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1.
Vaccine ; 41(3): 724-734, 2023 01 16.
Article in English | MEDLINE | ID: mdl-36564274

ABSTRACT

The candidate Adjuvant System AS37 contains a synthetic toll-like receptor agonist (TLR7a) adsorbed to alum. In a phase I study (NCT02639351), healthy adults were randomised to receive one dose of licensed alum-adjuvanted meningococcal serogroup C (MenC-CRM197) conjugate vaccine (control) or MenC-CRM197 conjugate vaccine adjuvanted with AS37 (TLR7a dose 12.5, 25, 50 or 100 µg). A subset of 66 participants consented to characterisation of peripheral whole blood transcriptomic responses, systemic cytokine/chemokine responses and multiple myeloid and lymphoid cell responses as exploratory study endpoints. Blood samples were collected pre-vaccination, 6 and 24 h post-vaccination, and 3, 7, 28 and 180 days post-vaccination. The gene expression profile in whole blood showed an early, AS37-specific transcriptome response that peaked at 24 h, increased with TLR7a dose up to 50 µg and generally resolved within one week. Five clusters of differentially expressed genes were identified, including those involved in the interferon-mediated antiviral response. Evaluation of 30 cytokines/chemokines by multiplex assay showed an increased level of interferon-induced chemokine CXCL10 (IP-10) at 24 h and 3 days post-vaccination in the AS37-adjuvanted vaccine groups. Increases in activated plasmacytoid dendritic cells (pDC) and intermediate monocytes were detected 3 days post-vaccination in the AS37-adjuvanted vaccine groups. T follicular helper (Tfh) cells increased 7 days post-vaccination and were maintained at 28 days post-vaccination, particularly in the AS37-adjuvanted vaccine groups. Moreover, most of the subjects that received vaccine containing 25, 50 and 100 µg TLR7a showed an increased MenC-specific memory B cell responses versus baseline. These data show that the adsorption of TLR7a to alum promotes an immune signature consistent with TLR7 engagement, with up-regulation of interferon-inducible genes, cytokines and frequency of activated pDC, intermediate monocytes, MenC-specific memory B cells and Tfh cells. TLR7a 25-50 µg can be considered the optimal dose for AS37, particularly for the adjuvanted MenC-CRM197 conjugate vaccine.


Subject(s)
Aluminum Hydroxide , Meningococcal Vaccines , Adult , Humans , Interferons , Toll-Like Receptor 7 , Antiviral Agents , Vaccines, Conjugate , Adjuvants, Immunologic , Cytokines , Systems Analysis
2.
Bioanalysis ; 14(11): 737-793, 2022 Jun.
Article in English | MEDLINE | ID: mdl-35578991

ABSTRACT

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.


Subject(s)
Receptors, Chimeric Antigen , Vaccines , Biomarkers/analysis , CRISPR-Cas Systems , Cell- and Tissue-Based Therapy , Humans , Immunotherapy, Active , Polymerase Chain Reaction
3.
Mol Ther ; 30(5): 1897-1912, 2022 05 04.
Article in English | MEDLINE | ID: mdl-34990810

ABSTRACT

RNA vaccines have demonstrated efficacy against SARS-CoV-2 in humans, and the technology is being leveraged for rapid emergency response. In this report, we assessed immunogenicity and, for the first time, toxicity, biodistribution, and protective efficacy in preclinical models of a two-dose self-amplifying messenger RNA (SAM) vaccine, encoding a prefusion-stabilized spike antigen of SARS-CoV-2 Wuhan-Hu-1 strain and delivered by lipid nanoparticles (LNPs). In mice, one immunization with the SAM vaccine elicited a robust spike-specific antibody response, which was further boosted by a second immunization, and effectively neutralized the matched SARS-CoV-2 Wuhan strain as well as B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta) variants. High frequencies of spike-specific germinal center B, Th0/Th1 CD4, and CD8 T cell responses were observed in mice. Local tolerance, potential systemic toxicity, and biodistribution of the vaccine were characterized in rats. In hamsters, the vaccine candidate was well-tolerated, markedly reduced viral load in the upper and lower airways, and protected animals against disease in a dose-dependent manner, with no evidence of disease enhancement following SARS-CoV-2 challenge. Therefore, the SARS-CoV-2 SAM (LNP) vaccine candidate has a favorable safety profile, elicits robust protective immune responses against multiple SARS-CoV-2 variants, and has been advanced to phase 1 clinical evaluation (NCT04758962).


