Characterization of Turbo, a TLR Ligand-based Adjuvant for Glycoconjugate Vaccines.

Many bacterial polysaccharide vaccines, including the typhoid Vi polysaccharide (ViPS) and tetravalent meningococcal polysaccharide conjugate (MCV4) vaccines, do not incorporate adjuvants and are not highly immunogenic, particularly in infants. I found that endotoxin, a TLR4 ligand in ViPS, contributes to the immunogenicity of typhoid vaccines. Because endotoxin is pyrogenic, and its levels are highly variable in vaccines, I developed monophosphoryl lipid A, a nontoxic TLR4 ligand-based adjuvant named Turbo. Admixing Turbo with ViPS and MCV4 vaccines improved their immunogenicity across all ages and eliminated booster requirement. To understand the characteristics of this adjuvanticity, I compared Turbo with alum. Unlike alum, which polarizes the response toward the IgG1 isotype, Turbo promoted Ab class switching to all IgG isotypes with affinity maturation; the magnitude of this IgG response is durable and accompanied by the presence of long-lived plasma cells in the mouse bone marrow. In striking contrast with the pathways employed by alum, Turbo adjuvanticity is independent of NLPR3, pyroptotic cell death effector Gasdermin D, and canonical and noncanonical inflammasome activation mediated by Caspase-1 and Caspase-11, respectively. Turbo adjuvanticity is primarily dependent on the MyD88 axis and is lost in mice deficient in costimulatory molecules CD86 and CD40, indicating that Turbo adjuvanticity includes activation of these pathways. Because Turbo formulations containing either monophosphoryl lipid A or TLR2 ligands, Pam2CysSerLys4, and Pam3CysSerLys4 help generate Ab response of all IgG isotypes, as an adjuvant Turbo can improve the immunogenicity of glycoconjugate vaccines against a wide range of bacterial pathogens whose elimination requires appropriate IgG isotypes.

Lipid A-modified Escherichia coli can produce porcine parvovirus virus-like particles with high immunogenicity and minimal endotoxin activity.

BACKGROUND: A cost-effective Escherichia coli expression system has gained popularity for producing virus-like particle (VLP) vaccines. However, the challenge lies in balancing the endotoxin residue and removal costs, as residual endotoxins can cause inflammatory reactions in the body. RESULTS: In this study, porcine parvovirus virus-like particles (PPV-VLPs) were successfully assembled from Decreased Endotoxic BL21 (BL21-DeE), and the effect of structural changes in the lipid A of BL21 on endotoxin activity, immunogenicity, and safety was investigated. The lipopolysaccharide purified from BL21-DeE produced lower IL-6 and TNF-α than that from wild-type BL21 (BL21-W) in both RAW264.7 cells and BALB/c mice. Additionally, mice immunized with PPV-VLP derived form BL21-DeE (BL21-DeE-VLP) showed significantly lower production of inflammatory factors and a smaller increase in body temperature within 3 h than those immunized with VLP from BL21-W (BL21-W-VLP) and endotoxin-removed VLP (ReE-VLP). Moreover, mice in the BL21-DeE-VLP immunized group had similar levels of serum antibodies as those in the BL21-W-VLP group but significantly higher levels than those in the ReE-VLP group. Furthermore, the liver, lungs, and kidneys showed no pathological damage compared with the BL21-W-VLP group. CONCLUSION: Overall, this study proposes a method for producing VLP with high immunogenicity and minimal endotoxin activity without chemical or physical endotoxin removal methods. This method could address the issue of endotoxin residues in the VLP and provide production benefits.

Tumor targeting peptide TMTP1 modified Antigen capture Nano-vaccine combined with chemotherapy and PD-L1 blockade effectively inhibits growth of ovarian cancer.

