitic life cycle - including egg, juvenile, and adult worm stages - in numerous animal models up to and including baboons. SchistoShield® has been shown safe in both preclinical toxicology studies in rabbits and in a Phase 1 clinical trial in the USA. A Phase 1b trial was initiated in 2023 in endemic regions of Africa, and to date no serious safety signals have been reported.MethodsIn preparation for large-scale Phase 2 clinical trials and eventual vaccine deployment, the Sm-p80 antigen production process has been transferred to a manufacturing organization, Quratis Corporation in South Korea, which specializes in preparation of vaccines for large-scale European and African trials. The process of scaling from our current production level of ~ 2000 vaccine doses, to a process that will generate more than 100 million doses has required multiple improvement steps in the process including fermentation, downstream purification of the protein antigen, lyophilization, and fill and finish.ResultsIn this study, we detail the large-scale production process of the SchistoShield® protein product by Quratis. In addition, an effort was made to analyze and compare the Quratis-made lot of Sm-p80, referred to as QTP-105, to the cGMP lot of Sm-p80 which is in use in human trials in the USA and Africa, referred to as Sm-p80 DP (made in USA). We show that QTP-105 demonstrates excellent potency, purity, identity, and endotoxin levels compared to our Phase 1 Sm-p80 DP and is suitable for use in Phase 2 studies and beyond.

Onchocerciasis remains a debilitating neglected tropical disease. Due to the many challenges of current control methods, an effective vaccine against the causative agent Onchocerca volvulus is urgently needed. Mice and cynomolgus macaque non-human primates (NHPs) were immunized with a vaccine consisting of a fusion of two O. volvulus protein antigens, Ov-103 and Ov-RAL-2 (Ov-FUS-1), and three different adjuvants: Advax-CpG, alum, and AlT4. All vaccine formulations induced high antigen-specific IgG titers in both mice and NHPs. Challenging mice with O. volvulus L3 contained within subcutaneous diffusion chambers demonstrated that Ov-FUS-1/Advax-CpG-immunized animals developed protective immunity, durable for at least 11 weeks. Passive transfer of sera, collected at several time points, from both mice and NHPs immunized with Ov-FUS-1/Advax-CpG transferred protection to naïve mice. These results demonstrate that Ov-FUS-1 with the adjuvant Advax-CpG induces durable protective immunity against O. volvulus in mice and NHPs that is mediated by vaccine-induced humoral factors.

This study was conducted to optimize a fusion protein vaccine for translational development as a vaccine against the human tropical parasitic infection, lymphatic filariasis (LF). The vaccine candidate, His-tagged rBmHAXT was developed previously in our laboratory and was tested in various animal models including mouse, gerbils and Rhesus macaque where it exhibited significant levels of vaccine-induced protection. However, for commercial manufacturing and for regulatory approval for human use, there was a need to modify the vaccine antigen and its production and analytical release methods. Therefore, the major focus of this study was to develop a process for manufacturing an affinity tag-free rBmHAXT and evaluate its immunogenicity, potency and protective efficacy in both inbred and outbred mouse models, as well as in outbred gerbil models. Our results demonstrate that the tag-free rBmHAXT vaccine produced with a process suitable for cGMP production had protective properties equivalent to the original His-tagged rBmHAXT.

Syphilis continues to be a significant public health concern worldwide. The disease is endemic in many low- and middle-income countries, and rates have risen sharply in high-income countries over the last decade. The continued prevalence of infectious and congenital syphilis worldwide highlights the need for the development of an effective syphilis vaccine to complement public health measures for syphilis control. The complex, multi-stage course of syphilis infection necessitates a holistic approach to the development of an effective vaccine, in which immunization prevents both the localized stage of infection (typified by the highly infectious chancre) and the disseminated stages of infection (typified by the secondary rash, neurosyphilis, and destructive tertiary lesions, as well as congenital syphilis). Inhibiting development of the infectious chancre would reduce transmission thus providing community- level protection, while preventing dissemination would provide individual-level protection by reducing serious sequelae and may also provide community level protection by reducing shedding during secondary syphilis. In the current study we build upon prior investigations which demonstrated that immunizations with individual, well characterized T. pallidum TprK, TprC, and Tp0751 peptides elicits partial protection against infection in the animal model. Specifically, we show here that immunization with a TprC/TprK/Tp0751 tri-antigen cocktail protects animals from progressive syphilis lesions and substantially inhibits dissemination of the infection.

Introduction Human onchocerciasis caused by the filarial nematode parasite Onchocerca volvulus remains a major cause of debilitating disease infecting millions primarily in Sub-Saharan Africa. The development of a prophylactic vaccine, along with mass drug administration, would facilitate meeting the goal of onchocerciasis elimination by 2030.Areas covered Models used to study immunity to Onchocerca include natural infection of cattle with Onchocerca ochengi and O. volvulus infective third-stage larvae implanted within diffusion chambers in mice. A vaccine, comprised of two adjuvanted recombinant antigens, induced protective immunity in genetically diverse mice suggesting that it will function similarly in diverse human populations. These antigens were recognized by immune humans and also induced protective immunity against Brugia malayi. We describe the development of a fusion protein composed of the two vaccine antigens with the plan to test the vaccine in cows and non-human primates as a prelude to the initiation of phase 1 clinical trials.Expert opinion The adjuvanted O. volvulus vaccine composed of two antigens Ov-103 and Ov-RAL-2 was shown to be consistently effective at inducing protective immunity using multiple immune mechanisms. The vaccine is ready for further evaluation in other animal models before moving to clinical trials in humans.