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/prevention & control , COVID-19 Vaccines , Cricetinae , Humans , Liposomes , Mice , Nanoparticles , RNA, Messenger , Rats , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Tissue Distribution
4.
Front Immunol ; 13: 1081156, 2022.
Article in English | MEDLINE | ID: mdl-36713458

ABSTRACT

The goal of this study was to utilize a multimodal magnetic resonance imaging (MRI) and positron emission tomography (PET) imaging approach to assess the local innate immune response in skeletal muscle and draining lymph node following vaccination in rats using two different vaccine platforms (AS01 adjuvanted protein and lipid nanoparticle (LNP) encapsulated Self-Amplifying mRNA (SAM)). MRI and 18FDG PET imaging were performed temporally at baseline, 4, 24, 48, and 72 hr post Prime and Prime-Boost vaccination in hindlimb with Cytomegalovirus (CMV) gB and pentamer proteins formulated with AS01, LNP encapsulated CMV gB protein-encoding SAM (CMV SAM), AS01 or with LNP carrier controls. Both CMV AS01 and CMV SAM resulted in a rapid MRI and PET signal enhancement in hindlimb muscles and draining popliteal lymph node reflecting innate and possibly adaptive immune response. MRI signal enhancement and total 18FDG uptake observed in the hindlimb was greater in the CMV SAM vs CMV AS01 group (↑2.3 - 4.3-fold in AUC) and the MRI signal enhancement peak and duration were temporally shifted right in the CMV SAM group following both Prime and Prime-Boost administration. While cytokine profiles were similar among groups, there was good temporal correlation only between IL-6, IL-13, and MRI/PET endpoints. Imaging mass cytometry was performed on lymph node sections at 72 hr post Prime and Prime-Boost vaccination to characterize the innate and adaptive immune cell signatures. Cell proximity analysis indicated that each follicular dendritic cell interacted with more follicular B cells in the CMV AS01 than in the CMV SAM group, supporting the stronger humoral immune response observed in the CMV AS01 group. A strong correlation between lymph node MRI T2 value and nearest-neighbor analysis of follicular dendritic cell and follicular B cells was observed (r=0.808, P<0.01). These data suggest that spatiotemporal imaging data together with AI/ML approaches may help establish whether in vivo imaging biomarkers can predict local and systemic immune responses following vaccination.


Subject(s)
Cytomegalovirus Infections , Fluorodeoxyglucose F18 , Rats , Animals , Vaccination , Magnetic Resonance Imaging/methods , Positron-Emission Tomography , Cytomegalovirus , Immunity, Innate , Muscle, Skeletal/diagnostic imaging , Multimodal Imaging , Lymph Nodes/diagnostic imaging
5.
Front Immunol ; 12: 757151, 2021.
Article in English | MEDLINE | ID: mdl-34777370

ABSTRACT

CD8+ T cells play a key role in mediating protective immunity after immune challenges such as infection or vaccination. Several subsets of differentiated CD8+ T cells have been identified, however, a deeper understanding of the molecular mechanism that underlies T-cell differentiation is lacking. Conventional approaches to the study of immune responses are typically limited to the analysis of bulk groups of cells that mask the cells' heterogeneity (RNA-seq, microarray) and to the assessment of a relatively limited number of biomarkers that can be evaluated simultaneously at the population level (flow and mass cytometry). Single-cell analysis, on the other hand, represents a possible alternative that enables a deeper characterization of the underlying cellular heterogeneity. In this study, a murine model was used to characterize immunodominant hemagglutinin (HA533-541)-specific CD8+ T-cell responses to nucleic- and protein-based influenza vaccine candidates, using single-cell sorting followed by transcriptomic analysis. Investigation of single-cell gene expression profiles enabled the discovery of unique subsets of CD8+ T cells that co-expressed cytotoxic genes after vaccination. Moreover, this method enabled the characterization of antigen specific CD8+ T cells that were previously undetected. Single-cell transcriptome profiling has the potential to allow for qualitative discrimination of cells, which could lead to novel insights on biological pathways involved in cellular responses. This approach could be further validated and allow for more informed decision making in preclinical and clinical settings.