The mortality of ovarian cancer (OC) has long been the highest among gynecological malignancies. Although OC is considered to be an immunogenic tumor, the effect of immunotherapy is not satisfactory. The immunosuppressive microenvironment is one reason for this, and the absence of recognized effective antigens for vaccines is another. Chemotherapy, as one of the most commonly used treatment for OC, can produce chemotherapy-associated antigens (CAAs) during treatment and show the effect of in situ vaccine. Herein, we designed an antigen capture nano-vaccine NP-TP1@M-M with tumor targeting peptide TMTP1 and dendritic cell (DC) receptor mannose assembled on the surface and adjuvant monophosphoryl lipid A (MPLA) encapsulated in the core of poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles. PLGA itself possessed the ability of antigen capture. TMTP1 was a tumor-homing peptide screened by our research team, which held extensive and excellent tumor targeting ability. After these modifications, NP-TP1@M-M could capture and enrich more tumor-specific antigens after chemotherapy, stimulate DC maturation, activate the adaptive immunity and combined with immune checkpoint blockade to maximize the release of the body's immune potential, providing an eutherapeutic strategy for the treatment of OC.

Identification of a Chimera Mass Spectrum of Isomeric Lipid A Species Using Negative Ion Tandem Mass Spectrometry.

The toxic nature of bacterial endotoxins is affected by the structural details of lipid A, including the variety and position of acyl chains and phosphate group(s) on its diglucosamine backbone. Negative-ion mode tandem mass spectrometry is a primary method for the structure elucidation of lipid A, used independently or in combination with separation techniques. However, it is challenging to accurately characterize constitutional isomers of lipid A extracts by direct mass spectrometry, as the elemental composition and molecular mass of these molecules are identical. Thus, their simultaneous fragmentation leads to a composite, so-called chimera mass spectrum. The present study focuses on the phosphopositional isomers of the classical monophosphorylated, hexaacylated Escherichia coli-type lipid A. Collision-induced dissociation (CID) was performed in an HPLC-ESI-QTOF system. Energy-resolved mass spectrometry (ERMS) was applied to uncover the distinct fragmentation profiles of the phosphorylation isomers. A fragmentation strategy applying multi-levels of collision energy has been proposed and applied to reveal sample complexity, whether it contains only a 4'-phosphorylated species or a mixture of 1- and 4'-phosphorylated variants. This comparative fragmentation study of isomeric lipid A species demonstrates the high potential of ERMS-derived information for the successful discrimination of co-ionized phosphorylation isomers of hexaacylated lipid A.

Adjuvant system AS01: from mode of action to effective vaccines.

INTRODUCTION: The use of novel adjuvants in human vaccines continues to expand as their contribution to preventing disease in challenging populations and caused by complex pathogens is increasingly understood. AS01 is a family of liposome-based vaccine Adjuvant Systems containing two immunostimulants: 3-O-desacyl-4'-monophosphoryl lipid A and the saponin QS-21. AS01-containing vaccines have been approved and administered to millions of individuals worldwide. AREAS COVERED: Here, we report advances in our understanding of the mode of action of AS01 that contributed to the development of efficacious vaccines preventing disease due to malaria, herpes zoster, and respiratory syncytial virus. AS01 induces early innate immune activation that induces T cell-mediated and antibody-mediated responses with optimized functional characteristics and induction of immune memory. AS01-containing vaccines appear relatively impervious to baseline immune status translating into high efficacy across populations. Currently licensed AS01-containing vaccines have shown acceptable safety profiles in clinical trials and post-marketing settings. EXPERT OPINION: Initial expectations that adjuvantation with AS01 could support effective vaccine responses and contribute to disease control have been realized. Investigation of the utility of AS01 in vaccines to prevent other challenging diseases, such as tuberculosis, is ongoing, together with efforts to fully define its mechanisms of action in different vaccine settings.

