Abstract Background: Irazu Oncology developed a tumor vaccine platform using a proprietary, attenuated Salmonella Typhi strain engineered to express human tumor antigens and shed antigen-decorated outer membrane vesicles (OMVs). This flexible system enables rapid development of mono- or multi-valent vaccines. IRZ-01 is an OMV-based vaccine displaying epitopes from two well-characterized and validated tumor-associated antigens (TAAs): Mucin-1 (MUC1) and CEACAM5 (CEA). Here we show that IRZ-01 administration to immunocompetent mice triggers potent antigen-specific humoral and cellular immunity and confers single-agent antitumor efficacy in models expressing CEA and/or MUC1. Methods: Salmonella Typhi CVD911ΔfliC (pPagL-CEA/MUC1) was cultured in animal-free soytone media to produce IRZ-01 OMVs. OMVs were purified by tangential flow filtration and size-exclusion chromatography. Characterization included DLS (size), TRPS (zeta potential), cryo-EM (morphology), immuno-gold EM, and Western blotting for CEA/MUC1 surface display. Toll-like receptor (TLR) activation was tested using HEK-Blue™ cells expressing human TLR2, 3, 4, 5, 7, 8, or 9 (InvivoGen). Immunogenicity was evaluated in C57BL/6 mice given two doses of IRZ-01 (0.25 µg IV or 2 µg IM, days 0 and 7) or 5×109 CFU live bacteria; controls received PBS. Serum IgG was quantified by ELISA (days -1, 6, 20); T-cell responses assessed by IFN-γ ELISPOT on day 21 splenocytes. Antitumor efficacy was assessed in MC38-CEA and MC38-MUC1 syngeneic models by monitoring tumor growth post-challenge with IRZ-01. Results: Purified IRZ-01 OMVs were 90-100 nm, zeta potential -13.9 mV, with confirmed CEA/MUC1 surface expression by Western blot and immuno-gold EM. IRZ-01 selectively activated TLR2 and TLR4, confirming self-adjuvanting properties due to native microbe-associated molecular patterns (MAMPs) naturally present in their outer membrane.Vaccination elicited rapid, high-titer antigen-specific IgG by day 6, strongly boosted after the second dose, with comparable responses via IV or IM routes and similar to the live vector. IFN-γ ELISPOT showed significant (p<0.05 vs PBS) CEA- and MUC1-specific T-cell responses that correlated closely with antibody levels.Encouraged by strong humoral and cellular responses, we conducted efficacy studies using a syngeneic C57BL/6 mouse model implanted with MC38-CEA and MC38-MUC1 cells, IRZ-01 induced marked tumor growth inhibition; volumes receded shortly after treatment and remained significantly lower than untreated PBS control groups for all experimental groups. Survival data demonstrated >90% survival in groups treated with IRZ-01 regardless of tumor line. Conclusion: IRZ-01 is a potent, self-adjuvanted OMV vaccine that induces robust CEA/MUC1-specific humoral and cellular immunity and delivers strong single-agent efficacy in syngeneic tumor models. Citation Format: Kevin Chen, Caleigh Fletcher, John Cowger, James E. Galen, Mayukh Das, Marcio Chedid. IRZ-01: A self-adjuvanted outer membrane vesicle vaccine targeting CEA and MUC1 for colorectal cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4380.

BACKGROUND:Invasive disease caused by typhoid and nontyphoidal Salmonella (NTS) is a threat confronting young children in sub-Saharan Africa. We are developing a trivalent Salmonella conjugate vaccine (TSCV) consisting of typhoid Vi conjugate and core-O-polysaccharide conjugates from 2 invasive NTS serovars (S. Typhimurium and S. Enteritidis). METHODS:We conducted a randomized-controlled Phase 1/2a trial assessing the safety and immunogenicity of a full-strength TSCV (FS; 25 µg of all 3 polysaccharides), half-strength TSCV (HS; 25 µg of Vi and 12.5 µg of both NTS polysaccharides), dilutional half-strength TSCV (dilHS; 12.5 µg of all 3 polysaccharides), or placebo. Local and systemic adverse events were recorded and blood was collected for serum antibody and antibody-secreting cells (ASCs). RESULTS:In total, 80 evaluable participants aged 20-47 years were enrolled. All vaccines were well tolerated; mild injection site pain and fatigue were the most common reactions reported. Serum antibody and ASC responses to the 3 primary polysaccharide antigens were robust, ranging between 85% and 100% response rates, with no statistically significant difference between the 3 formulations. No placebo recipients manifested significant antibody or ASC responses. CONCLUSIONS:These data encourage the further development of TSCV to address a pressing public health problem in young children of sub-Saharan Africa.