Subject(s)
Antigens, Viral/immunology , CD8-Positive T-Lymphocytes/metabolism , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza A Virus, H1N1 Subtype/immunology , Influenza Vaccines/pharmacology , Nucleic Acid-Based Vaccines/pharmacology , Single-Cell Analysis , T-Lymphocyte Subsets/metabolism , Transcriptome , Vaccines, Subunit/pharmacology , Adjuvants, Immunologic , Adoptive Transfer , Animals , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Gene Expression Regulation/drug effects , Mice , Mice, Inbred BALB C , T-Cell Antigen Receptor Specificity , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/immunology , Vaccination
6.
Bioanalysis ; 13(6): 415-463, 2021 Mar.
Article in English | MEDLINE | ID: mdl-33533276

ABSTRACT

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity). Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation) and Part 2B (Regulatory Input) are published in volume 13 of Bioanalysis, issues 4 and 5 (2020), respectively.


Subject(s)
Cell- and Tissue-Based Therapy , Flow Cytometry , Genetic Therapy , Real-Time Polymerase Chain Reaction , Vaccines/analysis , Humans , Quality Control , Receptors, Chimeric Antigen/analysis , United States , United States Food and Drug Administration
7.
Bioanalysis ; 13(5): 295-361, 2021 Mar.
Article in English | MEDLINE | ID: mdl-33511867

ABSTRACT

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 2A) BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation and (Part 2B) Regulatory Input. Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 4, and 6 (2021), respectively.


Subject(s)
Biological Assay , Biotechnology , Cell- and Tissue-Based Therapy , Genetic Therapy , Research Report , Biomarkers/analysis , Humans
8.
NPJ Vaccines ; 5: 88, 2020.
Article in English | MEDLINE | ID: mdl-33024579

ABSTRACT

Vaccines represent the most successful medical intervention in history, with billions of lives saved. Although multiple doses of the same vaccine are typically required to reach an adequate level of protection, it would be advantageous to develop vaccines that induce protective immunity with fewer doses, ideally just one. Single-dose vaccines would be ideal to maximize vaccination coverage, help stakeholders to greatly reduce the costs associated with vaccination, and improve patient convenience. Here we describe past attempts to develop potent single dose vaccines and explore the reasons they failed. Then, we review key immunological mechanisms of the vaccine-specific immune responses, and how innovative technologies and approaches are guiding the preclinical and clinical development of potent single-dose vaccines. By modulating the spatio-temporal delivery of the vaccine components, by providing the appropriate stimuli to the innate immunity, and by designing better antigens, the new technologies and approaches leverage our current knowledge of the immune system and may synergize to enable the rational design of next-generation vaccination strategies. This review provides a rational perspective on the possible development of future single-dose vaccines.

9.
J Control Release ; 316: 12-21, 2019 12 28.
Article in English | MEDLINE | ID: mdl-31678654

ABSTRACT

α-Tocopherol has been used as an immune supplement in humans, as an emulsion adjuvant component in several veterinary vaccines as well as an immunomodulatory component of AS03, an emulsion adjuvant that was used in an H1N1 pandemic vaccine (Pandemrix). AS03 is manufactured using microfluidization and high-pressure homogenization. Such high energy and complex manufacturing processes make it difficult and expensive to produce emulsion adjuvants on a large scale, especially in developing countries. In this study we have explored simpler, comparatively inexpensive methods, to formulate emulsion adjuvants containing α-tocopherol, that have the potential to be made in any well-established scale-up facility. This might facilitate producing and stock-piling adjuvant doses and therefore aide in pandemic preparedness. We used design of experiment as a tool to explore incorporating α-tocopherol into self-emulsified systems containing squalene oil and polysorbate 80. We created novel self-emulsified adjuvant systems (SE-AS) and evaluated their potency in vivo in BALB/c mice with inactivated quadrivalent influenza vaccine (QIV) and tested the cellular and humoral immune responses against the four vaccine strains.


Subject(s)
Adjuvants, Immunologic/administration & dosage , Influenza Vaccines/administration & dosage , Influenza, Human/prevention & control , alpha-Tocopherol/administration & dosage , Animals , Emulsions , Female , Humans , Influenza A Virus, H1N1 Subtype/immunology , Influenza Vaccines/immunology , Influenza, Human/immunology , Mice , Mice, Inbred BALB C , Polysorbates/chemistry , Squalene/chemistry , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/immunology , alpha-Tocopherol/immunology
10.
Front Immunol ; 9: 600, 2018.
Article in English | MEDLINE | ID: mdl-29628926

ABSTRACT

Despite advancements in immunotherapeutic approaches, influenza continues to cause severe illness, particularly among immunocompromised individuals, young children, and elderly adults. Vaccination is the most effective way to reduce rates of morbidity and mortality caused by influenza viruses. Frequent genetic shift and drift among influenza-virus strains with the resultant disparity between circulating and vaccine virus strains limits the effectiveness of the available conventional influenza vaccines. One approach to overcome this limitation is to develop a universal influenza vaccine that could provide protection against all subtypes of influenza viruses. Moreover, the development of a novel or improved universal influenza vaccines may be greatly facilitated by new technologies including virus-like particles, T-cell-inducing peptides and recombinant proteins, synthetic viruses, broadly neutralizing antibodies, and nucleic acid-based vaccines. This review discusses recent scientific advances in the development of next-generation universal influenza vaccines.