Adjuvants are added to vaccines to increase the immune response produced after vaccination. Adjuvant Systems contain two or more molecules that stimulate the immune system. AS01 is an Adjuvant System that contains two components, MPL and QS-21, that stimulate the immune system. AS01 is included in three approved vaccines: a malaria vaccine for children, a herpes zoster vaccine for older adults, and a respiratory syncytial virus vaccine also for older adults. Vaccines containing AS01 have been extensively evaluated in clinical trials and administered to millions of individuals during market use. These vaccines are effective in preventing disease and have acceptable safety in different age groups. Experiments have been done to investigate how AS01 works in vaccines to produce an efficient immune response that helps to protect against the disease being targeted. A key effect of AS01 is to encourage specific immune cells to produce chemicals that stimulate the immune system. We now know that this effect is due to co-operation between MPL and QS-21. Experiments have shown that AS01 induces a sophisticated immune 'gene signature' in blood within 24 h after vaccination, and people who developed this 'gene signature' had a stronger response to vaccination. AS01 seems to be able to stimulate the immune system of most people ­ even if they are older or have a weakened immune system. This means that AS01 could be included in other vaccines against other challenging diseases, such as tuberculosis, or could be used in the treatment of some disease, such as chronic hepatitis B.

Functional and epigenetic changes in monocytes from adults immunized with an AS01-adjuvanted vaccine.

The adjuvant AS01 plays a key role in the immunogenicity of several approved human vaccines with demonstrated high efficacy. Its adjuvant effect relies on activation of the innate immune system. However, specific effects of AS01-adjuvanted vaccines on innate cell function and epigenetic remodeling, as described for Bacille Calmette-Guérin (BCG) and influenza vaccines, are still unknown. We assessed the long-term functional and epigenetic changes in circulating monocytes and dendritic cells induced by a model vaccine containing hepatitis B surface antigen and AS01 in healthy adults (NCT01777295). The AS01-adjuvanted vaccine, but not an Alum-adjuvanted vaccine, increased the number of circulating monocytes and their expression of human leukocyte antigen (HLA)-DR, which correlated with the magnitude of the memory CD4+ T cell response. Single-cell analyses revealed epigenetic alterations in monocyte and dendritic cell subsets, affecting accessibility of transcription factors involved in cell functions including activator protein-1 (AP-1), GATA, C/EBP, and interferon regulatory factor. The functional changes were characterized by a reduced proinflammatory response to Toll-like receptor activation and an improved response to interferon-γ, a cytokine critical for the adjuvant's mode of action. Epigenetic changes were most evident shortly after the second vaccine dose in CD14+ monocytes, for which accessibility differences of some transcription factors could persist for up to 6 months postvaccination. Together, we show that reprogramming of monocyte subsets occurs after vaccination with an AS01-adjuvanted vaccine, an effect that may contribute to the impact of vaccination beyond antigen-specific protection.

Targeted Liposomal Co-delivery of an Immunogenic Cell Death Inducer and a Toll-Like Receptor 4 Agonist for Enhanced Cancer Chemo-immunotherapy.

Anticancer chemo-immunotherapy has gained considerable attention across various scientific domains as a prospective approach for the comprehensive eradication of malignant tumors. Recent research has particularly been focused on traditional anthracycline chemo drugs, such as doxorubicin and mitoxantrone. These compounds trigger apoptosis in tumor cells and evoke immunogenic cell death (ICD). ICD is a pivotal initiator of the cancer-immunity cycle by facilitating the release of damage-associated molecular patterns (DAMPs). The resultant DAMPs released from cancer cells effectively activate the immune system, resulting in an increase in tumor-infiltrating T cells. In this study, we have innovated a co-delivery strategy involving folate-modified liposomes to deliver doxorubicin and monophosphoryl lipid A (MPLA) simultaneously to tumor tissue. The engineered liposomes exploit the overexpression of folate receptors within the tumor tissues. Delivered doxorubicin initiates ICD at the tumor cells, further enhancing the immunogenic stimulus. Additionally, MPLA helps T cell priming by activating antigen-presenting cells. This intricate interplay culminates in a synergistic effect, ultimately resulting in an augmented and potentiated anticancer chemo-immunotherapeutic liposomal treatment.

Native mass spectrometry and structural studies reveal modulation of MsbA-nucleotide interactions by lipids.

