ABSTRACT
BACKGROUND: Protection induced by acellular pertussis (aP) vaccines is partial and short-lived, especially in teenagers, calling for novel immunization strategies. METHODS: We conducted an investigator-driven proof-of-concept randomized controlled trial in aP-primed adolescents in Geneva to assess the immunogenicity and reactogenicity of a novel recombinant aP (r-aP) vaccine including recombinant pertussis toxin (PT) and filamentous hemagglutinin (FHA) coadministered with tetanus-diphtheria toxoids (Td), compared to a licensed tetanus-diphtheria-aP vaccine containing chemically detoxified PT (cd/Tdap). The primary immunological endpoints were day 28/365 geometric mean concentrations (GMCs) of total and neutralizing anti-PT antibodies. Memory B cells were assessed. RESULTS: Sixty-two aP-primed adolescents were randomized and vaccinated with r-aP + Td or cd/Tdap. Reactogenicity, adverse events, and baseline GMCs were similar between the groups. Day 28 PT-neutralizing GMCs were low after cd/Tdap (73.91 [95% confidence interval {CI}, 49.88-109.52] IU/mL) and approximately 2-fold higher after r-aP + Td (127.68 [95% CI, 96.73-168.53] IU/mL; P = .0162). Anti-PT GMCs were also low after cd/Tdap (52.43 [95% CI, 36.41-75.50] IU/mL) and 2-fold higher after r-aP + Td (113.74 [95% CI, 88.31-146.50] IU/mL; P = .0006). Day 28 anti-FHA GMCs were similar in both groups. Day 365 anti-PT (but not PT-neutralizing) GMCs remained higher in r-aP + Td vaccinees. PT-specific memory B cells increased significantly after r-aP + Td but not cd/Tdap boosting. CONCLUSIONS: Boosting aP-primed adolescents with r-aP induced higher anti-PT and PT-neutralizing responses than cd/Tdap and increased PT-specific memory B cells. Despite this superior immunogenicity, r-aP may have to be given repeatedly, earlier, and/or with novel adjuvants to exert an optimal influence in aP-primed subjects. CLINICAL TRIALS REGISTRATION: NCT02946190.
Subject(s)
Antibodies, Neutralizing/blood , Immunization, Secondary/methods , Pertussis Toxin/immunology , Pertussis Vaccine/immunology , Whooping Cough/prevention & control , Adhesins, Bacterial/genetics , Adhesins, Bacterial/immunology , Adolescent , Antibodies, Bacterial/blood , Antitoxins/blood , B-Lymphocyte Subsets/immunology , Child , Female , Humans , Immunologic Memory , Male , Pertussis Toxin/genetics , Pertussis Vaccine/administration & dosage , Switzerland , Vaccines, Acellular/administration & dosage , Vaccines, Acellular/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunology , Virulence Factors, Bordetella/genetics , Virulence Factors, Bordetella/immunologyABSTRACT
We previously developed a negatively charged amino acid dendrimer to address the safety concerns associated with the constituent unit of these systems, which resulted in the formation of a sixth-generation glutamic acid-modified dendritic poly(L-lysine) system (KG6E). The aim of this study was to develop a nanocarrier for targeted drug delivery into cancer cells. In this study, we have synthesized a conjugate material consisting of anti-mucin 1 (MUC1) aptamer (anti-MUC1 apt) and KG6E (anti-MUC1 apt/KG6E) for targeted drug delivery to human lung adenocarcinoma A549 cells, which express high levels of the MUC1. The anti-MUC1 apt/KG6E was efficiently internalized by the A549 cells and subsequently transported to the endosomal and lysosomal compartments. In contrast, the cellular association of the sequence scrambled aptamer/KG6E conjugate (scrambled apt/KG6E) was much lower than that of the anti-MUC1 apt/KG6E in A549 cells. These results suggest that our newly developed anti-MUC1 apt/KG6E can be internalized in A549 cells via a MUC1 recognition pathway.
