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.

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.

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.

Protective efficacy of Hla-MntC-SACOL0723 fusion protein adjuvanted in alum and MPL against Staphylococcus aureus sepsis infection in mice.

To develop a suitable and effective vaccine against Staphylococcus aureus (S. aureus), we selected the Hla-MntC-SACOL0723 (HMS) recombinant protein with two different formulations of alum and Monophosphoryl lipid A (MPL) adjuvants. In this study, we aimed to evaluate the potentials of alum and MPL adjuvants in stimulating the immune response of HMS vaccine candidate against S. aureus. To evaluate the type of induced immune response, anti-HMS total IgG, IgG1, IgG2a, and IFN-γ, IL-2, IL-4, and IL-17 cytokines were determined after vaccination of mice with HMS-alum, HMS-MPL candidates. Mice were challenged with Methicillin-resistant Staphylococcus aureus (MRSA) was isolated from pressure sores and evaluated for bacterial load in the kidney homogenates and survival rate. It was observed that total IgG and isotypes (IgG1 and IgG2a), IL-4, and IL-17 were significantly increased in the group that received HMS-alum vaccine compared with the group that received HMS-MPL formulation. On the other hand, the levels of IFN-γ and IL-2 cytokines in the group that received HMS-MPL were higher than the group that received HMS-alum formulation. Bacterial load in the mice who received HMS protein formulated with alum adjuvant was reduced more than the mice who received HMS protein formulated with MPL adjuvant. Histopathological analysis showed more pathological changes in kidney tissues of the group received of HMS-MPL compared with the HMS-alum formulation. The survival rate was equal in both groups of immunized with HMS-alum and HMS-MPL formulations. Finally, it could be concluded that both adjuvants of alum and MPL are suitable immune response enhancers to HMS vaccine candidate.

Differential response induced by LPS and MPLA in immunocompetent and septic individuals.

Lipopolysaccharide (LPS) and monophosphoryl lipid A (MPLA) induce, overall, similar transcriptional profiles in healthy individuals, although LPS has been shown to more potently induce pro-inflammatory cytokines. We explore herein whether MPLA could be considered as a synthetic replacement of LPS in immune functional assays to study anergy of immune cells in septic patients. Ex vivo whole blood stimulation with MPLA revealed a lower induction of the TNFα secreted protein in 20 septic patients (SP) compared to 10 healthy volunteers (HV), in agreement with monocyte anergy. Principal component analysis of the 93-gene molecular response to MPLA and LPS stimulation found that the main variability was driven by stimulation in HV and by pathophysiology in SP. MPLA was a stronger inducer of the HLA family genes than LPS in both populations, arguing for divergent signalling pathways downstream of TLR-4. In addition, MPLA appeared to present a more informative stratification potential within the septic population.

Safety and immunogenicity of the adjunct therapeutic vaccine ID93 + GLA-SE in adults who have completed treatment for tuberculosis: a randomised, double-blind, placebo-controlled, phase 2a trial.