Subject(s)
Influenza A virus/immunology , Influenza Vaccines/immunology , Influenza, Human/immunology , Influenza, Human/prevention & control , Animals , Antibodies, Viral/immunology , Antibody Specificity/immunology , Antigens, Viral/genetics , Antigens, Viral/immunology , Humans , Influenza Vaccines/genetics , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Vaccination , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Vaccines, Virus-Like Particle/immunology
11.
Hum Vaccin Immunother ; 14(1): 45-58, 2018 01 02.
Article in English | MEDLINE | ID: mdl-29172945

ABSTRACT

Despite high vaccination coverage worldwide, pertussis has re-emerged in many countries. This randomized, controlled, observer-blind phase I study and extension study in Belgium (March 2012-June 2015) assessed safety and immunogenicity of investigational acellular pertussis vaccines containing genetically detoxified pertussis toxin (PT) (NCT01529645; NCT02382913). 420 healthy adults (average age: 26.8 ± 5.5 years, 60% female) were randomized to 1 of 10 vaccine groups: 3 investigational aP vaccines (containing pertussis antigens PT, filamentous hemagglutinin [FHA] and pertactin [PRN] at different dosages), 6 investigational TdaP (additionally containing tetanus toxoid [TT] and diphtheria toxoid [DT]), and 1 TdaP comparator containing chemically inactivated PT. Antibody responses were evaluated on days 1, 8, 30, 180, 365, and approximately 3 years post-booster vaccination. Cell-mediated immune responses and PT neutralization were evaluated in a subset of participants in pre-selected groups. Local and systemic adverse events (AEs), and unsolicited AEs were collected through day 7 and 30, respectively; serious AEs and AEs leading to study withdrawal were collected through day 365 post-vaccination. Antibody responses against pertussis antigens peaked at day 30 post-vaccination and then declined but remained above baseline level at approximately 3 years post-vaccination. Responses to FHA and PRN were correlated to antigen dose. Antibody responses specific to PT, toxin neutralization activity and persistence induced by investigational formulations were similar or significantly higher than the licensed vaccine, despite lower PT doses. Of 15 serious AEs, none were considered vaccination-related; 1 led to study withdrawal (premature labor, day 364; aP4 group). This study confirmed the potential benefits of genetically detoxified PT antigen. All investigational study formulations were well tolerated.


Subject(s)
Diphtheria-Tetanus-acellular Pertussis Vaccines/administration & dosage , Immunization, Secondary/methods , Pertussis Toxin/immunology , Pertussis Vaccine/administration & dosage , Vaccination/methods , Whooping Cough/prevention & control , Adult , Antibodies, Bacterial/analysis , Belgium , Diphtheria-Tetanus-acellular Pertussis Vaccines/adverse effects , Diphtheria-Tetanus-acellular Pertussis Vaccines/genetics , Diphtheria-Tetanus-acellular Pertussis Vaccines/immunology , Female , Humans , Immunity, Cellular , Immunogenicity, Vaccine , Male , Pertussis Toxin/genetics , Pertussis Vaccine/adverse effects , Pertussis Vaccine/genetics , Pertussis Vaccine/immunology , Treatment Outcome , Whooping Cough/blood , Whooping Cough/immunology , Young Adult
12.
Sci Rep ; 6: 38043, 2016 11 30.
Article in English | MEDLINE | ID: mdl-27901071

ABSTRACT

Staphylococcus aureus is the major cause of human septic arthritis and osteomyelitis, which deserve special attention due to their rapid evolution and resistance to treatment. The progression of the disease depends on both bacterial presence in situ and uncontrolled disruptive immune response, which is responsible for chronic disease. Articular and bone infections are often the result of blood bacteremia, with the knees and hips being the most frequently infected joints showing the worst clinical outcome. We report the development of a hematogenous model of septic arthritis in murine knees, which progresses from an acute to a chronic phase, similarly to what occurs in humans. Characterization of the local and systemic inflammatory and immune responses following bacterial infection brought to light specific signatures of disease. Immunization of mice with the vaccine formulation we have recently described (4C-Staph), induced a strong antibody response and specific CD4+ effector memory T cells, and resulted in reduced bacterial load in the knee joints, a milder general inflammatory state and protection against bacterial-mediated cellular toxicity. Possible correlates of protection are finally proposed, which might contribute to the development of an effective vaccine for human use.