Subject(s)
Aptamers, Nucleotide/administration & dosage , Dendrimers/administration & dosage , Drug Delivery Systems , Mucin-1/metabolism , A549 Cells , Adenocarcinoma/metabolism , Adenocarcinoma of Lung , Aptamers, Nucleotide/chemistry , Dendrimers/chemistry , Glutamic Acid/chemistry , Humans , Lung Neoplasms/metabolism , Polylysine/chemistryABSTRACT
AIM/OBJECTIVE: This study investigates placental antibody transfer following recombinant pertussis vaccination in pregnancy in a real-world setting. METHODS: This postmarketing observational study recruited pregnant women vaccinated with monovalent recombinant acellular pertussis (aP) vaccine (aPgen; n = 199) or combined to tetanus-diphtheria (TdaPgen; n = 200), or Td-vaccine only (n = 54). Pregnancy, delivery, and neonatal outcomes were assessed. Cord blood was collected postdelivery and pertussis toxin (PT)-IgG, filamentous hemagglutinin (FHA)-IgG, and PT-neutralizing antibodies (PT-Nab) were assessed. RESULTS: No adverse pregnancy, delivery, or neonatal outcomes attributed to aPgen, TdaPgen, or Td vaccination were reported. High anti-PT antibody levels were detected in cord samples from women vaccinated with aPgen (geometric mean concentration [GMC] PT-IgG 206.1 IU/ml, 95% confidence intervals [CI]: 164.3-258.6; geometric mean titer [GMT] PT-Nab 105.3 IU/ml, 95% CI: 81.7-135.8) or TdaPgen (GMC PT-IgG 153.1 IU/ml, 95% CI: 129.1-181.5; GMT PT-Nab 81.5 IU/ml, 95% CI: 66.4-100.0). In the Td-only group, anti-PT antibodies were low (GMC PT-IgG 6.5 IU/ml, 95% CI: 4.9-8.8; GMT PT-Nab 3.8 IU/ml, 95% CI: 2.8-5.1). The same was found for FHA-IgG. Recombinant pertussis vaccination at <27 or 27-36 weeks gestation induced similar cord pertussis antibody levels. CONCLUSION: This first real-world study confirms that recombinant pertussis vaccination in the second or third trimester of pregnancy results in high levels of passive immunity in infants. Thai Clinical Trial Registry: TCTR20200528006.
Subject(s)
Antibodies, Bacterial , Immunity, Maternally-Acquired , Whooping Cough , Humans , Female , Pregnancy , Adult , Antibodies, Bacterial/blood , Antibodies, Bacterial/immunology , Whooping Cough/prevention & control , Whooping Cough/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Fetal Blood/immunology , Vaccines, Synthetic/immunology , Vaccines, Synthetic/administration & dosage , Pertussis Vaccine/immunology , Pertussis Vaccine/administration & dosage , Young Adult , Maternal-Fetal Exchange/immunology , Diphtheria-Tetanus-acellular Pertussis Vaccines/immunology , Diphtheria-Tetanus-acellular Pertussis Vaccines/administration & dosage , Infant, Newborn , Pertussis Toxin/immunology , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Bordetella pertussis/immunology , VaccinationABSTRACT
ChulaCov19 mRNA vaccine demonstrated promising phase 1 results. Healthy adults aged 18-59 years were double-blind randomised 4:1 to receive two intramuscular doses of ChulaCov19 50 µg or placebo. Primary endpoints were safety and microneutralization antibody against-wild-type (Micro-VNT50) at day 50. One hundred fifty adults with median (IQR) age 37 (30-46) years were randomised. ChulaCov19 was well tolerated, and most adverse events were mild to moderate and temporary. Geometric mean titres (GMT) of neutralizing titre against wild-type for ChulaCov19 on day 50 were 1367 IU/mL. T-cell IFN-γ-ELISpot showed the highest responses at one week (Day29) after dose 2 then gradually declined. ChulaCov19 50 µg is well tolerated and elicited high neutralizing antibodies and strong T-cell responses in healthy adults.Trial registration number: ClinicalTrials.gov Identifier NCT04566276, 28/09/2020.
Subject(s)
COVID-19 Vaccines , COVID-19 , Adult , Humans , Middle Aged , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/prevention & control , COVID-19 Vaccines/adverse effects , Double-Blind Method , Immunogenicity, Vaccine , mRNA Vaccines , Adolescent , Young AdultABSTRACT
Establishment of an mRNA vaccine platform in low- and middle-income countries (LMICs) is important to enhance vaccine accessibility and ensure future pandemic preparedness. Here, we describe the preclinical studies of "ChulaCov19", a SARS-CoV-2 mRNA encoding prefusion-unstabilized ectodomain spike protein encapsulated in lipid nanoparticles (LNP). In female BALB/c mice, ChulaCov19 at 0.2, 1, 10, and 30 µg elicits robust neutralizing antibody (NAb) and T cell responses in a dose-dependent relationship. The geometric mean titers (GMTs) of NAb against wild-type (WT, Wuhan-Hu1) virus are 1,280, 11,762, 54,047, and 62,084, respectively. Higher doses induce better cross-NAb against Delta (B.1.617.2) and Omicron (BA.1 and BA.4/5) variants. This elicited immunogenicity is significantly higher than those induced by homologous CoronaVac or AZD1222 vaccination. In a heterologous prime-boost study, ChulaCov19 booster dose generates a 7-fold increase of NAb against Wuhan-Hu1 WT virus and also significantly increases NAb response against Omicron (BA.1 and BA.4/5) when compared to homologous CoronaVac or AZD1222 vaccination. Challenge studies show that ChulaCov19 protects human-ACE-2-expressing female mice from COVID-19 symptoms, prevents viremia and significantly reduces tissue viral load. Moreover, anamnestic NAb response is undetectable in challenge animals. ChulaCov19 is therefore a promising mRNA vaccine candidate either as a primary or boost vaccination and has entered clinical development.