The ATP-binding cassette (ABC) transporter, MsbA, plays a pivotal role in lipopolysaccharide (LPS) biogenesis by facilitating the transport of the LPS precursor lipooligosaccharide (LOS) from the cytoplasmic to the periplasmic leaflet of the inner membrane. Despite multiple studies shedding light on MsbA, the role of lipids in modulating MsbA-nucleotide interactions remains poorly understood. Here we use native mass spectrometry (MS) to investigate and resolve nucleotide and lipid binding to MsbA, demonstrating that the transporter has a higher affinity for adenosine 5'-diphosphate (ADP). Moreover, native MS shows the LPS-precursor 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A (KDL) can tune the selectivity of MsbA for adenosine 5'-triphosphate (ATP) over ADP. Guided by these studies, four open, inward-facing structures of MsbA are determined that vary in their openness. We also report a 2.7 Å-resolution structure of MsbA in an open, outward-facing conformation that is not only bound to KDL at the exterior site, but with the nucleotide binding domains (NBDs) adopting a distinct nucleotide-free structure. The results obtained from this study offer valuable insight and snapshots of MsbA during the transport cycle.

Potent activity of polymyxin B is associated with long-lived super-stoichiometric accumulation mediated by weak-affinity binding to lipid A.

Polymyxins are gram-negative antibiotics that target lipid A, the conserved membrane anchor of lipopolysaccharide in the outer membrane. Despite their clinical importance, the molecular mechanisms underpinning polymyxin activity remain unresolved. Here, we use surface plasmon resonance to kinetically interrogate interactions between polymyxins and lipid A and derive a phenomenological model. Our analyses suggest a lipid A-catalyzed, three-state mechanism for polymyxins: transient binding, membrane insertion, and super-stoichiometric cluster accumulation with a long residence time. Accumulation also occurs for brevicidine, another lipid A-targeting antibacterial molecule. Lipid A modifications that impart polymyxin resistance and a non-bactericidal polymyxin derivative exhibit binding that does not evolve into long-lived species. We propose that transient binding to lipid A permeabilizes the outer membrane and cluster accumulation enables the bactericidal activity of polymyxins. These findings could establish a blueprint for discovery of lipid A-targeting antibiotics and provide a generalizable approach to study interactions with the gram-negative outer membrane.

Genetic Analysis of the Plasmid-Based Temperature-Lethal Mutant pa1792|lpxH(Ts) in Pseudomonas aeruginosa.

Many enzymes in the Raetz pathway for lipid A biosynthesis in Escherichia coli are essential. A homologous protein Pa1792|LpxH in Pseudomonas aeruginosa is known to complement the loss of LpxH in E. coli. Genome-wide transposon-insertion sequencing analysis indicates that lpxH is essential in P. aeruginosa. However, genetic analysis of lpxH in P. aeruginosa has not been carried out, partly because the conditional alleles of essential genes are not readily constructed. In this study, we first constructed a plasmid-based temperature-sensitive mutant ΔlpxH/pTS-lpxH or lpxH(Ts) in P. aeruginosa PAO1. Spot-plating assay indicated that lpxH(Ts) was lethal at a restrictive temperature, confirming its essentiality for growth. Microscopic analysis revealed that lpxH(Ts) exhibited an oval-shaped morphology, suggesting that lpxH was required for rod-shape formation. SDS-PAGE and Western blotting analysis showed that lpxH(Ts) failed to synthesize lipid A, consistent with its function in lipid A biosynthesis. Strong expression of lpxH but not the non-homologous isoenzyme lpxI or lpxG impeded growth and caused cell lysis, implying that lpxH-specific cofactors were required for this toxic effect in P. aeruginosa. Together, our results demonstrate that lpxH is essential for lipid A biosynthesis, rod-shaped growth, and viability in P. aeruginosa. We propose that this plasmid-based conditional allele is a useful tool for the genetic study of essential genes in P. aeruginosa.

A Simple and Rapid Microscale Method for Isolating Bacterial Lipopolysaccharides.

Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of septic shock due to a Gram-negative bacterial infection; thus, the structure and functions of LPSs are of specific interest. Often, highly purified bacterial endotoxins must be isolated from small amounts of biological material. Each of the currently available methods for LPS extraction has certain limitations. Herein, we describe a rapid and simple microscale method for extracting LPSs. The method consists of the following steps: ultrasonic destruction of the bacterial material, LPS extraction via heating, LPS purification with organic solvents, and treatment with proteinase K. LPSs that were extracted by using this method contained less than 2-3% protein and 1% total nucleic acid. We also demonstrated the structural integrity of the O-antigen and lipid A via the sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) methods, respectively. We demonstrated the ability of the extracted LPSs to induce typical secretion of cytokines and chemokines by primary macrophages. Overall, this method may be used to isolate purified LPSs with preserved structures of both the O-antigen and lipid A and unchanged functional activity from small amounts of bacterial biomass.

Design, Synthesis, and Bioactivity Evaluation of a TF-Based Cancer Vaccine Candidate Using Lipid A Mimetics As a Built-In Adjuvant.

This study describes the design and synthesis of five TF-based cancer vaccine candidates using a lipid A mimetic as the carrier and a built-in adjuvant. All synthesized conjugates elicited robust and consistent TF-specific immune responses in mice without external adjuvants. Immunological studies subsequently conducted in wild-type and TLR4 knockout C57BL/6 mice demonstrated that the activation of TLR4 was the main reason that the synthesized lipid A mimetics increased the TF-specific immune responses. All antisera induced by these conjugates can specifically recognize, bind to, and induce the lysis of TF-positive cancer cells. Moreover, representative conjugates 2 and 3 could effectively reduce the growth of tumors and prolong the survival time of mice in vivo, and the efficacies were better than glycoprotein TF-CRM197 with alum adjuvant. Lipid A mimetics could therefore be a promising platform for the development of new carbohydrate-based vaccine carriers with self-adjuvanting properties for the treatment of cancer.

Genetic alterations in the pmrAB two-component system and lipid A biosynthesis genes of polymyxin-resistant Acinetobacter baumannii isolates.

The rate of pandrug-resistant Acinetobacter baumannii strains is on the rise in all continents. This bacterium can acquire resistance to all antibiotics, even to colistin. Alterations in the lipid A or/and the two-component pmrAB were earlier detected in colistin resistance. We investigated and analyzed two strains of A. baumannii (ABRC1 and ABRC2) isolated from two patients admitted to intensive care unit with a septic shock. Both strains were resistant to all tested antibiotics including colistin with a MIC >256 mg L-1. Colistin resistance genes (pmrA, pmrB, lpxA, lpxC, lpxD, and lpsB) of two strains (ABRC1 and ABRC2) were investigated by PCR and sequencing. Obtained nucleic acid sequences were aligned with reference sequences of ATCC 19606 and 17987. In this study two amino acid mutations, N287D in the lpxC gene and E117K in the lpxD gene, were detected in both ABRC1 and ABRC2 strains. ABRC1 had an additional H200L mutation in the pmrA gene. Both colistin resistant strains harbored the same A138T mutation in the pmrB gene. The ABRC2 strain also had an alteration in the kinase domain, specifically an R263S substitution of the histidine kinase domain. Three identical mutations were found in the lpsB gene of both A. baumannii strains: Q216K + H218G + S219E. As a result, a newly deduced protein sequence in both ABRC1 and ABRC2 strains differed from those described in ATCC 17978 and 19606 strains was determined. Colistin resistance is multifactorial in A. baumannii. In our study we detected novel mutations in colistin resistant A. baumannii clinical isolates.

Biomimetic Multilayered Lipid Nanovesicles for Potent Protein Vaccination.