Subject(s)
Arthritis, Infectious , Knee Joint , Staphylococcal Infections , Staphylococcal Vaccines , Staphylococcus aureus/immunology , Vaccination , Animals , Arthritis, Infectious/immunology , Arthritis, Infectious/microbiology , Arthritis, Infectious/pathology , Arthritis, Infectious/prevention & control , Female , Knee Joint/immunology , Knee Joint/microbiology , Knee Joint/pathology , Mice , Staphylococcal Infections/immunology , Staphylococcal Infections/pathology , Staphylococcal Infections/prevention & control , Staphylococcal Vaccines/immunology , Staphylococcal Vaccines/pharmacology
13.
PLoS One ; 11(8): e0161193, 2016.
Article in English | MEDLINE | ID: mdl-27525409

ABSTRACT

Current hemagglutinin (HA)-based seasonal influenza vaccines induce vaccine strain-specific neutralizing antibodies that usually fail to provide protection against mismatched circulating viruses. Inclusion in the vaccine of highly conserved internal proteins such as the nucleoprotein (NP) and the matrix protein 1 (M1) was shown previously to increase vaccine efficacy by eliciting cross-reactive T-cells. However, appropriate delivery systems are required for efficient priming of T-cell responses. In this study, we demonstrated that administration of novel self-amplifying mRNA (SAM®) vectors expressing influenza NP (SAM(NP)), M1 (SAM(M1)), and NP and M1 (SAM(M1-NP)) delivered with lipid nanoparticles (LNP) induced robust polyfunctional CD4 T helper 1 cells, while NP-containing SAM also induced cytotoxic CD8 T cells. Robust expansions of central memory (TCM) and effector memory (TEM) CD4 and CD8 T cells were also measured. An enhanced recruitment of NP-specific cytotoxic CD8 T cells was observed in the lungs of SAM(NP)-immunized mice after influenza infection that paralleled with reduced lung viral titers and pathology, and increased survival after homologous and heterosubtypic influenza challenge. Finally, we demonstrated for the first time that the co-administration of RNA (SAM(M1-NP)) and protein (monovalent inactivated influenza vaccine (MIIV)) was feasible, induced simultaneously NP-, M1- and HA-specific T cells and HA-specific neutralizing antibodies, and enhanced MIIV efficacy against a heterologous challenge. In conclusion, systemic administration of SAM vectors expressing conserved internal influenza antigens induced protective immune responses in mice, supporting the SAM® platform as another promising strategy for the development of broad-spectrum universal influenza vaccines.


Subject(s)
Antigens, Viral/genetics , Antigens, Viral/immunology , Conserved Sequence , Influenza A virus/immunology , Influenza A virus/physiology , Influenza Vaccines/genetics , Influenza Vaccines/immunology , Animals , Cell Line , Cricetinae , Gene Amplification , Gene Expression , Genetic Vectors/genetics , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H1N1 Subtype/physiology , Influenza A Virus, H3N2 Subtype/immunology , Influenza A Virus, H3N2 Subtype/physiology , Lung/immunology , Mice , RNA, Messenger/genetics , T-Lymphocytes, Cytotoxic/immunology , Vaccines, Inactivated/genetics , Vaccines, Inactivated/immunology , Viral Core Proteins/genetics , Viral Core Proteins/immunology , Viral Matrix Proteins/genetics , Viral Matrix Proteins/immunology
14.
PLoS One ; 11(6): e0157066, 2016.
Article in English | MEDLINE | ID: mdl-27336786