Subject(s)
COVID-19 Vaccines , COVID-19 , Female , Humans , Animals , Mice , ChAdOx1 nCoV-19 , COVID-19/prevention & control , SARS-CoV-2/genetics , Antibodies, Neutralizing , Mice, Inbred BALB C , RNA, Messenger/genetics , Antibodies, Viral , mRNA VaccinesABSTRACT
Effective mRNA SARS-CoV-2 vaccines are available but need to be stored in freezers, limiting their use to countries that have appropriate storage capacity. ChulaCov19 is a prefusion non-stabilized SARS-CoV-2 spike-protein-encoding, nucleoside-modified mRNA, lipid nanoparticle encapsulated vaccine that we report to be stable when stored at 2-8 °C for up to 3 months. Here we report safety and immunogenicity data from a phase I open-label, dose escalation, first-in-human trial of the ChulaCov19 vaccine (NCT04566276). Seventy-two eligible volunteers, 36 of whom were aged 18-55 (adults) and 36 aged 56-75 (elderly), were enroled. Two doses of vaccine were administered 21 d apart at 10, 25 or 50 µg per dose (12 per group). The primary outcome was safety and the secondary outcome was immunogenicity. All three dosages of ChulaCov19 were well tolerated and elicited robust dose-dependent and age-dependent B- and T-cell responses. Transient mild/moderate injection site pain, fever, chills, fatigue and headache were more common after the second dose. Four weeks after the second dose, in the adult cohort, MicroVNT-50 geometric mean titre against wild-type SARS-CoV-2 was 848 (95% CI, 483-1,489), 736 (459-1,183) and 1,140 (854-1,522) IU ml-1 at 10, 25 and 50 µg doses, respectively, versus 285 (196-413) IU ml-1 for human convalescent sera. All dose levels elicited 100% seroconversion, with geometric mean titre ratios 4-8-fold higher than for human convalescent sera (P < 0.01), and high IFNγ spot-forming cells per million peripheral blood mononuclear cells. The 50 µg dose induced better cross-neutralization against Alpha, Beta, Gamma and Delta variants than lower doses. ChulaCov19 at 50 µg is well tolerated and elicited higher neutralizing antibodies than human convalescent sera, with strong T-cell responses. These antibodies cross-neutralized four variants of concern. ChulaCov19 has proceeded to phase 2 clinical trials. We conclude that the mRNA vaccine expressing a prefusion non-stabilized spike protein is safe and highly immunogenic.
Subject(s)
COVID-19 Vaccines , COVID-19 , Adult , Aged , Humans , COVID-19 Vaccines/adverse effects , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , RNA, Messenger , Leukocytes, Mononuclear , Antibodies, Viral , COVID-19/prevention & control , COVID-19 Serotherapy , mRNA VaccinesABSTRACT
Dendrimers have well-organized high branches with a layered architecture providing a series of versatile chemical modification for various purposes. Consequently, this dendrimer nanotechnology explores a new promising class of nanoscale carriers for therapeutic drugs and imaging reagents using passive and active targeting approaches. By controlling dendritic structures, the biological fate of dendrimer/dendrimer-based drugs can be significantly altered based on their intrinsic physicochemical properties, including the hydrophilicity of the unit molecules, particle size, surface charge, and modification. Accordingly, pharmacokinetic aspects play an important role in the design and development of dendrimer systems for successful in vivo application and clinical translation. This review focuses on the recent progress regarding dendritic architectures, structure-related toxicity, and critical factors affecting the pharmacokinetics and biodistribution of dendrimer/dendrimer-based drugs. A better understanding of the basic aspects of dendritic systems and their pharmacokinetics will help to develop a rationale for the design of dendrimers for the controlled delivery of drugs and imaging reagents for therapeutic or diagnostic purposes.
Subject(s)
Chemistry, Pharmaceutical/trends , Dendrimers/chemistry , Diagnostic Imaging , Drug Carriers/chemistry , Dendrimers/pharmacokinetics , Drug Carriers/pharmacokineticsABSTRACT
More than 65 million people have been confirmed infection with SARS-CoV-2 and more than 1 million have died from COVID-19 and this pandemic remains critical worldwide. Effective vaccines are one of the most important strategies to limit the pandemic. Here, we report a construction strategy of DNA vaccine candidates expressing full length wild type SARS-CoV-2 spike (S) protein, S1 or S2 region and their immunogenicity in mice. All DNA vaccine constructs of pCMVkan-S, -S1 and -S2 induced high levels of specific binding IgG that showed a balance of IgG1/IgG2a response. However, only the sera from mice vaccinated with pCMKkan-S or -S1 DNA vaccines could inhibit viral RBD and ACE2 interaction. The highest neutralizing antibody (NAb) titer was found in pCMVkan-S group, followed by -S1, while -S2 showed the lowest PRNT50 titers. The geometric mean titers (GMTs) were 2,551, 1,005 and 291 for pCMVkan-S, -S1 and -S2, respectively. pCMVkan-S construct vaccine also induced the highest magnitude and breadth of T cells response. Analysis of IFN-γ positive cells after stimulation with SARS-CoV-2 spike peptide pools were 2,991, 1,376 and 1,885 SFC/106 splenocytes for pCMVkan-S, -S1 and -S2, respectively. Our findings highlighted that full-length S antigen is more potent than the truncated spike (S1 or S2) in inducing of neutralizing antibody and robust T cell responses.
