ABSTRACT
Accidents involving Brown spiders are reported throughout the world. In the venom, the major toxins involved in the deleterious effects are phospholipases D (PLDs). In this work, recombinant mutated phospholipases D from three endemic species medically relevant in South America (Loxosceles intermedia, L. laeta and L. gaucho) were tested as antigens in a vaccination protocol. In such isoforms, key amino acid residues involved in catalysis, magnesium-ion coordination, and binding to substrates were replaced by Alanine (H12A-H47A, E32A-D34A and W230A). These mutations eliminated the phospholipase activity and reduced the generation of skin necrosis and edema to residual levels. Molecular modeling of mutated isoforms indicated that the three-dimensional structures, topologies, and surface charges did not undergo significant changes. Mutated isoforms were recognized by sera against the crude venoms. Vaccination protocols in rabbits using mutated isoforms generated a serum that recognized the native PLDs of crude venoms and neutralized dermonecrosis and edema induced by L. intermedia venom. Vaccination of mice prevented the lethal effects of L. intermedia crude venom. Furthermore, vaccination of rabbits prevented the cutaneous lesion triggered by the three venoms. These results indicate a great potential for mutated recombinant PLDs to be employed as antigens in developing protective vaccines for Loxoscelism.
Subject(s)
Brown Recluse Spider , Mutant Proteins/immunology , Phospholipase D/immunology , Spider Bites/immunology , Spider Bites/therapy , Vaccines/immunology , Accidents , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antivenins/blood , Antivenins/immunology , Biomarkers , Disease Models, Animal , Immunogenicity, Vaccine , Leukocyte Count , Mice , Models, Molecular , Mutant Proteins/chemistry , Mutant Proteins/genetics , Neutralization Tests , Phospholipase D/chemistry , Phospholipase D/genetics , Rabbits , Spider Bites/diagnosis , Spider Bites/prevention & control , Spider Venoms/immunology , Structure-Activity Relationship , Treatment Outcome , Vaccination , Vaccines/administration & dosageABSTRACT
Antigen-adjuvant conjugation is known to enhance antigen-specific T-cell production in vaccine models, but scalable methods are required to generate site-specific conjugation for clinical translation of this technique. We report the use of the cell-free protein synthesis (CFPS) platform as a rapid method to produce large quantities (> 100 mg/L) of a model antigen, ovalbumin (OVA), with site-specific incorporation of p-azidomethyl-L-phenylalanine (pAMF) at two solvent-exposed sites away from immunodominant epitopes. Using copper-free click chemistry, we conjugated CpG oligodeoxynucleotide toll-like receptor 9 (TLR9) agonists to the pAMF sites on the mutant OVA protein. The OVA-CpG conjugates demonstrate enhanced antigen presentation in vitro and increased antigen-specific CD8+ T-cell production in vivo. Moreover, OVA-CpG conjugation reduced the dose of CpG needed to invoke antigen-specific T-cell production tenfold. These results highlight how site-specific conjugation and CFPS technology can be implemented to produce large quantities of covalently-linked antigen-adjuvant conjugates for use in clinical vaccines.