ABSTRACT

CD4+ T follicular helper cells (T(FH)) have been identified as the T-cell subset specialized in providing help to B cells for optimal activation and production of high affinity antibody. We recently demonstrated that the expansion of peripheral blood influenza-specific CD4(+)IL-21(+)ICOS1(+) T helper (T(H)) cells, three weeks after vaccination, associated with and predicted the rise of protective neutralizing antibodies to avian H5N1. In this study, healthy adults were vaccinated with plain seasonal trivalent inactivated influenza vaccine (TIIV), MF59(®)-adjuvanted TIIV (ATIIV), or saline placebo. Frequencies of circulating CD4(+) T(FH)1 ICOS(+) T(FH) cells and H1N1-specific CD4(+-)IL-21(+)ICOS(+) CXCR5(+) T(FH) and CXCR5(-) T(H) cell subsets were determined at various time points after vaccination and were then correlated with hemagglutination inhibition (HI) titers. All three CD4(+) T cell subsets expanded in response to TIIV and ATIIV, and peaked 7 days after vaccination. To demonstrate that these T(FH) cell subsets correlated with functional antibody titers, we defined an alternative endpoint metric, decorrelated HI (DHI), which removed any correlation between day 28/day 168 and day 0 HI titers, to control for the effect of preexisting immunity to influenza vaccine strains. The numbers of total circulating CD4(+)T(FH)1 ICOS(+) cells and of H1N1-specific CD4(+)IL-21(+)ICOS(+) CXCR5(+), measured at day 7, were significantly associated with day 28, and day 28 and 168 DHI titers, respectively. Altogether, our results show that CD4(+) T(FH) subsets may represent valuable biomarkers of vaccine-induced long-term functional immunity.


Subject(s)
Antibody Formation/immunology , Immunity , Lymphocyte Count , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Helper-Inducer/immunology , Vaccination , Adolescent , Adult , Antibodies, Viral/blood , Antibodies, Viral/immunology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Biomarkers , Hemagglutination Inhibition Tests , Humans , Immunophenotyping , Inducible T-Cell Co-Stimulator Protein/metabolism , Influenza A Virus, H1N1 Subtype/immunology , Influenza Vaccines/immunology , Lymphocyte Activation/immunology , Prognosis , Public Health Surveillance , Receptors, CXCR5/metabolism , T-Lymphocyte Subsets/metabolism , T-Lymphocytes, Helper-Inducer/metabolism , Time Factors , Young Adult
15.
Vaccine ; 34(32): 3697-701, 2016 07 12.
Article in English | MEDLINE | ID: mdl-27055022

ABSTRACT

BACKGROUND: New human immunodeficiency virus (HIV) infections continue to occur worldwide. Despite previous failures, there is renewed optimism about developing an efficacious HIV prophylactic vaccine following the 31.2% vaccine efficacy (modified intention to treat analysis) achieved in the RV-144 trial. Intense efforts at characterising the immune responses in the trial participants who appeared to gain some protection from the candidate vaccine are ongoing to delineate correlates of protection. However, the characteristics of a vaccine suitable for programmatic introduction in high prevalence areas remain undefined. AIMS: We set out to ascertain the vaccination policies and strategies that policy makers involved in vaccine introductions would advise were a candidate HIV vaccine to become available. METHODS: Structured questionnaires in both English and French were self-administered to consenting policy makers such as members of National Immunisation Technical Advisory Groups. Members from three out of the six WHO regional groups were purposively reached for their responses. RESULTS: Thirty-seven key opinion leaders were approached through self-administered questionnaires delivered by e-mail or in person. Nine responses were received, representing a 24.3% response rate. The responses received were from three [Africa (6), Americas (1) and Europe (2)] out of the six WHO regions. All respondents would prioritise the vaccination of commercial sex workers over other risk groups if there was an efficacious HIV vaccine. Vaccine efficacy was considered to be the most important factor, ahead of vaccine safety and cost, in determining the acceptability of a new prophylactic HIV vaccine. CONCLUSIONS: It is expected that the first generation HIV vaccines may be modestly efficacious. However, even a modestly efficacious vaccine might curtail the spread of HIV if universal or near-universal coverage is achieved. It is important to anticipate policy discussions which would influence how rapidly an HIV vaccine would be rolled-out programmatically to achieve maximum impact.


Subject(s)
AIDS Vaccines/therapeutic use , Health Policy , Vaccination/legislation & jurisprudence , Advisory Committees , HIV Infections/prevention & control , Humans , Sex Workers , Surveys and Questionnaires
16.
J Infect Dis ; 213(12): 1876-85, 2016 06 15.
Article in English | MEDLINE | ID: mdl-26908732

