Programming Bordetella pertussis lipid A to promote adjuvanticity.

BACKGROUND: Bordetella pertussis is the causative agent of whooping cough or pertussis. Although both acellular (aP) and whole-cell pertussis (wP) vaccines protect against disease, the wP vaccine, which is highly reactogenic, is better at preventing colonization and transmission. Reactogenicity is mainly attributed to the lipid A moiety of B. pertussis lipooligosaccharide (LOS). Within LOS, lipid A acts as a hydrophobic anchor, engaging with TLR4-MD2 on host immune cells to initiate both MyD88-dependent and TRIF-dependent pathways, thereby influencing adaptive immune responses. Lipid A variants, such as monophosphoryl lipid A (MPLA) can also act as adjuvants. Adjuvants may overcome the shortcomings of aP vaccines. RESULTS: This work used lipid A modifying enzymes from other bacteria to produce an MPLA-like adjuvant strain in B. pertussis. We created B. pertussis strains with distinct lipid A modifications, which were validated using MALDI-TOF. We engineered a hexa-acylated monophosphorylated lipid A that markedly decreased human TLR4 activation and activated the TRIF pathway. The modified lipooligosaccharide (LOS) promoted IRF3 phosphorylation and type I interferon production, similar to MPLA responses. We generated three other variants with increased adjuvanticity properties and reduced endotoxicity. Pyrogenicity studies using the Monocyte Activation Test (MAT) revealed that these four lipid A variants significantly decreased the IL-6, a marker for fever, response in peripheral blood mononuclear cells (PBMCs). CONCLUSION: These findings pave the way for developing wP vaccines that are possibly less reactogenic and designing adaptable adjuvants for current vaccine formulations, advancing more effective immunization strategies against pertussis.

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.

CD4+ and CD8+ T-cell multi-epitope chimeric protein associated with an MPLA adjuvant induce protective efficacy and long-term immunological memory for the immunoprophylaxis of American Tegumentary Leishmaniasis.

American Tegumentary Leishmaniasis (ATL) is a disease of high severity and incidence in Brazil, in addition to being a worldwide concern in public health. Leishmania amazonensis is one of the etiological agents of ATL, and the inefficiency of control measures, associated with the high toxicity of the treatment and the lack of effective immunoprophylactic strategies, makes the development of vaccines indispensable and imminent. In this light, the present study proposes to elaborate a chimeric protein (rChiP), based on the fusion of multiple epitopes of CD4+/CD8+ T cells, identified in the immunoproteome of the parasites L. amazonensis and L. braziliensis. The designed chimeric protein was tested in the L. amazonensis murine model of infection using the following formulations: 25 µg of the rChiP in saline (rChiP group) and 25 µg of the rChiP plus 25 µg of MPLA-PHAD® (rChiP+MPLA group). After completing immunization, CD4+ and CD8+ T cells, stimulated with SLa-Antigen or rChiP, showed an increased production of nitric oxide and intracytoplasmic pro-inflammatory cytokines, in addition to the generation of central and effector memory T cells. rChiP and rChiP+MPLA formulations were able to promote an effective protection against L. amazonensis infection determined by a reduction in the development of skin lesions and lower parasitic burden. Reduction in the development of skin lesions and lower parasitic burden in the vaccinated groups were associated with an increase of nitrite, CD4+/CD8+IFN-γ+TNF-α+ and CD4+/CD8+CD44highCD62Lhigh/low T cells, IgGTotal, IgG2a, and lower rates of IgG1 and CD4+/CD8+IL-10+. This data suggests that proposed formulations could be considered potential tools to prevent ATL.

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.

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.

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.

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.

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.

Monophosphoryl Lipid A-Rhamnose Conjugates as a New Class of Vaccine Adjuvants.

Adjuvant is an integral part of all vaccine formulations but only a few adjuvants with limited efficacies or application scopes are available. Thus, developing more robust and diverse adjuvants is necessary. To this end, a new class of adjuvants having α- and ß-rhamnose (Rha) attached to the 1- and 6'-positions of monophosphoryl lipid A (MPLA) was designed, synthesized, and immunologically evaluated in mice. The results indicated a synergistic effect of MPLA and Rha, two immunostimulators that function via interacting with toll-like receptor 4 and recruiting endogenous anti-Rha antibodies, respectively. All the tested MPLA-Rha conjugates exhibited potent adjuvant activities to promote antibody production against both protein and carbohydrate antigens. Overall, MPLA-α-Rha exhibited better activities than MPLA-ß-Rha, and 6'-linked conjugates were slightly better than 1-linked ones. Particularly, MPLA-1-α-Rha and MPLA-6'-α-Rha were the most effective adjuvants in promoting IgG antibody responses against protein antigen keyhole limpet hemocyanin and carbohydrate antigen sTn, respectively.