Subject(s)
Immunity, Humoral , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Th1 Cells/immunology , Vaccines, DNA/immunology , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Neutralizing/blood , COVID-19/prevention & control , COVID-19/virology , Cytokines/metabolism , Female , Immunoglobulin G/blood , Interferon-gamma/metabolism , Mice , Mice, Inbred ICR , Plasmids/genetics , Plasmids/metabolism , Protein Binding , Th1 Cells/cytology , Th1 Cells/metabolism , Vaccines, DNA/geneticsABSTRACT
The emergence of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected global public health and economy. Despite the substantial efforts, only few vaccines are currently approved and some are in the different stages of clinical trials. As the disease rapidly spreads, an affordable and effective vaccine is urgently needed. In this study, we investigated the immunogenicity of plant-produced receptor-binding domain (RBD) of SARS-CoV-2 in order to use as a subunit vaccine. In this regard, RBD of SARS-CoV-2 was fused with Fc fragment of human IgG1 and transiently expressed in Nicotiana benthamiana by agroinfiltration. The plant-produced RBD-Fc fusion protein was purified from the crude extract by using protein A affinity column chromatography. Two intramuscular administration of plant-produced RBD-Fc protein formulated with alum as an adjuvant have elicited high neutralization titers in immunized mice and cynomolgus monkeys. Further it has induced a mixed Th1/Th2 immune responses and vaccine-specific T-lymphocyte responses which was confirmed by interferon-gamma (IFN-γ) enzyme-linked immunospot assay. Altogether, our results demonstrated that the plant-produced SARS-CoV-2 RBD has the potential to be used as an effective vaccine candidate against SARS-CoV-2. To our knowledge, this is the first report demonstrating the immunogenicity of plant-produced SARS-CoV-2 RBD protein in mice and non-human primates.
ABSTRACT
BACKGROUND: Recombinant pertussis vaccines inducing long-lasting immune responses could help to control the rise in pertussis. We here report on persisting antibody responses 2 and 3 years after booster vaccination with a new generation recombinant acellular pertussis vaccine. METHODS: Participants of a phase 2/3 randomised-controlled clinical trial with a monovalent pertussis vaccine containing genetically inactivated pertussis toxin (aPgen) or its tetanus and diphtheria toxoids combination (TdaPgen), or a chemically detoxified comparator vaccine (Tdapchem), (originally conducted between July and August 2015) were invited to participate in observational studies of persisting antibody responses 2 and 3 years after vaccination. Serum IgG against pertussis toxin (PT-IgG) and filamentous hemagglutinin (FHA-IgG) were assessed by ELISA, and PT-neutralising antibodies (PT-Nab) by Chinese Hamster Ovary cell assay. FINDINGS: Waning of antibodies stabilised in aPgen and TdaPgen vaccinees 2 and 3 years after vaccination. Three years post-vaccination PT-neutralising antibodies remained 4·6-fold (95% Confidence Interval (CI) 2·6-8·1) and 3·7-fold (95% CI 2·2-6·1) higher, PT-IgG antibodies 3·0-fold (95% CI 2·2-4·1) and 2·5-fold (95% CI 1·9-3·3) higher, and FHA-IgG antibodies 1·8-fold (95% CI 1·3-2·5) and 1·6-fold (95% CI 1·2-2·1) higher than baseline in aPgen and TdaPgen recipients, respectively. In the Tdapchem group, PT-neutralising and PT-IgG and FHA-IgG antibodies were back at baseline levels 2 years post-vaccination. Three years post-vaccination seroconversion rates for PT-neutralising antibodies were 65·0% (95% CI 44·1-85·9) and 55·0% (95% CI 33·2-76·8) in aPgen and TdaPgen recipients, respectively. INTERPRETATION: Considering the persistence of elevated antibody responses 3 years post-booster vaccination, genetically detoxified monovalent aPgen and TdaPgen vaccines can be expected to induce longer-lasting protection than chemically inactivated Tdap vaccines. FUNDING: BioNet-Asia.
ABSTRACT
Trastuzumab, a humanized monoclonal antibody against human epidermal growth factor receptor 2 (HER2), offers a promising strategy of anticancer drug targeting to HER2-expressing cancer cells. Conjugation of trastuzumab to dendrimers, repeatedly branched polymers with a highly functionalized surface, can enhance the drug loading capacity. However, typical dendrimers such as cationic polyamidoamine dendrimers have exhibited a nonspecific cytotoxicity. In the present study, we developed a novel biocompatible amino acid dendrimer with potentially less toxicity by surface modification of the sixth generation lysine dendrimer with glutamate (KG6E). The synthesized KG6E showed a well-controlled particle size around 5-6 nm with low polydispersibility and negative surface potentials for negligible cytotoxicity. Next, the targeting efficiency of the fluorescent-labeled KG6E-trastuzumab conjugate was evaluated in HER2-positive (SKBR3) and -negative (MCF7) human breast cancer cell lines compared to free trastuzumab and KG6E dendrimers. The KG6E-trastuzumab conjugate was specifically bound to SKBR3 cells in a dose-dependent manner with low binding affinity to MCF7 cells. Furthermore, the conjugate was significantly internalized in SKBR3 cells and then trafficked to lysosomes. These results indicate the potential of KG6E-trastuzumab conjugates as HER2-targeting carriers for therapeutic and diagnostic approaches to cancer therapy.
Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Dendrimers/chemistry , Dendrimers/metabolism , Receptor, ErbB-2/metabolism , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal, Humanized , Cell Line, Tumor , Humans , Magnetic Resonance Spectroscopy , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , TrastuzumabABSTRACT
A new generation of recombinant acellular pertussis vaccine containing genetically inactivated pertussis toxin (PTgen) was licensed as a monovalent pertussis vaccine (aPgen; Pertagen®) and in combination with tetanus and reduced-dose diphtheria (TdaPgen; Boostagen®) for active immunization in individuals aged 11 years and older in Thailand in 2016. We here report post-marketing safety data on the use of the vaccines in individuals in the community obtained through active pharmacovigilance surveillance including pregnant women participating in a prospective observational study. Between May 2017 and February 2020 for TdaPgen and between June 2018 and February 2020 for aPgen, participating health care providers vaccinated and collected safety data for 11,429 exposed adolescents and adults. This included 1778 pregnant women. The incidence rate of adverse events following immunization (AEFIs) was 11.5 per 1000 of vaccinated individuals (95% confidence interval (95% CI) 9.7-13.6). AEFIs mostly concerned local pain at the injection site and muscle pain, and symptoms were mild and mostly resolved within a few days with no complications. The incidence rate of AEFIs in women vaccinated during pregnancy was 1.1 per 1000 (95% CI 0.3-4.1). Of 833 pregnant women vaccinated with recombinant aPgen or TdaPgen, 91.4% (95% CI 89.3-93.3) had uncomplicated pregnancies and 98.7% (95% CI 97.7-99.4) of the 855 babies delivered by these women were born healthy, which exceeds rates generally reported in Thailand. There were no vaccine-related serious adverse events reported during the surveillance period. In conclusion, active pharmacovigilance confirms that the recombinant pertussis vaccines aPgen (Pertagen) and TdaPgen (Boostagen) are safe in adolescents and adults, including pregnant women vaccinated in the second or third trimester of pregnancy.
Subject(s)
Diphtheria-Tetanus-acellular Pertussis Vaccines , Diphtheria , Tetanus , Whooping Cough , Adolescent , Adult , Antibodies, Bacterial , Child , Diphtheria-Tetanus-acellular Pertussis Vaccines/adverse effects , Female , Humans , Immunization , Immunization, Secondary , Pertussis Vaccine , Pregnancy , Thailand , Vaccination , Whooping Cough/prevention & controlABSTRACT
The skin is an immune organ comprised of a large network of antigen-presenting cells such as dendritic cells, making it an attractive target for the development of new vaccines and immunotherapies. Recently, we developed a new innovative and non-invasive vaccination method without adjuvant based on epicutaneous vaccine patches on which antigen forms a dry deposit. Here we describe in mice a method for potentiating the efficacy of our epicutaneous vaccination approach using a minimally invasive and epidermis-limited skin preparation based on laser-induced micro-perforation. Our results showed that epidermal micro-perforation increased trans-epidermal water loss, resulting in an enhancement of antigen solubilization from the surface of the patch, and increased the quantity of antigen delivered to the epidermis. Importantly, this was not associated with an increase in systemic passage of the antigen. Skin micro-perforation slightly activated keratinocytes without inducing an excessive level of local inflammation. Moreover, epidermal micro-perforation improved antigen capture by epidermal dendritic cells and specifically increased the level of Langerhans cells activation. Finally, we observed that epidermal micro-perforation significantly increased the level of the specific antibody response induced by our epicutaneous Pertussis vaccine candidate containing non-adsorbed recombinant Pertussis Toxin and reduced the amount of antigen dose required. Overall, these data confirm the benefit of a minimal and controlled epidermal preparation for improving the effectiveness of an epicutaneous patch-based vaccine, without adversely affecting the safety of the method.