ABSTRACT

BACKGROUND: Most preclinical studies assess vaccine effectiveness in single-pathogen infection models. This is unrealistic given that humans are continuously exposed to different commensals and pathogens in sequential and mixed infections. Accordingly, complications from secondary bacterial infection are a leading cause of influenza-associated morbidity and mortality. New vaccination strategies are needed to control infections on simultaneous fronts. METHODS: We compared different anti-influenza vaccines for their protective potential in a model of viral infection with bacterial superinfection. Mice were immunized with H1N1/A/California/7/2009 subunit vaccines, formulated with different adjuvants inducing either T-helper type 1 (Th1) (MF59 plus CpG)-, Th1/2 (MF59)-, or Th17 (LTK63)-prone immune responses and were sequentially challenged with mouse-adapted influenza virus H1N1/A/Puerto Rico/8/1934 and Staphylococcus aureus USA300, a clonotype emerging as a leading contributor in postinfluenza pneumonia in humans. RESULTS: Unadjuvanted vaccine controlled single viral infection, yet mice had considerable morbidity from viral disease and bacterial superinfection. In contrast, all adjuvanted vaccines efficiently protected mice in both conditions. Interestingly, the Th1-inducing formulation was superior to Th1/2 or Th17 inducers. CONCLUSIONS: Our studies should help us better understand how differential immunity to influenza skews immune responses toward coinfecting bacteria and discover novel modes to prevent bacterial superinfections in the lungs of persons with influenza.


Subject(s)
Influenza A Virus, H1N1 Subtype/immunology , Influenza Vaccines/immunology , Influenza, Human/prevention & control , Staphylococcal Infections/prevention & control , Staphylococcus aureus/immunology , Superinfection/prevention & control , Adjuvants, Immunologic/administration & dosage , Animals , Bacterial Toxins/administration & dosage , Enterotoxins/administration & dosage , Escherichia coli Proteins/administration & dosage , Female , Humans , Immunization , Influenza Vaccines/administration & dosage , Influenza, Human/complications , Influenza, Human/microbiology , Mice , Mice, Inbred BALB C , Oligodeoxyribonucleotides/administration & dosage , Polysorbates/administration & dosage , Specific Pathogen-Free Organisms , Squalene/administration & dosage , Staphylococcal Infections/complications , Staphylococcal Infections/microbiology , Superinfection/microbiology
17.
PLoS One ; 11(1): e0147767, 2016.
Article in English | MEDLINE | ID: mdl-26812180

ABSTRACT

A rapidly acting, single dose vaccine against Staphylococcus aureus would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent S. aureus candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4+ T cells not only reduced survival but also increased S. aureus load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by in vivo neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against S. aureus through the combined actions of antibodies, CD4+ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units.


Subject(s)
Antibodies, Bacterial/immunology , CD4-Positive T-Lymphocytes/immunology , Interleukin-17/metabolism , Staphylococcal Infections/prevention & control , Staphylococcal Vaccines/immunology , Staphylococcus aureus/immunology , Toll-Like Receptor 7/metabolism , Adjuvants, Immunologic , Animals , Antibodies, Neutralizing/immunology , CD4-Positive T-Lymphocytes/cytology , Cytokines/metabolism , Female , Mice , Mice, Inbred C57BL , Spleen/metabolism , Spleen/pathology , Staphylococcal Infections/immunology , Staphylococcal Infections/mortality , Staphylococcus aureus/genetics , Survival Rate , Th1 Cells/immunology , Th17 Cells/immunology , Toll-Like Receptor 7/immunology
18.
J Virol ; 90(1): 332-44, 2016 01 01.
Article in English | MEDLINE | ID: mdl-26468547

ABSTRACT

UNLABELLED: Seasonal influenza is a vaccine-preventable disease that remains a major health problem worldwide, especially in immunocompromised populations. The impact of influenza disease is even greater when strains drift, and influenza pandemics can result when animal-derived influenza virus strains combine with seasonal strains. In this study, we used the SAM technology and characterized the immunogenicity and efficacy of a self-amplifying mRNA expressing influenza virus hemagglutinin (HA) antigen [SAM(HA)] formulated with a novel oil-in-water cationic nanoemulsion. We demonstrated that SAM(HA) was immunogenic in ferrets and facilitated containment of viral replication in the upper respiratory tract of influenza virus-infected animals. In mice, SAM(HA) induced potent functional neutralizing antibody and cellular immune responses, characterized by HA-specific CD4 T helper 1 and CD8 cytotoxic T cells. Furthermore, mice immunized with SAM(HA) derived from the influenza A virus A/California/7/2009 (H1N1) strain (Cal) were protected from a lethal challenge with the heterologous mouse-adapted A/PR/8/1934 (H1N1) virus strain (PR8). Sera derived from SAM(H1-Cal)-immunized animals were not cross-reactive with the PR8 virus, whereas cross-reactivity was observed for HA-specific CD4 and CD8 T cells. Finally, depletion of T cells demonstrated that T-cell responses were essential in mediating heterologous protection. If the SAM vaccine platform proves safe, well tolerated, and effective in humans, the fully synthetic SAM vaccine technology could provide a rapid response platform to control pandemic influenza. IMPORTANCE: In this study, we describe protective immune responses in mice and ferrets after vaccination with a novel HA-based influenza vaccine. This novel type of vaccine elicits both humoral and cellular immune responses. Although vaccine-specific antibodies are the key players in mediating protection from homologous influenza virus infections, vaccine-specific T cells contribute to the control of heterologous infections. The rapid production capacity and the synthetic origin of the vaccine antigen make the SAM platform particularly exploitable in case of influenza pandemic.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza Vaccines/immunology , Orthomyxoviridae Infections/prevention & control , RNA, Messenger/genetics , RNA, Messenger/metabolism , Vaccines, DNA/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cross Protection , Disease Models, Animal , Female , Ferrets , Influenza Vaccines/administration & dosage , Influenza Vaccines/genetics , Leukocyte Reduction Procedures , Mice, Inbred BALB C , Orthomyxoviridae Infections/immunology , Respiratory System/virology , Survival Analysis , Treatment Outcome , Vaccines, DNA/administration & dosage , Vaccines, DNA/genetics , Viral Load
19.
Front Immunol ; 6: 439, 2015.
Article in English | MEDLINE | ID: mdl-26441955