Subject(s)
Adjuvants, Immunologic/metabolism , Antigen Presentation , Antigens/immunology , CD8-Positive T-Lymphocytes/immunology , Mutant Proteins/immunology , Oligodeoxyribonucleotides/immunology , Ovalbumin/immunology , Animals , Antigen-Presenting Cells/immunology , Antigens/genetics , Cell-Free System , Click Chemistry/methods , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Models, Animal , Oligodeoxyribonucleotides/metabolism , Oligodeoxyribonucleotides/pharmacology , Ovalbumin/genetics , Toll-Like Receptor 9/agonists , Toll-Like Receptor 9/genetics , Toll-Like Receptor 9/metabolism , Transfection , Vaccination/methods , Vaccines, Conjugate/administration & dosage , Vaccines, Conjugate/immunology , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/immunologyABSTRACT
Mutated isocitrate dehydrogenase 1 (IDH1) defines a molecularly distinct subtype of diffuse glioma1-3. The most common IDH1 mutation in gliomas affects codon 132 and encodes IDH1(R132H), which harbours a shared clonal neoepitope that is presented on major histocompatibility complex (MHC) class II4,5. An IDH1(R132H)-specific peptide vaccine (IDH1-vac) induces specific therapeutic T helper cell responses that are effective against IDH1(R132H)+ tumours in syngeneic MHC-humanized mice4,6-8. Here we describe a multicentre, single-arm, open-label, first-in-humans phase I trial that we carried out in 33 patients with newly diagnosed World Health Organization grade 3 and 4 IDH1(R132H)+ astrocytomas (Neurooncology Working Group of the German Cancer Society trial 16 (NOA16), ClinicalTrials.gov identifier NCT02454634). The trial met its primary safety endpoint, with vaccine-related adverse events restricted to grade 1. Vaccine-induced immune responses were observed in 93.3% of patients across multiple MHC alleles. Three-year progression-free and death-free rates were 0.63 and 0.84, respectively. Patients with immune responses showed a two-year progression-free rate of 0.82. Two patients without an immune response showed tumour progression within two years of first diagnosis. A mutation-specificity score that incorporates the duration and level of vaccine-induced IDH1(R132H)-specific T cell responses was associated with intratumoral presentation of the IDH1(R132H) neoantigen in pre-treatment tumour tissue. There was a high frequency of pseudoprogression, which indicates intratumoral inflammatory reactions. Pseudoprogression was associated with increased vaccine-induced peripheral T cell responses. Combined single-cell RNA and T cell receptor sequencing showed that tumour-infiltrating CD40LG+ and CXCL13+ T helper cell clusters in a patient with pseudoprogression were dominated by a single IDH1(R132H)-reactive T cell receptor.
Subject(s)
Cancer Vaccines/immunology , Cancer Vaccines/therapeutic use , Glioma/diagnosis , Glioma/therapy , Isocitrate Dehydrogenase/genetics , Isocitrate Dehydrogenase/immunology , Mutation , Adult , Cells, Cultured , Disease Progression , Female , Glioma/genetics , Glioma/immunology , Humans , Male , Mutant Proteins/genetics , Mutant Proteins/immunology , Phenotype , Receptors, Antigen, T-Cell/immunology , Survival Rate , T-Lymphocytes/immunologyABSTRACT
Mutations in the RAS oncogenes occur in multiple cancers, and ways to target these mutations has been the subject of intense research for decades. Most of these efforts are focused on conventional small-molecule drugs rather than antibody-based therapies because the RAS proteins are intracellular. Peptides derived from recurrent RAS mutations, G12V and Q61H/L/R, are presented on cancer cells in the context of two common human leukocyte antigen (HLA) alleles, HLA-A3 and HLA-A1, respectively. Using phage display, we isolated single-chain variable fragments (scFvs) specific for each of these mutant peptide-HLA complexes. The scFvs did not recognize the peptides derived from the wild-type form of RAS proteins or other related peptides. We then sought to develop an immunotherapeutic agent that was capable of killing cells presenting very low levels of these RAS-derived peptide-HLA complexes. Among many variations of bispecific antibodies tested, one particular format, the single-chain diabody (scDb), exhibited superior reactivity to cells expressing low levels of neoantigens. We converted the scFvs to this scDb format and demonstrated that they were capable of inducing T cell activation and killing of target cancer cells expressing endogenous levels of the mutant RAS proteins and cognate HLA alleles. CRISPR-mediated alterations of the HLA and RAS genes provided strong genetic evidence for the specificity of the scDbs. Thus, this approach could be applied to other common oncogenic mutations that are difficult to target by conventional means, allowing for more specific anticancer therapeutics.
Subject(s)
Antibodies, Bispecific/pharmacology , Antigens, Neoplasm , Biomarkers, Tumor/antagonists & inhibitors , Mutant Proteins/antagonists & inhibitors , ras Proteins/antagonists & inhibitors , Amino Acid Sequence , Animals , Antibodies, Bispecific/immunology , Antigens, Neoplasm/chemistry , Antigens, Neoplasm/immunology , Biomarkers, Tumor/chemistry , Biomarkers, Tumor/genetics , Biomarkers, Tumor/immunology , Cell Line , Cross Reactions , HLA Antigens/immunology , Humans , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Mutant Proteins/chemistry , Mutant Proteins/immunology , Mutation , Peptide Fragments , Protein Binding/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , ras Proteins/chemistry , ras Proteins/genetics , ras Proteins/immunologyABSTRACT
The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals.