Subject(s)
Antigens, Bacterial/immunology , Epidermis/immunology , Pertussis Vaccine/administration & dosage , Vaccination/methods , Animals , Antibody Formation/immunology , Dendritic Cells/immunology , Langerhans Cells/immunology , Mice , Mice, Inbred BALB C , Pertussis Vaccine/immunology , Transdermal PatchABSTRACT
Inhalation of bacterial endotoxin induces pulmonary inflammation by activation of nuclear factor kappaB (NFkappaB), production of cytokines and chemokines, and neutrophil activation. Although glucocorticoids are the drugs of choice, administration of free drugs results in adverse effects as a result of a lack of selectivity for the inflammatory effector cells. Because alveolar macrophages play a key role in the inflammatory response in the lung, selective targeting of glucocorticoids to alveolar macrophages offers efficacious pharmacological intervention with minimal side effects. We have demonstrated previously the selective targeting of mannosylated liposomes to alveolar macrophages via mannose receptor-mediated endocytosis after intratracheal administration. In this study, the anti-inflammatory effects of dexamethasone palmitate incorporated in mannosylated liposomes (DPML) at 0.5 mg/kg via intratracheal administration were investigated in lipopolysaccharide-induced lung inflammation in rats. DPML significantly inhibited tumor necrosis factor alpha, interleukin-1beta, and cytokine-induced neutrophil chemoattractant-1 levels, suppressed neutrophil infiltration and myeloperoxidase activity, and inhibited NFkappaB and p38 mitogen-activated protein kinase activation in the lung. These results prove the value of inhaled mannosylated liposomes as powerful targeting systems for the delivery of anti-inflammatory drugs to alveolar macrophages to improve their efficacy against lung inflammation.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Dexamethasone/pharmacology , Lipopolysaccharides/toxicity , Liposomes , Mannose/chemistry , Pneumonia/chemically induced , Administration, Inhalation , Animals , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/therapeutic use , Bronchoalveolar Lavage Fluid , Dexamethasone/administration & dosage , Dexamethasone/therapeutic use , Electrophoretic Mobility Shift Assay , Enzyme-Linked Immunosorbent Assay , Macrophages, Alveolar/drug effects , Macrophages, Alveolar/metabolism , Pneumonia/drug therapy , Rats , Rats, Wistar , Tissue DistributionABSTRACT
BACKGROUND: The immunogenicity of acellular pertussis vaccines and persistence of immunity after vaccination might be improved by using genetically inactivated pertussis toxin (PTgen) instead of chemically inactivated pertussis toxin (PTchem) because of the preservation of conformational epitopes. We assessed the safety and immunogenicity of two vaccines containing PTgen 1 year after vaccination. METHODS: We did a phase 2/3 non-inferiority, randomised, controlled trial involving 450 adolescents (age 12-17 years) enrolled between July 6, 2015, and Aug 20, 2015. Participants were randomised 1:1:1 to receive one dose of vaccine containing PTgen and filamentous haemagglutinin (FHA) either in a monovalent formulation (aP[PTgen/FHA]) or in a combined formulation with tetanus and reduced-dose diphtheria toxoids (TdaP[PTgen/FHA]) or to receive a commercial vaccine containing reduced-dose PTchem (Tdap) as a comparator. We report a secondary trial outcome, namely antibody persistence 1 year after vaccination, assessed per protocol in 150 randomly preselected participants (50 per group). Seroconversion was defined as antibody titres at least four times greater than at baseline. Safety was assessed in all trial participants. This study is registered in the Thai Clinical Trial Registry, number TCTR20150703002. FINDINGS: Between June 5, 2016, and Aug 9, 2016, 442 (98%) of 450 enrolled participants attended a 1-year follow-up visit. After 1 year, persistent seroconversion for pertussis toxin neutralising antibodies was seen in 38 (76%, 95% CI 64-88) participants in the aP(PTgen/FHA) group and 41 (81%, 70-92) in the TdaP(PTgen/FHA) group, but in only four (8%, 1-16) in the Tdap comparator group. Seroconversion rates for IgG antibodies against pertussis toxin and FHA were also greater in the aP(PTgen/FHA) group (82%, 95% CI 71-93 and 64%, 51-77, respectively) and TdaP(PTgen/FHA) group (75%, 63-87 and 56%, 42-70, respectively) than in the Tdap group (4%, 0-9, p<0·0001, and 28%, 16-41, p=0·0007, respectively). 13 serious adverse events were reported in 12 participants and all were judged to be unrelated to the study vaccines. Five pregnancies were reported during follow-up, none of which had any maternal or neonatal complications. INTERPRETATION: A monovalent and a combined recombinant acellular pertussis vaccine containing PTgen induced antibody responses that were greater and sustained for longer than those achieved with the Tdap comparator vaccine. New recombinant pertussis vaccines containing PTgen might offer new opportunities to limit pertussis resurgence and can be widely used, including in pregnant women. FUNDING: BioNet-Asia.