Lipid vesicles are widely used for drug and gene delivery, but their structural instability reduces in vivo efficacy and requires specialized handling. To address these limitations, strategies like lipid cross-linking and polymer-lipid conjugation are suggested to enhance stability and biological efficacy. However, the in vivo metabolism of these altered lipids remains unclear, necessitating further studies. A new stabilization technique without chemical modification is urgently needed. Here, a bio-mimetic approach for fabricating robust multilamellar lipid vesicles to enhance in vivo delivery and stabilization of protein antigens is presented. This method leverages 1-O-acylceramide, a natural skin lipid, to facilitate the self-assembly of lipid nanovesicles. Incorporating 1-O-acylceramide, anchoring lipid bilayers akin to its role in the stratum corneum, provides excellent stability under environmental stresses, including freeze-thaw cycles. Encapsulating ovalbumin as a model antigen and the adjuvant monophosphoryl lipid A demonstrates the vesicle's potential as a nanovaccine platform. In vitro studies show enhanced immune responses with both unilamellar and multilamellar vesicles, but in vivo analyses highlight the superior efficiency of multilamellar vesicles in inducing higher antibody and cytokine levels. This work suggests ceramide-induced multilamellar lipid vesicles as an effective nanovaccine platform for enhanced antigen delivery and stability.

Lipid A modification of colistin-resistant Klebsiella pneumoniae does not alter innate immune response in a mouse model of pneumonia.

Polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae bacteria is associated with high morbidity and mortality in vulnerable populations throughout the world. Ineffective antimicrobial activity by these last resort therapeutics can occur by transfer of mcr-1, a plasmid-mediated resistance gene, causing modification of the lipid A portion of lipopolysaccharide (LPS) and disruption of the interactions between polymyxins and lipid A. Whether this modification alters the innate host immune response or carries a high fitness cost in the bacteria is not well established. To investigate this, we studied infection with K. pneumoniae (KP) ATCC 13883 harboring either the mcr-1 plasmid (pmcr-1) or the vector control (pBCSK) ATCC 13883. Bacterial fitness characteristics of mcr-1 acquisition were evaluated. Differentiated human monocytes (THP-1s) were stimulated with KP bacterial strains or purified LPS from both parent isolates and isolates harboring mcr-1. Cell culture supernatants were analyzed for cytokine production. A bacterial pneumonia model in WT C57/BL6J mice was used to monitor immune cell recruitment, cytokine induction, and bacterial clearance in the bronchoalveolar lavage fluid (BALF). Isolates harboring mcr-1 had increased colistin MIC compared to the parent isolates but did not alter bacterial fitness. Few differences in cytokines were observed with purified LPS from mcr-1 expressing bacteria in vitro. However, in a mouse pneumonia model, no bacterial clearance defect was observed between pmcr-1-harboring KP and parent isolates. Consistently, no differences in cytokine production or immune cell recruitment in the BALF were observed, suggesting that other mechanisms outweigh the effect of these lipid A mutations in LPS.

SARS-CoV-2 recombinant spike ferritin nanoparticle vaccine adjuvanted with Army Liposome Formulation containing monophosphoryl lipid A and QS-21: a phase 1, randomised, double-blind, placebo-controlled, first-in-human clinical trial.