BACKGROUND: A therapeutic vaccine that prevents recurrent tuberculosis would be a major advance in the development of shorter treatment regimens. We aimed to assess the safety and immunogenicity of the ID93 + GLA-SE vaccine at various doses and injection schedules in patients with previously treated tuberculosis. METHODS: This randomised, double-blind, placebo-controlled, phase 2a trial was conducted at three clinical sites near Cape Town, South Africa. Patients were recruited at local clinics after receiving 4 months of tuberculosis treatment, and screened for eligibility after providing written informed consent. Participants were aged 18-60 years, BCG-vaccinated, HIV-uninfected, and diagnosed with drug-sensitive pulmonary tuberculosis. Eligible patients had completed standard treatment for pulmonary tuberculosis in the past 28 days. Participants were enrolled after completing standard treatment and randomly assigned sequentially to receive vaccine or placebo in three cohorts: 2 µg intramuscular ID93 + 2 µg GLA-SE on days 0 and 56 (cohort 1); 10 µg ID93 + 2 µg GLA-SE on days 0 and 56 (cohort 2); 2 µg ID93 + 5 µg GLA-SE on days 0 and 56 and placebo on day 28 (cohort 3); 2 µg ID93 + 5 µg GLA-SE on days 0, 28, and 56 (cohort 3); or placebo on days 0 and 56 (cohorts 1 and 2), with the placebo group for cohort 3 receiving an additional injection on day 28. Randomisation was in a ratio of 3:1 for ID93 + GLA-SE and saline placebo in cohorts 1 and 2, and in a ratio of 3:3:1 for (2 ×) ID93 + GLA-SE, (3 ×) ID93 + GLA-SE, and placebo in cohort 3. The primary outcomes were safety and immunogenicity (vaccine-specific antibody response and T-cell response). For the safety outcome, participants were observed for 30 min after each injection, injection site reactions and systemic adverse events were monitored until day 84, and serious adverse events and adverse events of special interest were monitored for 6 months after the last injection. Vaccine-specific antibody responses were measured by serum ELISA, and T-cell responses after stimulation with vaccine antigens were measured in cryopreserved peripheral blood mononuclear cells specimens using intracellular cytokine staining followed by flow cytometry. This study is registered with ClinicalTrials.gov, number NCT02465216. FINDINGS: Between June 17, 2015, and May 30, 2016, we assessed 177 patients for inclusion. 61 eligible patients were randomly assigned to receive: saline placebo (n=5) or (2 ×) 2 µg ID93 + 2 µg GLA-SE (n=15) on days 0 and 56 (cohort 1); saline placebo (n=2) or (2 ×) 10 µg ID93 + 2 µg GLA-SE (n=5) on days 0 and 56 (cohort 2); saline placebo (n=5) on days 0, 28 and 56, or 2 µg ID93 + 5 µg GLA-SE (n=15) on days 0 and 56 and placebo injection on day 28, or (3 ×) 2 µg ID93 + 5 µg GLA-SE (n=14) on days 0, 28, and 56 (cohort 3). ID93 + GLA-SE induced robust and durable antibody responses and specific, polyfunctional CD4 T-cell responses to vaccine antigens. Two injections of the 2 µg ID93 + 5 µg GLA-SE dose induced antigen-specific IgG and CD4 T-cell responses that were significantly higher than those with placebo and persisted for the 6-month study duration. Mild to moderate injection site pain was reported after vaccination across all dose combinations, and induration and erythema in patients given 2 µg ID93 + 5 µg GLA-SE in two or three doses. One participant had grade 3 erythema and induration at the injection site. No vaccine-related serious adverse events were observed. INTERPRETATION: Vaccination with ID93 + GLA-SE was safe and immunogenic for all tested regimens. These data support further evaluation of ID93 + GLA-SE in therapeutic vaccination strategies to improve tuberculosis treatment outcomes. FUNDING: Wellcome Trust (102028/Z/13/Z).

SARS-CoV-2 spike protein binds to bacterial lipopolysaccharide and boosts proinflammatory activity.

There is a link between high lipopolysaccharide (LPS) levels in the blood and the metabolic syndrome, and metabolic syndrome predisposes patients to severe COVID-19. Here, we define an interaction between SARS-CoV-2 spike (S) protein and LPS, leading to aggravated inflammation in vitro and in vivo. Native gel electrophoresis demonstrated that SARS-CoV-2 S protein binds to LPS. Microscale thermophoresis yielded a KD of ∼47 nM for the interaction. Computational modeling and all-atom molecular dynamics simulations further substantiated the experimental results, identifying a main LPS-binding site in SARS-CoV-2 S protein. S protein, when combined with low levels of LPS, boosted nuclear factor-kappa B (NF-κB) activation in monocytic THP-1 cells and cytokine responses in human blood and peripheral blood mononuclear cells, respectively. The in vitro inflammatory response was further validated by employing NF-κB reporter mice and in vivo bioimaging. Dynamic light scattering, transmission electron microscopy, and LPS-FITC analyses demonstrated that S protein modulated the aggregation state of LPS, providing a molecular explanation for the observed boosting effect. Taken together, our results provide an interesting molecular link between excessive inflammation during infection with SARS-CoV-2 and comorbidities involving increased levels of bacterial endotoxins.

Early evolutionary loss of the lipid A modifying enzyme PagP resulting in innate immune evasion in Yersinia pestis.