ABSTRACT

Staphylococcus aureus (S. aureus) is an important opportunistic pathogen that may cause invasive life-threatening infections, like sepsis and pneumonia. Due to the increasing antibiotic resistance, the development of an effective vaccine against S. aureus is needed. Although a correlate of protection against staphylococcal diseases is not yet established, several findings suggest that both antibodies and CD4 T cells might contribute to optimal immunity. In this study, we show that adjuvanting a multivalent vaccine (4C-Staph) with MF59, an oil-in-water emulsion licensed in human vaccines, further potentiated antigen-specific IgG titers and CD4 T-cell responses compared to alum and conferred protection in the peritonitis model of S. aureus infection. Moreover, we showed that MF59- and alum-adjuvanted 4C-Staph vaccines induced persistent antigen-specific humoral and T-cell responses, and protected mice from infection up to 4 months after immunization. Furthermore, 4C-Staph formulated with MF59 was used to investigate which immune compartment is involved in vaccine-induced protection. Using CD4 T cell-depleted mice or B cell-deficient mice, we demonstrated that both T and B-cell responses contributed to 4C-Staph vaccine-mediated protective immunity. However, the role of CD4 T cells seemed more evident in the presence of low-antibody responses. This study provides preclinical data further supporting the use of the adjuvanted 4C-Staph vaccines against S. aureus diseases, and provides critical insights on the correlates of protective immunity necessary to combat this pathogen.

20.
PLoS One ; 10(8): e0135474, 2015.
Article in English | MEDLINE | ID: mdl-26280677

ABSTRACT

Developing a universal influenza vaccine that induces broad spectrum and longer-term immunity has become an important potentially achievable target in influenza vaccine research and development. Hemagglutinin (HA) and neuraminidase (NA) are the two major influenza virus antigens. Although antibody responses against influenza virus are mainly directed toward HA, NA is reported to be more genetically stable; hence NA-based vaccines have the potential to be effective for longer time periods. NA-specific immunity has been shown to limit the spread of influenza virus, thus reducing disease symptoms and providing cross-protection against heterosubtypic viruses in mouse challenge experiments. The production of large quantities of highly pure and stable NA could be beneficial for the development of new antivirals, subunit-based vaccines, and novel diagnostic tools. In this study, recombinant NA (rNA) was produced in mammalian cells at high levels from both swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/01/2005 (H5N1) influenza viruses. Biochemical, structural, and immunological characterizations revealed that the soluble rNAs produced are tetrameric, enzymatically active and immunogenic, and finally they represent good alternatives to conventionally used sources of NA in the Enzyme-Linked Lectin Assay (ELLA).


Subject(s)
Antigens, Viral/immunology , Lectins/immunology , Neuraminidase/immunology , Recombinant Proteins/immunology , Viral Proteins/immunology , Animals , Antibodies, Viral/immunology , Antibody Formation/immunology , Birds , Cell Line , Cross Protection/immunology , Cross Reactions/immunology , Enzyme-Linked Immunospot Assay/methods , Female , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H5N1 Subtype/immunology , Influenza Vaccines/immunology , Influenza in Birds/immunology , Mice , Orthomyxoviridae Infections/immunology , Swine
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