BACKGROUND: A self-assembling SARS-CoV-2 WA-1 recombinant spike ferritin nanoparticle (SpFN) vaccine co-formulated with Army Liposomal Formulation (ALFQ) adjuvant containing monophosphoryl lipid A and QS-21 (SpFN/ALFQ) has shown protective efficacy in animal challenge models. This trial aims to assess the safety and immunogenicity of SpFN/ALFQ in a first-in-human clinical trial. METHODS: In this phase 1, randomised, double-blind, placebo-controlled, first-in-human clinical trial, adults were randomly assigned (5:5:2) to receive 25 µg or 50 µg of SpFN/ALFQ or saline placebo intramuscularly at day 1 and day 29, with an optional open-label third vaccination at day 181. Enrolment and randomisation occurred sequentially by group; randomisation was done by an interactive web-based randomisation system and only designated unmasked study personnel had access to the randomisation code. Adults were required to be seronegative and unvaccinated for inclusion. Local and systemic reactogenicity, adverse events, binding and neutralising antibodies, and antigen-specific T-cell responses were quantified. For safety analyses, exact 95% Clopper-Pearson CIs for the probability of any incidence of an unsolicited adverse event was computed for each group. For immunogenicity results, CIs for binary variables were computed using the exact Clopper-Pearson methodology, while CIs for geometric mean titres were based on 10 000 empirical bootstrap samples. Post-hoc, paired one-sample t tests were used to assess the increase in mean log-10 neutralising antibody titres between day 29 and day 43 (after the second vaccination) for the primary SARS-CoV-2 targets of interest. This trial is registered at ClinicalTrials.gov, NCT04784767, and is closed to new participants. FINDINGS: Between April 7, and June 29, 2021, 29 participants were enrolled in the study. 20 individuals were assigned to receive 25 µg SpFN/ALFQ, four to 50 µg SpFN/ALFQ, and five to placebo. Neutralising antibody responses peaked at day 43, 2 weeks after the second dose. Neutralisation activity against multiple omicron subvariants decayed more slowly than against the D614G or beta variants until 5 months after second vaccination for both dose groups. CD4+ T-cell responses were elicited 4 weeks after the first dose and were boosted after a second dose of SpFN/ALFQ for both dose groups. Neutralising antibody titres against early omicron subvariants and clade 1 sarbecoviruses were detectable after two immunisations and peaked after the third immunisation for both dose groups. Neutralising antibody titres against XBB.1.5 were detected after three vaccinations. Passive IgG transfer from vaccinated volunteers into Syrian golden hamsters controlled replication of SARS-CoV-1 after challenge. INTERPRETATION: SpFN/ALFQ was well tolerated and elicited robust and durable binding antibody and neutralising antibody titres against a broad panel of SARS-CoV-2 variants and other sarbecoviruses. FUNDING: US Department of Defense, Defense Health Agency.

Decellularized skin pretreatment by monophosphoryl lipid A and lactobacillus casei supernatant accelerate skin recellularization.

BACKGROUND: Bioscaffolds and cells are two main components in the regeneration of damaged tissues via cell therapy. Umbilical cord stem cells are among the most well-known cell types for this purpose. The main objective of the present study was to evaluate the effect of the pretreatment of the foreskin acellular matrix (FAM) by monophosphoryl lipid A (MPLA) and Lactobacillus casei supernatant (LCS) on the attraction of human umbilical cord mesenchymal stem cells (hucMSC). METHODS AND RESULTS: The expression of certain cell migration genes was studied using qRT-PCR. In addition to cell migration, transdifferentiation of these cells to the epidermal-like cells was evaluated via immunohistochemistry (IHC) and immunocytochemistry (ICC) of cytokeratin 19 (CK19). The hucMSC showed more tissue tropism in the presence of MPLA and LCS pretreated FAM compared to the untreated control group. We confirmed this result by scanning electron microscopy (SEM) analysis, glycosaminoglycan (GAG), collagen, and DNA content. Furthermore, IHC and ICC data demonstrated that both treatments increase the protein expression level of CK19. CONCLUSION: Pretreatment of acellular bioscaffolds by MPLA or LCS can increase the migration rate of cells and also transdifferentiation of hucMSC to epidermal-like cells without growth factors. This strategy suggests a new approach in regenerative medicine.

A TLR4 ligand-based adjuvant for promoting the immunogenicity of typhoid subunit vaccines.