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.

Chemical Synthesis of Acetobacter pasteurianus Lipid A with a Unique Tetrasaccharide Backbone and Evaluation of Its Immunological Functions.

Lipopolysaccharide (LPS), a cell surface component of Gram-negative bacteria, activates innate immunity. Its active principle is the terminal glycolipid lipid A. Acetobacter pasteurianus is a Gram-negative bacterium used in the fermentation of traditional Japanese black rice vinegar (kurozu). In this study, we focused on A. pasteurianus lipid A, which is a potential immunostimulatory component of kurozu. The active principle structure of A. pasteurianus lipid A has not yet been identified. Herein, we first systematically synthesized three types of A. pasteurianus lipid As containing a common and unique tetrasaccharide backbone. We developed an efficient method for constructing the 2-trehalosamine skeleton utilizing borinic acid-catalyzed glycosylation to afford 1,1'-α,α-glycoside in high yield and stereoselectivity. A common tetrasaccharide intermediate with an orthogonal protecting group pattern was constructed via [2+2] glycosylation. After introducing various fatty acids, all protecting groups were removed to achieve the first chemical synthesis of three distinct types of A. pasteurianus lipid As. After evaluating their immunological function using both human and murine cell lines, we identified the active principles of A. pasteurianus LPS. We also found the unique anomeric structure of A. pasteurianus lipid A contributes to its high chemical stability.

Monophosphoryl lipid A and poly I:C combination enhances immune responses of equine influenza virus vaccine.

Equine influenza is a contagious respiratory disease caused by H3N8 type A influenza virus. Vaccination against equine influenza is conducted regularly; however, infection still occurs globally because of the short immunity duration and suboptimal efficacy of current vaccines. Hence the objective of this study was to investigate whether an adjuvant combination can improve immune responses to equine influenza virus (EIV) vaccines. Seventy-two mice were immunized with an EIV vaccine only or with monophosphoryl lipid A (MPL), polyinosinic-polycytidylic acid (Poly I:C), or MPL + Poly I:C. Prime immunization was followed by boost immunization after 2 weeks. Mice were euthanized at 4, 8, and 32 weeks post-prime immunization, respectively. Sera were collected to determine humoral response. Bone marrow, spleen, and lung samples were harvested to determine memory cell responses, antigen-specific T-cell proliferation, and lung viral titers. MPL + Poly I:C resulted in the highest IgG, IgG1, and IgG2a antibodies and hemagglutination inhibition titers among the groups and sustained their levels until 32 weeks post-prime immunization. The combination enhanced memory B cell responses in the bone marrow and spleen. At 8 weeks post-prime immunization, the combination induced higher CD8+ central memory T cell frequencies in the lungs and CD8+ central memory T cells in the spleen. In addition, the combination group exhibited enhanced antigen-specific T cell proliferation, except for CD4+ T cells in the lungs. Our results demonstrated improved immune responses when using MPL + Poly I:C in EIV vaccines by inducing enhanced humoral responses, memory cell responses, and antigen-specific T cell proliferation.

Monophosphoryl lipid A as a co-adjuvant in methicillin-resistant Staphylococcus aureus vaccine development: improvement of immune responses in a mouse model of infection.

To increase the effectiveness of methicillin-resistant Staphylococcus aureus vaccines (MRSA), a new generation of immune system stimulating adjuvants is necessary, along with other adjuvants. In some vaccines, monophosphoryl lipid A (MPLA) as a toll-like receptor 4 agonist is currently used as an adjuvant or co-adjuvant. MPLA could increase the immune response and vaccine immunogenicity. The current investigation assessed the immunogenicity and anti-MRSA efficacy of recombinant autolysin formulated in MPLA and Alum as co-adjuvant/adjuvant. r-Autolysin was expressed and purified by Ni-NTA affinity chromatography and characterized by SDS-PAGE. Then, the vaccine candidate formulation in MPLAs and Alum was prepared. To investigate the immunogenic responses, total IgG, isotype (IgG1 and IgG2a) levels, and cytokines (IL-4, IL-12, TNF-α, and IFN-γ) profiles were evaluated by ELISA. Also, the bacterial burden in internal organs, opsonophagocytosis, survival rate, and pathobiology changes was compared among the groups. Results demonstrated that mice immunized with the r-Autolysin + Alum + MPLA Synthetic and r-Autolysin + Alum + MPLA Biologic led to increased levels of opsonic antibodies, IgG1, IgG2a isotype as well as increased levels of cytokines profiles, as compared with other experimental groups. More importantly, mice immunized with MPLA and r-Autolysin exhibited a decrease in mortality and bacterial burden, as compared with the control group. The highest level of survival was seen in the r-Autolysin + Alum + MPLA Synthetic group. We concluded that both MPLA forms, synthetic and biological, are reliable candidates for immune response improvement against MRSA infection.