Subject(s)
COVID-19 Drug Treatment , COVID-19/therapy , COVID-19/virology , Evolution, Molecular , Mutagenesis/drug effects , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Adenosine Monophosphate/therapeutic use , Aged , Alanine/analogs & derivatives , Alanine/pharmacology , Alanine/therapeutic use , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Chronic Disease , Genome, Viral/drug effects , Genome, Viral/genetics , High-Throughput Nucleotide Sequencing , Humans , Immune Evasion/drug effects , Immune Evasion/genetics , Immune Evasion/immunology , Immune Tolerance/drug effects , Immune Tolerance/immunology , Immunization, Passive , Immunosuppression Therapy , Male , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/immunology , Mutation , Phylogeny , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Time Factors , Viral Load/drug effects , Virus Shedding , COVID-19 SerotherapyABSTRACT
Dendritic cells (DCs) are professional antigen presenting cells with a great capacity for cross-presentation of exogenous antigens from which robust anti-tumor immune responses ensue. However, this function is not always available and requires DCs to first be primed to induce their maturation. In particular, in the field of DC vaccine design, currently available methodologies have been limited in eliciting a sustained anti-tumor immune response. Mechanistically, part of the maturation response is influenced by the presence of stimulatory receptors relying on ITAM-containing activating adaptor molecules like DAP12, that modulates their function. We hypothesize that activating DAP12 in DC could force their maturation and enhance their potential anti-tumor activity for therapeutic intervention. For this purpose, we developed constitutively active DAP12 mutants that can promote activation of monocyte-derived DC. Here we demonstrate its ability to induce the maturation and activation of monocyte-derived DCs which enhances migration, and T cell stimulation in vitro using primary human cells. Moreover, constitutively active DAP12 stimulates a strong immune response in a murine melanoma model leading to a reduction of tumor burden. This provides proof-of-concept for investigating the pre-activation of antigen presenting cells to enhance the effectiveness of anti-tumor immunotherapies.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Dendritic Cells/immunology , Immunity, Cellular/immunology , Melanoma, Experimental/immunology , Membrane Proteins/genetics , Adaptor Proteins, Signal Transducing/immunology , Animals , Antigen-Presenting Cells/immunology , Cancer Vaccines/immunology , Cell Movement/genetics , Cell Proliferation/genetics , Humans , Immunity, Cellular/genetics , Melanoma, Experimental/pathology , Melanoma, Experimental/therapy , Membrane Proteins/immunology , Mice , Monocytes/immunology , Mutant Proteins/genetics , Mutant Proteins/immunology , Tumor Burden/immunologyABSTRACT
In antibody light chain amyloidosis (AL), mutant light chains (LCs) or their variable domains (VLs) form fibrils, which accumulate in organs and lead to their failure. The molecular mechanism of this disease is still poorly understood. One of the key open issues is whether the mutant VLs and LCs differ in fibril formation. We addressed this question studying the effects of the VL mutations S20N and R61A within the isolated VL domain and in the full-length LC scaffold. Both VL variants readily form fibrils. Here, we find that in the LC context, the S20N variant is protected from fibril formation while for LC R61A fibril formation is even accelerated compared to VL R61A. Our analyses revealed that the partially unfolded state of the VL R61A domain destabilizes the CL domain by non-native interactions, in turn leading to a further unfolding of the VL domain. In contrast, the folded mutant VL S20N and VL wt form native interactions with CL. These are beneficial for LC stability and promote amyloid resistance. Thus the effects of specific mutations on the VL fold can have opposing effects on LC domain interactions, stability and amyloidogenicity.