In sub-Saharan Africa, typhoidal and non-typhoidal Salmonella (NTS) are leading causes of invasive disease among young children. Trivalent Salmonella conjugate vaccine (TSCV) consists of Vi capsule polysaccharide conjugated to tetanus toxoid and core-plus-O-polysaccharides from the two most prevalent invasive NTS serovars conjugated to flagellin subunits. We conducted a first-in-human, randomized, placebo-controlled, phase 1 trial evaluating the safety and immunogenicity of TSCV. A total of 22 healthy adults aged 18-45 years were randomly allocated to 6.25-µg TSCV (n = 8), 12.5-µg TSCV (n = 10) or placebo (n = 4). The primary objective was the assessment of safety. The co-primary immunogenicity objective was the serum IgG response against the three vaccine polysaccharides and two flagellin carrier proteins. Here we show that TSCV was safe and well tolerated, meeting the prespecified safety endpoints, with the most common solicited symptom being short-lived injection site pain. For each of the three polysaccharides, immune responses, as demonstrated by ≥4-fold increases over baseline, were observed among all (100%) vaccinees, and no responses were elicited in the placebo group, meeting the prespecified immunogenicity endpoints. The two flagellin components elicited 88% (7/8) and 100% (8/8) responses among 6.25-µg and 12.5-µg TSCV recipients and no placebo recipients. These data warrant further evaluation of TSCV for protection against invasive Salmonella disease. ClinicalTrial.gov identifier: NCT03981952 .

In sub-Saharan Africa, non-typhoidal Salmonella (NTS) and Salmonella Typhi are leading causes of invasive disease among young children. Trivalent Salmonella Conjugate Vaccine (TSCV) consists of Vi capsule polysaccharide conjugated to tetanus toxoid and core-plus-O-polysaccharides from the two most prevalent invasive non-typhoidal serovars (Typhimurium, Enteritidis) conjugated to serovar-homologous flagellin subunits. We conducted a first-in-human, randomized, placebo-controlled, stepwise-dose-escalation phase 1 trial (NCT03981952) evaluating TSCV safety and immunogenicity; 22 healthy adults aged 18-45 years were randomly allocated 6.25 μg TSCV (n=8), 12.5 μg TSCV (n=10), or placebo (n=4). TSCV was safe and well-tolerated, with the most common solicited symptom being short-lived injection site pain. For each of the 3 polysaccharides, serum IgG and IgA ELISA antibody responses, as demonstrated by four-fold or greater increases over baseline, were observed among all vaccinees but among no placebo recipients. Binding and functional antibodies, gut-homing antibody secreting cells, and polysaccharide-specific memory B cells responses were also elicited. ClinicalTrials.gov Registry NCT03981952.

Salmonella enterica is a diverse species that infects both humans and animals. S. enterica subspecies enterica consists of more than 1,500 serovars. Unlike typhoidal Salmonella serovars which are human host-restricted, non-typhoidal Salmonella (NTS) serovars are associated with foodborne illnesses worldwide and are transmitted via the food chain. Additionally, NTS serovars can cause disease in livestock animals causing significant economic losses. Salmonella is a well-studied model organism that is easy to manipulate and evaluate in animal models of infection. Advances in genetic engineering approaches in recent years have led to the development of Salmonella vaccines for both humans and animals. In this review, we focus on current progress of recombinant live-attenuated Salmonella vaccines, their use as a source of antigens for parenteral vaccines, their use as live-vector vaccines to deliver foreign antigens, and their use as therapeutic cancer vaccines in humans. We also describe development of live-attenuated Salmonella vaccines and live-vector vaccines for use in animals.