Recombinant Full-length Plasmodium falciparum Circumsporozoite Protein-Based Vaccine Adjuvanted With Glucopyranosyl Lipid A-Liposome Quillaja saponaria 21: Results of Phase 1 Testing With Malaria Challenge.

BACKGROUND: Malaria is preventable yet causes >600 000 deaths annually. RTS,S, the first marketed malaria vaccine, has modest efficacy, but improvements are needed for eradication. METHODS: We conducted an open-label, dose escalation phase 1 study of a full-length recombinant circumsporozoite protein vaccine (rCSP) administered with adjuvant glucopyranosyl lipid A-liposome Quillaja saponaria 21 formulation (GLA-LSQ) on days 1, 29, and 85 or 1 and 490 to healthy, malaria-naive adults. The primary end points were safety and reactogenicity. The secondary end points were antibody responses and Plasmodium falciparum parasitemia after homologous controlled human malaria infection. RESULTS: Participants were enrolled into 4 groups receiving rCSP/GLA-LSQ: 10 µg × 3 (n = 20), 30 µg × 3 (n = 10), 60 µg × 3 (n = 10), or 60 µg × 2 (n = 9); 10 participants received 30 µg rCSP alone × 3, and there were 6 infectivity controls. Participants experienced no serious adverse events. Rates of solicited and unsolicited adverse events were similar among groups. All 26 participants who underwent controlled human malaria infection 28 days after final vaccinations developed malaria. Increasing vaccine doses induced higher immunoglobulin G titers but did not achieve previously established RTS,S benchmarks. CONCLUSIONS: rCSP/GLA-LSQ had favorable safety results. However, tested regimens did not induce protective immunity. Further investigation could assess whether adjuvant or schedule adjustments improve efficacy. CLINICAL TRIALS REGISTRATION: NCT03589794.

Remodeling Yersinia pseudotuberculosis to generate a highly immunogenic outer membrane vesicle vaccine against pneumonic plague.

SignificanceYersinia pestis, the etiologic agent of plague, has been responsible for high mortality in several epidemics throughout human history. This plague bacillus has been used as a biological weapon during human history and is currently one of the deadliest biological threats. Currently, no licensed plague vaccines are available in the Western world. Since an array of immunogens are enclosed in outer membrane vesicles (OMVs), immune responses elicited by OMVs against a diverse range of antigens may reduce the likelihood of antigen circumvention. Therefore, self-adjuvanting OMVs from a remodeled Yersinia pseudotuberculosis strain as a type of plague vaccine could diversify prophylactic choices and solve current vaccine limitations.

MPLA-Adjuvanted Liposomes Encapsulating S-Trimer or RBD or S1, but Not S-ECD, Elicit Robust Neutralization Against SARS-CoV-2 and Variants of Concern.

Safe and effective vaccines are the best method to defeat worldwide SARS-CoV-2 and its circulating variants. The SARS-CoV-2 S protein and its subunits are the most attractive targets for the development of protein-based vaccines. In this study, we evaluated three lipophilic adjuvants, monophosphoryl lipid A (MPLA), Toll-like receptor (TLR) 1/2 ligand Pam3CSK4, and α-galactosylceramide (α-GalCer), in liposomal and nonliposomal vaccines. The immunological results showed that the MPLA-adjuvanted liposomal vaccine induced the strongest humoral and cellular immunity. Therefore, we further performed a systematic comparison of S-trimer, S-ECD, S1, and RBD as antigens in MPLA-adjuvanted liposomes and found that, although these four vaccines all induced robust specific antibody responses, only S-trimer, S1, and RBD liposomes, but not S-ECD, elicited potent neutralizing antibody responses. Moreover, RBD, S-trimer, and S1 liposomes effectively neutralized variants (B.1.1.7/alpha, B.1.351/beta, P.1/gamma, B.1.617.2/delta, and B.1.1.529/omicron). These results provide important information for the subunit vaccine design against SARS-CoV-2 and its variants.

A two-dose optimum for recombinant S1 protein-based COVID-19 vaccination.