Subject(s)
Amyloid/genetics , Amyloidogenic Proteins/genetics , Immunoglobulin Light Chains/immunology , Protein Aggregation, Pathological/genetics , Amino Acid Sequence/genetics , Amyloid/immunology , Amyloidogenic Proteins/immunology , Amyloidosis/genetics , Amyloidosis/immunology , Humans , Immunoglobulin Light Chains/genetics , Models, Molecular , Mutant Proteins/genetics , Mutant Proteins/immunology , Protein Aggregation, Pathological/immunology , Protein ConformationABSTRACT
Interleukin-2 (IL-2) controls the homeostasis and function of regulatory T (Treg) cells, and defects in the IL-2 pathway contribute to multiple autoimmune diseases. Although recombinant IL-2 therapy has been efficacious in certain inflammatory conditions, the capacity for IL-2 to also activate inflammatory effector responses highlights the need for IL-2-based therapeutics with improved Treg cell specificity. From a panel of rationally designed murine IL-2 variants, we identified IL-2 muteins with reduced potency and enhanced Treg cell selectivity due to increased dependence on the IL-2 receptor component CD25. As an Fc-fused homodimer, the optimal Fc.IL-2 mutein induced selective Treg cell enrichment and reduced agonism of effector cells across a wide dose range. Furthermore, despite being a weaker agonist, overall Treg cell growth was greater and more sustained due to reduced receptor-mediated clearance of the Fc.IL-2 mutein compared with Fc-fused wild-type IL-2. Preferential Treg cell enrichment was also observed in the presence of activated pathogenic T cells in the pancreas of nonobese diabetic (NOD) mice, despite a loss of Treg cell selectivity in an IL-2R proximal response. These properties facilitated potent and extended resolution of NOD diabetes with infrequent dosing schedules.
Subject(s)
Autoimmunity , Interleukin-2/pharmacology , Mutant Proteins/pharmacology , Receptors, Fc/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Blood Glucose , Diabetes Mellitus, Type 1/blood , Diabetes Mellitus, Type 1/immunology , Female , Genetic Engineering , Genetic Variation , HEK293 Cells , Humans , Interleukin-2/genetics , Interleukin-2/immunology , Male , Mice, Inbred C57BL , Mice, Inbred NOD , Mutant Proteins/genetics , Mutant Proteins/immunology , Pancreas/immunology , Receptors, Fc/genetics , Recombinant Proteins/immunology , Recombinant Proteins/pharmacologyABSTRACT
Immunodominance is recognized as a key factor in the antigenic drift of seasonal influenza viruses. In the immunodominance model, each individual in a population predominantly responds to a single epitope among the five antigenic epitopes of the viral hemagglutinin (HA), driving escape mutations one at a time, and sequential mutations in multiple individuals who respond to different epitopes eventually generate a drifted strain with mutations in epitopes that are targeted by a majority of the population. A focused antibody response to the Sa epitope in people born between 1965 and 1979 was believed to contribute to a mutation at HA residue 163 and the first antigenic drift of the 2009 pandemic influenza A H1N1 virus. A serine-to-threonine mutation at HA residue 185 in the Sb epitope emerged in 2010 even before the 163 mutation. We show here that a large fraction of the population in 2010-2011 had responses to the Sb epitope, as shown by 47% of tested sera having altered titers to the S185T mutant. Responses to the Sb epitope showed an age-specific trend similar to that found for the response to Sa epitope in these subjects. Together, the focused responses to Sa and Sb epitopes may have driven the first antigenic drift of the 2009 pandemic H1N1 virus.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Antigenic Variation , Evolution, Molecular , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza A Virus, H1N1 Subtype/immunology , Animals , Dogs , Epitope Mapping , Hemagglutination Inhibition Tests , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Humans , Influenza A Virus, H1N1 Subtype/genetics , Mutant Proteins/genetics , Mutant Proteins/immunology , RNA, Viral/genetics , Selection, Genetic , Sequence Analysis, DNA , Virus CultivationABSTRACT
Antibodies targeting the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) present a promising approach to combat the coronavirus disease 2019 (COVID-19) pandemic; however, concerns remain that mutations can yield antibody resistance. We investigated the development of resistance against four antibodies to the spike protein that potently neutralize SARS-CoV-2, individually as well as when combined into cocktails. These antibodies remain effective against spike variants that have arisen in the human population. However, novel spike mutants rapidly appeared after in vitro passaging in the presence of individual antibodies, resulting in loss of neutralization; such escape also occurred with combinations of antibodies binding diverse but overlapping regions of the spike protein. Escape mutants were not generated after treatment with a noncompeting antibody cocktail.
Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Betacoronavirus/immunology , Coronavirus Infections/immunology , Pneumonia, Viral/immunology , Spike Glycoprotein, Coronavirus/immunology , Betacoronavirus/chemistry , Betacoronavirus/genetics , COVID-19 , Epitopes , Genome, Viral , Humans , Mutant Proteins/chemistry , Mutant Proteins/immunology , Mutation , Neutralization Tests , Pandemics , Protein Interaction Domains and Motifs , SARS-CoV-2 , Selection, Genetic , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/geneticsABSTRACT
The global coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made the development of a vaccine a top biomedical priority. In this study, we developed a series of DNA vaccine candidates expressing different forms of the SARS-CoV-2 spike (S) protein and evaluated them in 35 rhesus macaques. Vaccinated animals developed humoral and cellular immune responses, including neutralizing antibody titers at levels comparable to those found in convalescent humans and macaques infected with SARS-CoV-2. After vaccination, all animals were challenged with SARS-CoV-2, and the vaccine encoding the full-length S protein resulted in >3.1 and >3.7 log10 reductions in median viral loads in bronchoalveolar lavage and nasal mucosa, respectively, as compared with viral loads in sham controls. Vaccine-elicited neutralizing antibody titers correlated with protective efficacy, suggesting an immune correlate of protection. These data demonstrate vaccine protection against SARS-CoV-2 in nonhuman primates.
Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Spike Glycoprotein, Coronavirus/immunology , Vaccines, DNA/immunology , Viral Vaccines/immunology , Adjuvants, Immunologic , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Betacoronavirus/physiology , Bronchoalveolar Lavage Fluid/virology , COVID-19 , COVID-19 Vaccines , Coronavirus Infections/immunology , Coronavirus Infections/virology , Disease Models, Animal , Female , Humans , Immunity, Cellular , Immunity, Humoral , Immunization, Secondary , Immunogenicity, Vaccine , Immunologic Memory , Macaca mulatta , Male , Mutant Proteins/chemistry , Mutant Proteins/immunology , Nasal Mucosa/virology , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , Protein Domains , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Vaccination , Vaccines, DNA/administration & dosage , Viral Load , Viral Vaccines/administration & dosageABSTRACT
BACKGROUND: Early introduction of food allergens into children's diet is considered as a strategy for the prevention of food allergy. The major fish allergen parvalbumin exhibits high stability against gastrointestinal digestion. We investigated whether resistance of carp parvalbumin to digestion affects oral tolerance induction. METHODS: Natural Cyp c 1, nCyp c 1, and a gastrointestinal digestion-sensitive recombinant Cyp c 1 mutant, mCyp c 1, were analyzed for their ability to induce oral tolerance in a murine model. Both antigens were compared by gel filtration, circular dichroism measurement, in vitro digestion, and splenocyte proliferation assays using synthetic Cyp c 1-derived peptides. BALB/c mice were fed once with high doses of nCyp c 1 or mCyp c 1, before sensitization to nCyp c 1. Immunological tolerance was studied by measuring Cyp c 1-specific antibodies and cellular responses by ELISA, basophil activation, splenocyte proliferations, and intragastric allergen challenge. RESULTS: Wild-type and mCyp c 1 showed the same physicochemical properties and shared the same major T-cell epitope. However, mCyp c 1 was more sensitive to enzymatic digestion in vitro than nCyp c 1. A single high-dose oral administration of nCyp c 1 but not of mCyp c 1 induced long-term oral tolerance, characterized by lack of parvalbumin-specific antibody and cellular responses. Moreover, mCyp c 1-fed mice, but not nCyp c 1-fed mice developed allergic symptoms upon challenge with nCyp c 1. CONCLUSION: Sensitivity to digestion in the gastrointestinal tract influences the capacity of an allergen to induce prophylactic oral tolerance.