Subject(s)
Antibodies, Bacterial/blood , Antitoxins/blood , Pertussis Toxin/immunology , Pertussis Vaccine/immunology , Adolescent , Asia , Drug-Related Side Effects and Adverse Reactions/epidemiology , Drug-Related Side Effects and Adverse Reactions/pathology , Female , Humans , Male , Pertussis Toxin/genetics , Pertussis Vaccine/administration & dosage , Pertussis Vaccine/adverse effects , Pertussis Vaccine/genetics , Seroconversion , Single-Blind Method , Time Factors , Vaccines, Acellular/administration & dosage , Vaccines, Acellular/adverse effects , Vaccines, Acellular/genetics , Vaccines, Acellular/immunology , Vaccines, Combined/administration & dosage , Vaccines, Combined/adverse effects , Vaccines, Combined/genetics , Vaccines, Combined/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/adverse effects , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunologyABSTRACT
CRM197 is a diphtheria toxin (DT) mutant (G52E) which has been used as a carrier protein for conjugate vaccines. However, it still possesses cytotoxicity toward mammalian cells. The goal of this project was to produce a non-toxic and soluble CRM197EK through introduction of triple amino acid substitutions (K51E/G52E/E148K) in Escherichia coli. The expression of CRM197EKTrxHis was optimized and co-expressed with different molecular chaperones. The soluble CRM197EKTrxHis was produced at a high concentration (97.33 ± 17.47 µg/ml) under the optimal condition (induction with 0.1 mM IPTG at 20 °C for 24 h). Cells containing pG-Tf2, expressing trigger factor and GroEL-GroES, accumulated the highest amount of soluble CRM197EKTrxHis at 111.24 ± 10.40 µg/ml after induction for 24 h at 20 °C. The soluble CRM197EKTrxHis still possesses nuclease activity and completely digest λDNA at 25 and 37 °C with 8- and 4-h incubation, respectively. Molecular modeling of diphtheria toxin, CRM197 and CRM197EK indicated that substitutions of two amino acids (K51E/E148K) may cause poor NAD binding, consistent with the lack of toxicity. Therefore, CRM197EK might be used as a new potential carrier protein. However, further in vivo study is required to confirm its roles as functional carrier protein in conjugate vaccines.
Subject(s)
Bacterial Proteins/biosynthesis , Bacterial Proteins/chemistry , Cloning, Molecular/methods , Escherichia coli/physiology , Models, Chemical , Models, Molecular , Bacterial Proteins/genetics , Binding Sites , Computer Simulation , Metabolic Engineering/methods , Molecular Chaperones/biosynthesis , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Chaperones/ultrastructure , Protein Binding , Protein Conformation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SolubilityABSTRACT
BACKGROUND: An acellular Pertussis (aP) vaccine containing recombinant genetically detoxified Pertussis Toxin (PTgen), Filamentous Hemagglutinin (FHA) and Pertactin (PRN) has been developed by BioNet-Asia (BioNet). We present here the results of the first clinical study of this recombinant aP vaccine formulated alone or in combination with tetanus and diphtheria toxoids (TdaP). METHODS: A phase I/II, observer-blind, randomized controlled trial was conducted at Mahidol University in Bangkok, Thailand in healthy adult volunteers aged 18-35 y. The eligible volunteers were randomized to receive one dose of either BioNet's aP or Tetanus toxoid-reduced Diphtheria toxoid-acellular Pertussis (TdaP) vaccine, or the Tdap Adacel® vaccine in a 1:1:1 ratio. Safety follow-up was performed for one month. Immunogenicity was assessed at baseline, at 7 and 28 d after vaccination. Anti-PT, anti-FHA, anti-PRN, anti-tetanus and anti-diphtheria IgG antibodies were assessed by ELISA. Anti-PT neutralizing antibodies were assessed also by CHO cell assay. RESULTS: A total of 60 subjects (20 per each vaccine group) were enrolled and included in the safety analysis. Safety laboratory parameters, incidence of local and systemic post-immunization reactions during 7 d after vaccination and incidence of adverse events during one month after vaccination were similar in the 3 vaccine groups. One month after vaccination, seroresponse rates of anti-PT, anti-FHA and anti-PRN IgG antibodies exceeded 78% in all vaccine groups. The anti-PT IgG, anti-FHA IgG, and anti-PT neutralizing antibody geometric mean titers (GMTs) were significantly higher following immunization with BioNet's aP and BioNet's TdaP than Adacel® (P< 0.05). The anti-PRN IgG, anti-tetanus and anti-diphtheria GMTs at one month after immunization were comparable in all vaccine groups. All subjects had seroprotective titers of anti-tetanus and anti-diphtheria antibodies at baseline. CONCLUSION: In this first clinical study, PTgen-based BioNet's aP and TdaP vaccines showed a similar tolerability and safety profile to Adacel® and elicited significantly higher immune responses to PT and FHA.
Subject(s)
Diphtheria-Tetanus-acellular Pertussis Vaccines/adverse effects , Diphtheria-Tetanus-acellular Pertussis Vaccines/immunology , Diphtheria/prevention & control , Tetanus/prevention & control , Whooping Cough/prevention & control , Adolescent , Adult , Antibodies, Bacterial/blood , Cell Survival/drug effects , Diphtheria-Tetanus-acellular Pertussis Vaccines/administration & dosage , Double-Blind Method , Drug-Related Side Effects and Adverse Reactions/epidemiology , Drug-Related Side Effects and Adverse Reactions/pathology , Enzyme-Linked Immunosorbent Assay , Female , Healthy Volunteers , Humans , Immunoglobulin G/blood , Injections, Intramuscular , Male , Neutralization Tests , Thailand , Young AdultABSTRACT
Targeted gene delivery via selective cellular receptors has been realized as a crucial strategy for successful gene therapy by maximizing therapeutic efficiency in target cells and minimizing systemic toxicity. The membrane carbohydrate-binding proteins (membrane lectins) with different carbohydrate specificities are differentially expressed on the cellular and intracellular membranes of a number of cells. Their multiplicity, high affinity, and effective endocytosis after receptor binding as well as the biocompatibility of carbohydrate ligands endow them as potential ligands for glycosylated carriers in cell-selective delivery of nucleic acids. To achieve the in vivo application, glycosylated carriers/nucleic acid complexes have to fulfill certain conditions, including having a suitable size, minimal nonspecific interactions, low immunogenicity, and high uptake in target cells. Accordingly, the effective nuclear delivery of nucleic acids is the paramount important step for efficient gene transfer. This review summarizes the recent progress regarding application of glycosylated carriers for cell-selective and nuclear delivery of nucleic acids and their critical factors for efficient gene transfer. In addition, the development of new materials, such as carbon nanotubes, carbon nanospheres, and gold nanoparticles, as innovative carriers will be discussed with regards to glycosylation-mediated delivery of nucleic acids.