The use of live-attenuated bacterial vaccines as carriers for the mucosal delivery of foreign antigens to stimulate the mucosal immune system was first proposed over three decades ago. This novel strategy aimed to induce immunity against at least two distinct pathogens using a single bivalent carrier vaccine. It was first tested using a live-attenuated Salmonella enterica serovar Typhi strain in clinical trials in 1984, with excellent humoral immune responses against the carrier strain but only modest responses elicited against the foreign antigen. Since then, clinical trials with additional Salmonella-based carrier vaccines have been conducted. As with the original trial, only modest foreign antigen-specific immunity was achieved in most cases, despite the incorporation of incremental improvements in antigen expression technologies and carrier design over the years. In this review, we will attempt to deconstruct carrier vaccine immunogenicity in humans by examining the basis of bacterial immunity in the human gastrointestinal tract and how the gut detects and responds to pathogens versus benign commensal organisms. Carrier vaccine design will then be explored to determine the feasibility of retaining as many characteristics of a pathogen as possible to elicit robust carrier and foreign antigen-specific immunity, while avoiding over-stimulation of unacceptably reactogenic inflammatory responses.

Although immunity induced by typhoid fever is moderated and short-lived, typhoid vaccination with the attenuated Ty21a oral vaccine generates long-lasting protection rates reaching up to 92%. Thus, there are important differences on how wild-type Salmonella and typhoid vaccine strains stimulate host immunity. We hypothesize that vaccine strains with different mutations might affect gut inflammation and intestinal permeability by different mechanisms. To test this hypothesis, we used an in vitro organotypic model of the human intestinal mucosa composed of human intestinal epithelial cells, lymphocytes/monocytes, endothelial cells, and fibroblasts. We also used six Salmonella enterica serovar Typhi (S. Typhi) strains: the licensed Ty21a oral vaccine, four typhoid vaccine candidates (i.e., CVD 908, CVD 909, CVD 910, and CVD 915) and the wild-type Ty2 strain. We found that genetically engineered S. Typhi vaccine strains elicit differential host changes not only in the intestinal permeability and secretion of inflammatory cytokines, but also in the phenotype and activation pathways of innate cells. These changes were distinct from those elicited by the parent wild-type S. Typhi and depended on the genetic manipulation. In sum, these results emphasize the importance of carefully selecting specific manipulations of the Salmonella genome in the development of typhoid vaccines.

Poor antigen delivery is one of the major limitations of modern cancer vaccine vectors. To overcome this challenge, we exploited Salmonella Pathogenicity Island 2 and type III secretion system to deliver tumor-associated antigen (TAAs) into the cytosol of antigen-presenting cells in situ. In a recent report, we demonstrated that an attenuated strain of S. typhimurium, which was engineered to express SPI2-regulated oncoprotein survivin (SVN), induced potent CD8 T-cell-mediated antitumor response that was curative in a murine model of highly aggressive lymphoma. In further development of this technology for clinical use, the vaccine has been transferred from an experimental strain to a clinically validated strain of S. typhi, CVD908. To adapt CVD908 to stably express recombinant antigens without antibiotic-dependent selection, we used a recently reported plasmid stabilization system that encodes the single-stranded binding protein (SSB), an essential protein in DNA metabolism, which was deleted from the bacterial chromosome. The SPI2-regulated expression cassette was then cloned into the SSB plasmid, so that the resultant construct maintained bacterial vector stability while expressing and translocating TAAs. We found that CVD908Δssb vector effectively infects human dendritic cells in vitro and translocates recombinant SVN and MYCN oncoproteins into their cytosol. Furthermore, CVD908Δssb remains stable in mice and induces generation of antigen-specific CD8 T cells. Therapeutic vaccination of tumor-bearing mice with SVN or MYCN vaccine produced potent antitumor activity in murine models of lymphoma or neuroblastoma. The results justify clinical testing of CVD908Δssb-based SVN and MYCN vaccines in cancer patients.

Invasive infections associated with non-typhoidal Salmonella (NTS) serovars Enteritidis (SE), Typhimurium (STm) and monophasic variant 1,4,[5],12:i:- are a major health problem in infants and young children in sub-Saharan Africa, and currently, there are no approved human NTS vaccines. NTS O-polysaccharides and flagellin proteins are protective antigens in animal models of invasive NTS infection. Conjugates of SE core and O-polysaccharide (COPS) chemically linked to SE flagellin have enhanced the anti-COPS immune response and protected mice against fatal challenge with a Malian SE blood isolate. We report herein the development of a STm glycoconjugate vaccine comprised of STm COPS conjugated to the homologous serovar phase 1 flagellin protein (FliC) with assessment of the role of COPS O-acetyls for functional immunity. Sun-type COPS conjugates linked through the polysaccharide reducing end to FliC were more immunogenic and protective in mice challenged with a Malian STm blood isolate than multipoint lattice conjugates (>95% vaccine efficacy [VE] versus 30-43% VE). Immunization with de-O-acetylated STm-COPS conjugated to CRM197 provided significant but reduced protection against STm challenge compared to mice immunized with native STm-COPS:CRM197 (63-74% VE versus 100% VE). Although OPS O-acetyls were highly immunogenic, post-vaccination sera that contained various O-acetyl epitope-specific antibody profiles displayed similar in vitro bactericidal activity when equivalent titers of anti-COPS IgG were assayed. In-silico molecular modeling further indicated that STm OPS forms a single dominant conformation, irrespective of O-acetylation, in which O-acetyls extend outward and are highly solvent exposed. These preclinical results establish important quality attributes for an STm vaccine that could be co-formulated with an SE-COPS:FliC glycoconjugate as a bivalent NTS vaccine for use in sub-Saharan Africa.