Immune evasion through membrane remodeling is a hallmark of Yersinia pestis pathogenesis. Yersinia remodels its membrane during its life cycle as it alternates between mammalian hosts (37 °C) and ambient (21 °C to 26 °C) temperatures of the arthropod transmission vector or external environment. This shift in growth temperature induces changes in number and length of acyl groups on the lipid A portion of lipopolysaccharide (LPS) for the enteric pathogens Yersinia pseudotuberculosis (Ypt) and Yersinia enterocolitica (Ye), as well as the causative agent of plague, Yersinia pestis (Yp). Addition of a C16 fatty acid (palmitate) to lipid A by the outer membrane acyltransferase enzyme PagP occurs in immunostimulatory Ypt and Ye strains, but not in immune-evasive Yp Analysis of Yp pagP gene sequences identified a single-nucleotide polymorphism that results in a premature stop in translation, yielding a truncated, nonfunctional enzyme. Upon repair of this polymorphism to the sequence present in Ypt and Ye, lipid A isolated from a Yp pagP+ strain synthesized two structures with the C16 fatty acids located in acyloxyacyl linkage at the 2' and 3' positions of the diglucosamine backbone. Structural modifications were confirmed by mass spectrometry and gas chromatography. With the genotypic restoration of PagP enzymatic activity in Yp, a significant increase in lipid A endotoxicity mediated through the MyD88 and TRIF/TRAM arms of the TLR4-signaling pathway was observed. Discovery and repair of an evolutionarily lost lipid A modifying enzyme provides evidence of lipid A as a crucial determinant in Yp infectivity, pathogenesis, and host innate immune evasion.

Differential Immunogenicity and Protective Efficacy Elicited by MTO- and DMT-Adjuvanted CMFO Subunit Vaccines against Mycobacterium tuberculosis Infection.

Tuberculosis (TB) remains a major and global problem of public health. An effective TB subunit vaccine is urgently needed. Proper selection of the delivery system for the vaccine is crucial for inducing an appropriate immune response tailored to control the target pathogen. In this study, we compared the immunogenicity and protective efficacy of CMFO subunit vaccines against primary progressive TB in two different adjuvant systems: the MTO oil-in-water (O/W) emulsion composed of monophosphoryl lipid A (MPL), trehalose-6,60-dibehenate (TDB), and oil in water emulsion MF59 and the DMT liposome containing dimethyldioctadecylammonium bromide (DDA), monophosphoryl lipid A (MPL), and trehalose-6,60-dibehenate (TDB). Our results demonstrated that the DMT-adjuvanted CMFO could confer more significant protection against M. tuberculosis infection than the CMFO/MTO did in mice. In particular, the adjuvant DMT showed a stronger ability than the O/W emulsion to adjuvant CMFO subunit vaccine and enhanced protection, attributed to elicit Th1-biased responses, strong Th1/Th17 cytokine responses, and IFN-γ + or IL-2+ T cell responses. Therefore, our findings demonstrate that the liposome delivery system shows more effectiveness to adjuvant TB subunit vaccine than O/W emulsion and highlight the importance of adjuvant formulation for the better efficacy of a protein vaccine.

Recombination Monophosphoryl Lipid A-Derived Vacosome for the Development of Preventive Cancer Vaccines.

Recently, there has been an increasing interest for utilizing the host immune system to fight against cancer. Moreover, cancer vaccines, which can stimulate the host immune system to respond to cancer in the long term, are being investigated as a promising approach to induce tumor-specific immunity. In this work, we prepared an effective cancer vaccine (denoted as "vacosome") by reconstructing the cancer cell membrane, monophosphoryl lipid A as a toll-like receptor 4 agonist, and egg phosphatidylcholine. The vacosome triggered and enhanced bone marrow dendritic cell maturation as well as stimulated the antitumor response against breast cancer 4T1 cells in vitro. Furthermore, an immune memory was established in BALB/c mice after three-time preimmunization with the vacosome. After that, the immunized mice showed inhibited tumor growth and prolonged survival period (longer than 50 days). Overall, our results demonstrate that the vacosome can be a potential candidate for clinical translation as a cancer vaccine.

Particle-based, Pfs230 and Pfs25 immunization is effective, but not improved by duplexing at fixed total antigen dose.