BACKGROUND: Recombinant protein subunit vaccination is considered to be a safe, fast and reliable technique when combating emerging and re-emerging diseases such as coronavirus disease 2019 (COVID-19). Typically, such subunit vaccines require the addition of adjuvants to attain adequate immunogenicity. AS01, which contains adjuvants MPL and saponin QS21, is a liposome-based vaccine adjuvant system that is one of the leading candidates. However, the adjuvant effect of AS01 in COVID-19 vaccines is not well described yet. METHODS: In this study, we utilized a mixture of AS01 as the adjuvant for an S1 protein-based COVID-19 vaccine. RESULTS: The adjuvanted vaccine induced robust immunoglobulin G (IgG) binding antibody and virus-neutralizing antibody responses. Importantly, two doses induced similar levels of IgG binding antibody and neutralizing antibody responses compared with three doses and the antibody responses weakened only slightly over time up to six weeks after immunization. CONCLUSION: These results suggested that two doses may be enough for a clinical vaccine strategy design using MPL & QS21 adjuvanted recombinant protein, especially in consideration of the limited production capacity of COVID-19 vaccine in a public health emergency.

Lipopolysaccharide of the Yersinia pseudotuberculosis Complex.

Lipopolysaccharide (LPS), localized in the outer leaflet of the outer membrane, serves as the major surface component of the Gram-negative bacterial cell envelope responsible for the activation of the host's innate immune system. Variations of the LPS structure utilized by Gram-negative bacteria promote survival by providing resistance to components of the innate immune system and preventing recognition by TLR4. This review summarizes studies of the biosynthesis of Yersinia pseudotuberculosis complex LPSs, and the roles of their structural components in molecular mechanisms of yersiniae pathogenesis and immunogenesis.

Small Molecule Potentiator of Adjuvant Activity Enhancing Survival to Influenza Viral Challenge.

As viruses continue to mutate the need for rapid high titer neutralizing antibody responses has been highlighted. To meet these emerging threats, agents that enhance vaccine adjuvant activity are needed that are safe with minimal local or systemic side effects. To respond to this demand, we sought small molecules that would sustain and improve the protective effect of a currently approved adjuvant, monophosphoryl lipid A (MPLA), a Toll-like receptor 4 (TLR4) agonist. A lead molecule from a high-throughput screen, (N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide, was identified as a hit compound that sustained NF-κB activation by a TLR4 ligand, lipopolysaccharide (LPS), after an extended incubation (16 h). In vitro, the resynthesized compound (2D216) enhanced TLR4 ligand-induced innate immune activation and antigen presenting function in primary murine bone marrow-derived dendritic cells without direct activation of T cells. In vivo murine vaccination studies demonstrated that compound 2D216 acted as a potent co-adjuvant when used in combination with MPLA that enhanced antigen-specific IgG equivalent to that of AS01B. The combination adjuvant MPLA/2D216 produced Th1 dominant immune responses and importantly protected mice from lethal influenza virus challenge. 2D216 alone or 2D216/MPLA demonstrated minimal local reactogenicity and no systemic inflammatory response. In summary, 2D216 augmented the beneficial protective immune responses of MPLA as a co-adjuvant and showed an excellent safety profile.

Yeast Shells Encapsulating Adjuvant AS04 as an Antigen Delivery System for a Novel Vaccine against Toxoplasma Gondii.

Toxoplasma gondii (T. gondii) infection causes severe zoonotic toxoplasmosis, which threatens the safety of almost one-third of the human population globally. However, there is no effective protective vaccine against human toxoplasmosis. This necessitates anti-T. gondii vaccine development, which is a main priority of public health. In this study, we optimized the adjuvant system 04 (AS04), a vaccine adjuvant constituted by 3-O-desacyl-4'-monophosphoryl lipid A (a TLR4 agonist) and aluminum salts, by packing it within natural extracts of ß-glucan particles (GPs) from Saccharomyces cerevisiae to form a GP-AS04 hybrid adjuvant system. Through a simple mixing procedure, we loaded GP-AS04 particles with the total extract (TE) of T. gondii lysate, forming a novel anti-T. gondii vaccine GP-AS04-TE. Results indicated that the hybrid adjuvant can efficiently and stably load antigens, mediate antigen delivery, facilitate the dendritic uptake of antigens, boost dendritic cell maturation and stimulation, and increase the secretion of pro-inflammatory cytokines. In the mouse inoculation model, GP-AS04-TE significantly stimulated the function of dendritic cells, induced a very strong TE-specific humoral and cellular immune response, and finally showed a strong and effective protection against toxoplasma chronic and acute infections. This work proves the potential of GP-AS04 for exploitation as a vaccine against a range of pathogens.