Space exploration programs have long been interested in the effects of spaceflight on biology. This research is important not only in its relevance to future deep space exploration, but also because it has allowed investigators to ask questions about how gravity impacts cell behavior here on Earth. In the 1980s, scientists designed and built the first rotating wall vessel, capable of mimicking the low shear environment found in space. This vessel has since been used to investigate growth of both microorganisms and human tissue cells in low shear modeled microgravity conditions. Bacterial behavior has been shown to be altered both in space and under simulated microgravity conditions. In some cases, bacteria appear attenuated, whereas in others virulence is enhanced. This has consequences not only for manned spaceflight, but poses larger questions about the ability of bacteria to sense the world around them. By using the microgravity environment as a tool, we can exploit this phenomenon in the search for new therapeutics and preventatives against pathogenic bacteria for use both in space and on Earth.

Salmonellosis, caused by members of the genus Salmonella, is responsible for considerable global morbidity and mortality in both animals and humans. In this review, we will discuss the pathogenesis of Salmonella enterica serovar Typhi and Salmonella enterica serovar Typhimurium, focusing on human Salmonella infections. We will trace the path of Salmonella through the body, including host entry sites, tissues and organs affected, and mechanisms involved in both pathogenesis and stimulation of host immunity. Careful consideration of the natural progression of disease provides an important context in which attenuated live oral vaccines can be rationally designed and developed. With this in mind, we will describe a series of attenuated live oral vaccines that have been successfully tested in clinical trials and demonstrated to be both safe and highly immunogenic. The attenuation strategies summarized in this review offer important insights into further development of attenuated vaccines against other Salmonella for which live oral candidates are currently unavailable.

Inadequate antigen delivery is one of the major limitations of modern cancer vaccine vectors. To overcome this challenge, we exploited Salmonella Pathogenicity Island 2 (SPI2) and its type III secretion system (T3SS) to deliver a tumor-associated antigen (TAA) of choice into the cytosol of antigen-presenting cells (APC) in situ. The goal of this study was to explore and exploit the potential of SPI2-encoded T3SS of clinically validated S. typhi strain CVD908 for construction of an effective cancer vaccine. We engineered the clinically validated S. typhi strain CVD908 to express SPI2-regulated dominant-negative oncoproteins survivin and MYCN. To adapt CVD908 to stably express recombinant antigens without antibiotic-dependent selection, we used a recently reported plasmid stabilization system that encodes the single-stranded binding protein (SSB), an essential protein in DNA metabolism, which was deleted from the bacterial chromosome. The SPI2-regulated expression cassette was then cloned into the SSB plasmid, so that the resultant construct maintained bacterial vector stability. We found that CVD908Δssb vector could effectively infects dendritic cells and induce antigen-specific CD8 T cell responses in vitro and in vivo. Furthermore, therapeutic vaccination with CVD908Δssb vector expressing survivin or MYCN produced potent antitumor activity in murine models of neuroblastoma and lymphoma. Thus, oral antigen delivery via SPI2-encoded T3SS of Salmonella typhi may provide the foundation of an effective cancer vaccine platform. Supported by grants from CPRIT (RP121035) and The Leukemia and Lymphoma Society.