BACKGROUND: The Plasmodium falciparum sexual-stage surface proteins Pfs25 and Pfs230 are antigen candidates for a malaria transmission-blocking vaccine (TBV), and have been widely investigated as such. It is not clear whether simultaneously presenting these two antigens in a particulate vaccine would enhance the transmission reducing activity (TRA) of induced antibodies. To assess this, immunization was carried out with liposomes containing synthetic lipid adjuvant monophosphoryl lipid A (MPLA), and cobalt-porphyrin-phospholipid (CoPoP), which rapidly converts recombinant, his-tagged antigens into particles. METHODS: His-tagged, recombinant Pfs25 and Pfs230C1 were mixed with CoPoP liposomes to form a bivalent vaccine. Antigens were fluorescently labelled to infer duplex particleization serum-stability and binding kinetics using fluorescence resonance energy transfer. Mice and rabbits were immunized with individual or duplexed particleized Pfs25 and Pfs230C1, at fixed total antigen doses. The resulting antibody responses were assessed for magnitude and TRA. RESULTS: Pfs230C1 and Pfs25 rapidly bound CoPoP liposomes to form a serum-stable, bivalent particle vaccine. In mice, immunization with 5 ng of total antigen (individual antigen or duplexed) elicited functional antibodies against Pfs25 and Pfs230. Compared to immunization with the individual antigen, Pfs25 antibody production was moderately lower for the bivalent CoPoP vaccine, whereas Pfs230C1 antibody production was not impacted. All antibodies demonstrated at least 92% inhibition in oocyst density at 750 µg/mL purified mouse IgG in the standard membrane feeding assay (SMFA). At lower IgG concentrations, the bivalent vaccine did not improve TRA; antibodies induced by particleized Pfs25 alone showed stronger function in these conditions. In rabbits, immunization with a 20 µg total antigen dose with the duplexed antigens yielded similar antibody production against Pfs25 and Pfs230 compared to immunization with a 20 µg dose of individual antigens. However, no enhanced TRA was observed with duplexing. CONCLUSIONS: Pfs25, Pfs230 or the duplexed combination can readily be prepared as particulate vaccines by mixing CoPoP liposomes with soluble, recombinant antigens. This approach induces potent transmission-reducing antibodies following immunization in mice and rabbits. Immunization with bivalent, particleized, Pfs230 and Pfs25 did not yield antibodies with superior TRA compared to immunization with particleized Pfs25 as a single antigen. Altogether, duplexing antigens is straightforward and effective using CoPoP liposomes, but is likely to be more useful for targeting distinct parasite life stages.

Optimized dose of synthetic analogues of Monophosphoryl lipid A as an effective alternative for formulating recombinant human papillomavirus vaccine.

Adjuvants are a crucial component of recombinant vaccines such as the human papillomavirus (HPV) vaccine. Monophosphoryl lipid A (MPL) extracted from Salmonella Minnesota lipopolysaccharide is used as an adjuvant for the HPV vaccine. Due to the limitations in accessibility and reproducibility of MPL, investigating synthetic analogues of MPL (synMPL) is urgently needed to overcome these limitations. In this study, female BALB/c mice were vaccinated by HPV vaccine formulated with synMPL and aluminum hydroxide gel in which the concentration of synMPL ranged from 0 to 100 µg/dose. Anti-HPV L1 VLP antibody was measured for each group through Indirect ELISA and compared with Cervarix and Gardasil vaccines as approved anti-HPV vaccines. SynMPL showed a concentration-dependent increase up to 50 µg/dose in the immunogenicity of the vaccine. Therefore, synMPL at concentration of 50 µg/dose was selected as optimum concentration. The GMT profiling of synMPL-formulated vaccine (named Papilloguard) and Cervarix was not statistically different (Mann-Whitney test). The Gardasil vaccine showed 10-fold lower GMT for anti-HPV 18 L1 VLP antibody but anti-HPV 16 L1 VLP antibody was similar to Cervarix and Papilloguard. The current findings suggest that the synMPL in combination with aluminum hydroxide could be used as a potential adjuvant candidate for human vaccine.

Immunogenicity of Antigen Adjuvanted with AS04 and Its Deposition in the Upper Respiratory Tract after Intranasal Administration.