Subject(s)
Drug Carriers , Genetic Therapy/methods , Nucleic Acids/administration & dosage , Nucleic Acids/genetics , Animals , Cell Nucleus/genetics , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Drug Carriers/chemistry , Endosomes/metabolism , Gene Transfer Techniques , Glycosylation , Hepatic Stellate Cells/drug effects , Hepatic Stellate Cells/metabolism , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Lung/cytology , Lung/drug effects , Lung/metabolism , Macrophages/drug effects , Macrophages/metabolism , Models, Biological , Neoplasms/genetics , Neoplasms/therapy , Targeted Gene Repair/methodsABSTRACT
The nuclear factor kappa B (NFκB) signaling pathway is a key mechanism in the pathophysiology of lung inflammation. NFκB is critically responsible for the expression of pro-inflammatory mediators following activation. The specific inhibition of NFκB by a NFκB decoy via inhalation appears to improve therapeutic effects. However, administration of naked NFκB decoy limits the efficacy of the decoy strategy due to low targeting ability to immune cells such as alveolar macrophages. In this study, we have assessed the effect of alveolar macrophage-targeted NFκB decoy by mannosylated (Man) cationic liposomes in a LPS-induced lung inflammation model after intratracheal administration. The complex of Man-cationic liposome/NFκB decoy was physically stable during spraying. Man-cationic liposome/NFκB decoy complex was selectively delivered to alveolar macrophages for subsequent localization of NFκB decoy in the cytoplasm and to a lesser extent in the nucleus. In the LPS-induced lung inflammation model, pre-treatment with Man-cationic liposome/50µg NFκB decoy complex significantly inhibited the release of TNF-α, IL-1ß and CINC-1, neutrophil infiltration and NFκB activation compared with naked NFκB decoy, cationic liposome/NFκB decoy complex and Man-cationic liposome/scrambled decoy complex treatments. This study demonstrates the sufficient targeting of NFκB decoy using Man-cationic liposomes in a novel effective anti-inflammatory therapy for lung inflammation.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Lung/drug effects , Mannose/chemistry , NF-kappa B/antagonists & inhibitors , Oligodeoxyribonucleotides/therapeutic use , Pneumonia/drug therapy , Administration, Inhalation , Animals , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/pharmacokinetics , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , Cations , Cells, Cultured , Cytokines/immunology , Disease Models, Animal , Electrophoretic Mobility Shift Assay , Epithelial Cells/drug effects , Epithelial Cells/immunology , Epithelial Cells/metabolism , Lipopolysaccharides/toxicity , Liposomes , Lung/immunology , Lung/metabolism , Macrophages, Alveolar/drug effects , Macrophages, Alveolar/immunology , Macrophages, Alveolar/metabolism , Male , Microscopy, Confocal , NF-kappa B/immunology , Neutrophil Infiltration/drug effects , Neutrophil Infiltration/immunology , Oligodeoxyribonucleotides/administration & dosage , Oligodeoxyribonucleotides/pharmacokinetics , Peroxidase/metabolism , Pneumonia/immunology , Pneumonia/metabolism , Rats , Rats, Wistar , Tissue DistributionABSTRACT
AIM: Nascent high-density lipoprotein (HDL) capable of intracellularly delivering anticancer drugs was developed to potentiate antitumor activities. MATERIALS & METHODS: Apolipoprotein A-I, a major component protein of HDL, was genetically fused to TAT peptide, a protein transduction domain. Nascent HDL was prepared with this mutant and phospholipids. RESULTS & DISCUSSION: Intracellular delivery of doxorubicin (DXR) by TAT-fused HDL was confirmed by confocal microscopy. Treatment of cancer cells with TAT-fused HDL-DXR complex resulted in enhanced growth inhibition. Furthermore, TAT-fused HDL-DXR complex suppressed tumor growth in mice more efficiently than HDL-DXR complex. No bodyweight loss was observed for the TAT complex. These results clearly demonstrate the usefulness of TAT fusion to nascent HDL to potentiate the antitumor activity of DXR. CONCLUSION: The genetic fusion of apoA-I with biologically active peptides potentially enables a simple assembly of biocompatible and versatile drug carriers.