ABSTRACT Invasive nontyphoidal Salmonella (NTS) infections constitute a major health problem among infants and toddlers in sub-Saharan Africa; these infections also occur in infants and the elderly in developed countries. We genetically engineered a Salmonella enterica serovar Typhimurium strain of multilocus sequence type 313, the predominant genotype circulating in sub-Saharan Africa. We evaluated the capacities of S. Typhimurium and Salmonella enterica serovar Enteritidis ΔguaBA ΔclpX live oral vaccines to protect mice against a highly lethal challenge dose of the homologous serovar and determined protection against other group B and D serovars circulating in sub-Saharan Africa. The vaccines S. Typhimurium CVD 1931 and S. Enteritidis CVD 1944 were immunogenic and protected BALB/c mice against 10,000 50% lethal doses (LD50) of S. Typhimurium or S. Enteritidis, respectively. S. Typhimurium CVD 1931 protected mice against the group B serovar Salmonella enterica serovar Stanleyville (91% vaccine efficacy), and S. Enteritidis CVD 1944 protected mice against the group D serovar Salmonella enterica serovar Dublin (85% vaccine efficacy). High rates of survival were observed when mice were infected 12 weeks postimmunization, indicating that the vaccines elicited long-lived protective immunity. Whereas CVD 1931 did not protect against S. Enteritidis R11, CVD 1944 did mediate protection against S. Typhimurium D65 (81% efficacy). These findings suggest that a bivalent (S. Typhimurium and S. Enteritidis) vaccine would provide broad protection against the majority of invasive NTS infections in sub-Saharan Africa.

Invasive Salmonella infections for which improved or new vaccines are being developed include enteric fever caused by Salmonella enterica serovars Typhi, Paratyphi A, and Paratyphi B and sepsis and meningitis in young children in sub-Saharan Africa caused by nontyphoidal Salmonella (NTS) serovars, particularly S. enterica serovars Typhimurium and Enteritidis. Assays are needed to measure functional antibodies elicited by the new vaccines to assess their immunogenicities and potential protective capacities. We developed in vitro assays to quantify serum bactericidal antibody (SBA) activity induced by S. Typhi, S. Paratyphi A, S. Typhimurium, and S. Enteritidis vaccines in preclinical studies. Complement from various sources was tested in assays designed to measure antibody-dependent complement-mediated killing. Serum from rabbits 3 to 4 weeks of age provided the best complement source compared to serum from pigs, goats, horses, bovine calves, or rabbits 8 to 12 weeks of age. For S. Enteritidis, S. Typhimurium, and S. Typhi SBA assays to be effective, bacteria had to be harvested at log phase. In contrast, S. Paratyphi A was equally susceptible to killing whether it was grown to the stationary or log phase. The typhoidal serovars were more susceptible to complement-mediated killing than were the nontyphoidal serovars. Lastly, the SBA endpoint titers correlated with serum IgG anti-lipopolysaccharide (LPS) titers in mice immunized with mucosally administered S. Typhimurium, S. Enteritidis, and S. Paratyphi A but not S. Typhi live attenuated vaccines. The SBA assay described here is a useful tool for measuring functional antibodies elicited by Salmonella vaccine candidates.

ABSTRACT Live attenuated bacteria hold great promise as multivalent mucosal vaccines against a variety of pathogens. A major challenge of this approach has been the successful delivery of sufficient amounts of vaccine antigens to adequately prime the immune system without overattenuating the live vaccine. Here we used a live attenuated Salmonella enterica serovar Typhi strain to create a bivalent mucosal plague vaccine that produces both the protective F1 capsular antigen of Yersinia pestis and the LcrV protein required for secretion of virulence effector proteins. To reduce the metabolic burden associated with the coexpression of F1 and LcrV within the live vector, we balanced expression of both antigens by combining plasmid-based expression of F1 with chromosomal expression of LcrV from three independent loci. The immunogenicity and protective efficacy of this novel vaccine were assessed in mice by using a heterologous prime-boost immunization strategy and compared to those of a conventional strain in which F1 and LcrV were expressed from a single low-copy-number plasmid. The serum antibody responses to lipopolysaccharide (LPS) induced by the optimized bivalent vaccine were indistinguishable from those elicited by the parent strain, suggesting an adequate immunogenic capacity maintained through preservation of bacterial fitness; in contrast, LPS titers were 10-fold lower in mice immunized with the conventional vaccine strain. Importantly, mice receiving the optimized bivalent vaccine were fully protected against lethal pulmonary challenge. These results demonstrate the feasibility of distributing foreign antigen expression across both chromosomal and plasmid locations within a single vaccine organism for induction of protective immunity.

•Describes recent advances in the rational engineering of live bacterial vaccines.•Explores the challenge of achieving the proper balance between attenuation and immunogenicity.•The availability of powerful genetic strategies suggests a bright future in vaccine development.