Subject(s)
Allergens/immunology , Calcium-Binding Proteins/immunology , Digestion/immunology , Fish Proteins/immunology , Food Hypersensitivity/prevention & control , Gastrointestinal Absorption/immunology , Immune Tolerance , Immunization/methods , Parvalbumins/immunology , Pre-Exposure Prophylaxis/methods , Allergens/genetics , Amino Acid Sequence , Animals , Calcium-Binding Proteins/genetics , Carps/metabolism , Cell Line, Tumor , Disease Models, Animal , Epitopes, T-Lymphocyte/immunology , Female , Fish Proteins/genetics , Food Hypersensitivity/immunology , Immunoglobulin E/blood , Immunoglobulin E/immunology , Mice , Mice, Inbred BALB C , Mutant Proteins/immunology , Parvalbumins/genetics , RatsABSTRACT
BACKGROUND: T cell therapy for cancer involves genetic introduction of a target-binding feature into autologous T cells, ex vivo expansion and single large bolus administration back to the patient. These reprogrammed T cells can be highly effective in killing cells, but tumor heterogeneity results in regrowth of cells that do not sufficiently express the single antigen being targeted. We describe a cell-based therapy that simultaneously targets multiple tumor-specific antigens. METHODS: High-affinity polyclonal rabbit antibodies were generated against nine different surface-related tumor-specific mutations on B16F10 cells. Unsorted splenic effector cells from syngeneic mice were incubated with a cocktail of the nine anti-B16F10 antibodies. These 'armed' effector cells were used to treat mice previously inoculated with B16F10 melanoma cells. RESULTS: The cocktail of nine antibodies resulted in dense homogeneous binding to histological sections of B16F10 cells. Five treatments with the armed effector cells and PD1 inhibition inhibited tumor growth and improved survival. Shortening the interval of the five treatments from every three days to every day increased survival. Arming effector cells with the four antibodies showing best binding to B16F10 cells even further increased survival. CONCLUSIONS: This study demonstrates that ex vivo arming a mixed population of immune effector cells with antibodies targeting multiple tumor-specific mutated proteins in conjunction with PD1 inhibition delayed tumor growth and prolonged survival in mice inoculated with an aggressive melanoma. A remarkably low total antibody dose of less than 5 µg was sufficient to accomplish tumor inhibition. Scaling up to clinical level may be feasible.
Subject(s)
Antibodies, Neoplasm/therapeutic use , Antigens, Neoplasm/immunology , Immunotherapy, Adoptive/methods , Leukocytes/immunology , Melanoma, Experimental/therapy , Skin Neoplasms/therapy , Tumor Burden , Animals , Antigens, Neoplasm/genetics , Melanoma, Experimental/immunology , Melanoma, Experimental/pathology , Mice , Mutant Proteins/genetics , Mutant Proteins/immunology , Mutation , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Skin Neoplasms/immunology , Skin Neoplasms/pathology , Spleen/cytology , Survival RateABSTRACT
BACKGROUND: Nonstructural protein 1 (NS1) is a virulence factor encoded by influenza A virus (IAV) that is expressed in the nucleus and cytoplasm of host cells during the earliest stages of infection. NS1 is a multifunctional protein that plays an important role in virus replication, virulence and inhibition of the host antiviral immune response. However, to date, the phosphorylation sites of NS1 have not been identified, and the relationship between phosphorylation and protein function has not been thoroughly elucidated. METHOD: In this study, potential phosphorylation sites in the swine influenza virus (SIV) NS1 protein were bioinformatically predicted and determined by Phos-tag SDS-PAGE analysis. To study the role of NS1 phosphorylation sites, we rescued NS1 mutants (Y73F and S83A) of A/swine/Shanghai/3/2014(H1N1) strain and compared their replication ability, cytokine production as well as the intracellular localization in cultured cells. Additionally, we used small interfering RNA (siRNA) assay to explore whether changes in the type I IFN response with dephosphorylation at positions 73 and 83 were mediated by the RIG-I pathway. RESULTS: We checked 18 predicted sites in 30 SIV NS1 genes to exclude strain-specific sites, covering H1N1, H1N2 and H3N2 subtypes and identified two phosphorylation sites Y73 and S83 in the H1N1 SIV protein by Phos-tag SDS-PAGE analysis. We found that dephosphorylation at positions 73 and 83 of the NS1 protein attenuated virus replication and reduced the ability of NS1 to antagonize IFN-ß expression but had no effect on nuclear localization. Knockdown of RIG-I dramatically impaired the induction of IFN-ß and ISG56 in NS1 Y73F or S83A mutant-infected cells, indicating that RIG-I plays a role in the IFN-ß response upon rSIV NS1 Y73F and rSIV NS1 S83A infection. CONCLUSION: We first identified two functional phosphorylation sites in the H1N1 SIV protein: Y73 and S83. We found that dephosphorylation at positions 73 and 83 of the NS1 protein affected the antiviral state in the host cells, partly through the RIG-I pathway.