Adjuvant system 04 (AS04) is in injectable human vaccines. AS04 contains two known adjuvants, 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and insoluble aluminum salts. Data from previous studies showed that both MPL and insoluble aluminum salts have nasal mucosal vaccine adjuvant activity. The present study was designed to test the feasibility of using AS04 as an adjuvant to help nasally administered antigens to induce specific mucosal and systemic immunity as well as to evaluate the deposition of antigens in the upper respiratory tract when adjuvanted with AS04. Alhydrogel, an aluminum (oxy)hydroxide suspension, was mixed with MPL to form AS04, which was then mixed with ovalbumin (OVA) or 3× M2e-HA2, a synthetic influenza virus hemagglutinin fusion protein, as an antigen to prepare OVA/AS04 and 3× M2e-HA2/AS04 vaccines, respectively. In mice, AS04 enabled antigens, when given intranasally, to induce specific IgA response in nasal and lung mucosal secretions as well as specific IgG response in the serum samples of the immunized mice, whereas subcutaneous injection of the same vaccine induced specific antibody responses only in the serum samples but not in the mucosal secretions. Splenocytes isolated from mice intranasally immunized with the OVA/AS04 also proliferated and released cytokines (i.e., IL-4 and IFN-γ) after in vitro stimulation with the antigen. In the immunogenicity test, intranasal OVA/AS04 was not more effective than intranasal OVA/MPL at the dosing regimens tested. However, when compared to OVA/MPL, OVA/AS04 showed a different atomized droplet size distribution and more importantly a more favorable OVA deposition profile when atomized into a nasal cast that was 3-D printed based on the computer tomography scan of the nose of a child. It is concluded that AS04 has mucosal adjuvant activity when given intranasally. In addition, there is a reason to be optimistic about using AS04 as an adjuvant to target an antigen of interest to the right region of the nasal cavity in humans for immune response induction.

Live attenuated Salmonella Typhimurium with monophosphoryl lipid A retains ability to induce T-cell and humoral immune responses against heterologous polysaccharide of Shigella flexneri 2a.

Shigella flexneri 2a (Sf2a) is one of the most frequently isolated Shigella strains that causes the endemic shigellosis in developing countries. In this study, we used recombinant attenuated Salmonella vaccine (RASV) strains to deliver Sf2a O-antigen and characterized the immune responses induced by the vectored O-antigen. First, we identified genes sufficient for biosynthesis of Sf2a O-antigen. A plasmid containing the identified genes was then introduced into the RASV strains, which were manipulated to produce only the heterologous O-antigen and modified lipid A. After oral immunization of mice, we demonstrated that RASV strains could induce potent humoral immune responses as well as robust CD4+ T-cell responses against Sf2a Lipopolysaccharide (LPS) and protect mice against virulent Sf2a challenge. The induced serum antibodies mediated high levels of Shigella-specific serum bactericidal activity and C3 deposition. Moreover, the IgG+ B220low/int BM cell and T follicular helper (Tfh) cell responses could also be triggered effectively. The live attenuated Salmonella with the modified lipid A delivering Sf2a O-antigen polysaccharide showed the same ability to induce immune responses against Sf2a LPS as the strain with the original lipid A. These findings underscore the potential of RASV delivered Sf2a O-antigen for induction of robust CD4+ T-cell and IgG responses and warrant further studies toward the development of Shigella vaccine candidates with RASV strains.

Characterization of Bacillus anthracis Spore Proteins Using a Nanoscaffold Vaccine Platform.

Subunit vaccines are theoretically safe and easy to manufacture but require effective adjuvants and delivery systems to yield protective immunity, particularly at critical mucosal sites such as the lung. We investigated nanolipoprotein particles (NLPs) containing the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) as a platform for intranasal vaccination against Bacillus anthracis. Modified lipids enabled attachment of disparate spore and toxin protein antigens. Intranasal vaccination of mice with B. anthracis antigen-MPLA-NLP constructs induced robust IgG and IgA responses in serum and in bronchoalveolar and nasal lavage. Typically, a single dose sufficed to induce sustained antibody titers over time. When multiple immunizations were required for sustained titers, specific antibodies were detected earlier in the boost schedule with MPLA-NLP-mediated delivery than with free MPLA. Administering combinations of constructs induced responses to multiple antigens, indicating potential for a multivalent vaccine preparation. No off-target responses to the NLP scaffold protein were detected. In summary, the NLP platform enhances humoral and mucosal responses to intranasal immunization, indicating promise for NLPs as a flexible, robust vaccine platform against B. anthracis and potentially other inhalational pathogens.