None of the typhoid Vi Polysaccharide (ViPS) subunit vaccines incorporate adjuvants, and the immunogenicity of ViPS vaccines (e.g. Typbar TCV® and Typhim Vi®) is in part due to associated TLR4 ligands such as endotoxin present in these vaccines. Since endotoxin content in vaccines is variable and kept very low due to inherent toxicity, it was hypothesized that incorporating a defined amount of a non-toxic TLR4-ligand such as monophosphoryl lipid A in ViPS vaccines would improve their immunogenicity. To test this hypothesis, a monophosphoryl lipid A-based adjuvant formulation named Turbo was developed. Admixing Turbo with Typbar TCV® (ViPS-conjugated to tetanus toxoid) increased the levels of anti-ViPS IgM, IgG1, IgG2b, IgG2a/c, and IgG3 in inbred and outbred mice. In infant mice, a single immunization with Turbo adjuvanted Typbar TCV® resulted in a significantly increased and durable IgG response and improved the control of bacterial burden compared to mice immunized without Turbo. Similarly, when adjuvanted with Turbo, the antibody response and control of bacteremia were also improved in mice immunized with Typhim Vi®, an unconjugated vaccine. The immunogenicity of unconjugated ViPS is inefficient in young mice and is lost in adult mice when immunostimulatory ligands in ViPS are removed. Nevertheless, when adjuvanted with Turbo, poorly immunogenic ViPS induced a robust IgG response in young and adult mice, and this was observed even under antigen-limiting conditions. These data suggest that incorporation of Turbo as an adjuvant will make typhoid vaccines more immunogenic regardless of their intrinsic immunogenicity or conjugation status and maximize the efficacy across all ages.

A comprehensive review of recent developments in the gram-negative bacterial UDP-2,3-diacylglucosamine hydrolase (LpxH) enzyme.

The emergence of multidrug resistance has provided a great challenge to treat nosocomial infections, which have become a major health threat around the globe. Lipid A (an active endotoxin component), the final product of the Raetz lipid A metabolism pathway, is a membrane anchor of lipopolysaccharide (LPS) of the gram-negative bacterial outer membrane. It shields bacterial cells and serves as a protective barrier from antibiotics, thereby eliciting host response and making it difficult to destroy. UDP-2,3-diacylglucosamine pyrophosphate hydrolase (LpxH), a crucial peripheral membrane enzyme of the Raetz pathway, turned out to be the potential target to inhibit the production of Lipid A. This review provides a comprehensive compilation of information regarding the structural and functional aspects of LpxH, as well as its analogous LpxI and LpxG. In addition, apart from by providing a broader understanding of the enzyme-inhibitor mechanism, this review facilitates the development of novel drug candidates that can inhibit the pathogenicity of the lethal bacterium.

Pseudomonas aeruginosa Lipid A Structural Variants Induce Altered Immune Responses.

Pseudomonas aeruginosa causes chronic lung infection in cystic fibrosis (CF), resulting in structural lung damage and progressive pulmonary decline. P. aeruginosa in the CF lung undergoes numerous changes, adapting to host-specific airway pressures while establishing chronic infection. P. aeruginosa undergoes lipid A structural modification during CF chronic infection that is not seen in any other disease state. Lipid A, the membrane anchor of LPS (i.e., endotoxin), comprises the majority of the outer membrane of Gram-negative bacteria and is a potent Toll-like receptor 4 (TLR4) agonist. The structure of P. aeruginosa lipid A is intimately linked with its recognition by TLR4 and subsequent immune response. Prior work has identified P. aeruginosa strains with altered lipid A structures that arise during chronic CF lung infection; however, the impact of the P. aeruginosa lipid A structure on airway disease has not been investigated. Here, we show that P. aeruginosa lipid A lacks PagL-mediated deacylation during human airway infection using a direct-from-sample mass spectrometry approach on human BAL fluid. This structure triggers increased proinflammatory cytokine production by primary human macrophages. Furthermore, alterations in lipid A 2-hydroxylation impact cytokine response in a site-specific manner, independent of CF transmembrane conductance regulator function. It is interesting that there is a CF-specific reduction in IL-8 secretion within the epithelial-cell compartment that only occurs in CF bronchial epithelial cells when infected with CF-adapted P. aeruginosa that lacks PagL-mediated lipid A deacylation. Taken together, we show that P. aeruginosa alters its lipid A structure during acute lung infection and that this lipid A structure induces stronger signaling through TLR4.