Subject(s)
Influenza A Virus, H1N1 Subtype/growth & development , Interferon-beta/metabolism , Protein Processing, Post-Translational , Serine/metabolism , Tyrosine/metabolism , Viral Nonstructural Proteins/metabolism , Virus Replication , Animals , Cytokines/metabolism , Dogs , HEK293 Cells , Host-Pathogen Interactions , Humans , Immunologic Factors/metabolism , Influenza A Virus, H1N1 Subtype/immunology , Madin Darby Canine Kidney Cells , Mutant Proteins/immunology , Mutant Proteins/metabolism , Phosphorylation , Viral Nonstructural Proteins/immunology , Virulence Factors/immunology , Virulence Factors/metabolismABSTRACT
BACKGROUND: Vaccination and the use of neuraminidase inhibitors (NAIs) are currently the front lines of defense against seasonal influenza. The activity of influenza vaccines and antivirals drugs such as the NAIs can be affected by mutations in the influenza hemagglutinin (HA) protein. Numerous HA substitutions have been identified in nonclinical NAI resistance-selection experiments as well as in clinical specimens from NAI treatment or surveillance studies. These mutations are listed in the prescribing information (package inserts) for FDA-approved NAIs, including oseltamivir, zanamivir, and peramivir. METHODS: NAI treatment-emergent H1 HA mutations were mapped onto the H1N1 HA1 trimeric crystal structure and most of them localized to the HA antigenic sites predicted to be important for anti-influenza immunity. Recombinant A/California/04/09 (H1N1)-like viruses carrying HA V152I, G155E, S162 N, S183P, and D222G mutations were generated. We then evaluated the impact of these mutations on the immune reactivity and replication potential of the recombinant viruses in a human respiratory epithelial cell line, Calu- 3. RESULTS: We found that the G155E and D222G mutations significantly increased viral titers ~ 13-fold compared to the wild-type virus. The hemagglutination and microneutralization activity of goat and ferret antisera, monoclonal antibodies, and human serum samples raised against pandemic A(H1N1)pdm09 viruses was ~ 100-fold lower against mutants carrying G155E or D222G compared to the wild-type virus. CONCLUSIONS: Although the mechanism by which HA mutations emerge during NAI treatment is uncertain, some NAI treatment-emergent HA mutations correlate with decreased immunity to influenza virus.
Subject(s)
Drug Resistance, Viral , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Mutant Proteins/genetics , Mutant Proteins/immunology , Mutation, Missense , Acids, Carbocyclic , Antiviral Agents/pharmacology , Cell Line , Crystallography, X-Ray , Cyclopentanes/pharmacology , Epithelial Cells/virology , Epitopes/genetics , Guanidines/pharmacology , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Humans , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H1N1 Subtype/immunology , Mutant Proteins/chemistry , Neuraminidase/antagonists & inhibitors , Oseltamivir/pharmacology , Protein Conformation , Selection, Genetic , Viral Proteins/antagonists & inhibitors , Virus Replication , Zanamivir/pharmacologyABSTRACT
BACKGROUND: Supercharged GFP proteins were known as effective carriers for delivery of macromolecules into eukaryotic cells as well as fluorescent fusion tags for in vitro and in vivo detection. OBJECTIVE: Herein, anti-viral effects of +36 GFP and its anti-tumor effects were studied in vitro and in vivo, respectively. METHODS: We evaluated anti-HIV, anti-HSV, and anti-HCV effects of +36 GFP in vitro using ELISA, and real time PCR as common techniques for their detection, respectively. Moreover, we assessed the role of +36 GFP for eliciting HPV-related anti-tumor effects in mice due to the lack of HPV replication in vitro. RESULTS: Our data showed that +36 GFP efficiently enter the cells and augment the transfection rate of HPV16E7 antigen, as well. Furthermore, +36 GFP significantly reduced HCV, HIV and HSV replication up to 75%, 49% and 43% in HCV-infected Huh7.5 cells, HIV-infected Hela cells and HSV-infected Vero cells, respectively. On the other hand, mice immunization with +36 GFP complexed with HPV16 E7 antigen (+36GFP + E7) or fused to HPV16 E7 antigen (+36GFP-E7) elicited a higher Th1 cellular immune response with the predominant IgG2a, IgG2b, IFN-γ and Granzyme B levels than those induced by other groups. These regimens protected mice against TC- 1 tumor challenge (~ 67%) compared to E7 protein alone (~ 33%). These data suggested that +36 GFP can act as an anti-viral agent at certain dose due to its high efficiency in cell penetration in vitro and in vivo. CONCLUSION: Generally, +36 GFP targets viral replication in vitro as well as helps to suppress the growth of HPV-related tumors in vivo.
Subject(s)
Antiviral Agents/pharmacology , Green Fluorescent Proteins/genetics , Mutant Proteins/genetics , Papillomavirus Vaccines/genetics , Recombinant Fusion Proteins/genetics , Animals , Antineoplastic Agents/immunology , Antineoplastic Agents/pharmacology , Cell Line , Chlorocebus aethiops , Female , Granzymes/metabolism , Green Fluorescent Proteins/immunology , HIV/drug effects , HIV Infections/immunology , HIV Infections/prevention & control , Hepacivirus/drug effects , Human papillomavirus 16/drug effects , Humans , Immunity, Cellular , Interferon-gamma/metabolism , Mice, Inbred C57BL , Mutant Proteins/immunology , Mutant Proteins/pharmacology , Papillomavirus E7 Proteins/genetics , Papillomavirus E7 Proteins/immunology , Papillomavirus Vaccines/immunology , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/pharmacology , Virus Replication/drug effectsABSTRACT
C2 toxin produced from Clostridium botulinum serotypes C and D has a potential role in many pathophysiological mechanisms in birds and animals. It has encompassed an ADP ribosyltransferase subunit (C2I) and a translocation/binding subunit (C2II). In the present study, we intended to produce C2I mutant proteins as recombinant subunit vaccines by using glutathione-S-transferase-gene fusion system. The mutants of this study were previously evaluated from their evolutionary imprints and suggested as suitable candidates for subunit vaccines. A synthetic C2 gene was inserted in a pGEX-2T vector, cloned and expressed in Escherichia coli BL21 host. The expressed mutant proteins were purified by using glutathione-agarose column and then examined for their ADP ribosyltransferase activity and vaccinogenic characteristics. The pGEX-2T-C2I constructs with Y298F, S347A and S350A substitutions have shown effective transformation efficiencies in E. coli XL10 competent cells but their mutagenesis efficiency was relatively low. Gene expression analysis indicated the rate of gene expression was depended on the fused mutant genes. A high-level expression was achieved for Y298F, S347A and S350A mutant proteins. All purified protein exhibited a molecular mass of 49 kDa. C2I mutant proteins exhibited a reduced ADP ribosyltransferase activity with retained immunogenic and vaccinogenic characteristics compared to the wild-type C2I subunit. The overall analysis of our study suggested the recombinant C2I proteins (S197A and Y298F) are the most promising candidates for the development of subunit vaccine or immunogen for C2 mutants mediated diseases in birds and animals.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/immunology , Clostridium botulinum/genetics , Clostridium botulinum/immunology , Mutant Proteins/immunology , ADP Ribose Transferases/genetics , ADP Ribose Transferases/immunology , Cloning, Molecular , Escherichia coli/genetics , Gene Expression , Genetic Vectors , Mutagenesis , Vaccines, Subunit/genetics , Vaccines, Subunit/immunology , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunologyABSTRACT
MenB-4C (Bexsero; GlaxoSmithKline Biologicals) is a licensed meningococcal vaccine for capsular B strains. The vaccine contains detergent-extracted outer membrane vesicles (dOMV) and three recombinant proteins, of which one is factor H binding protein (FHbp). In previous studies, overexpression of FHbp in native OMV (NOMV) with genetically attenuated endotoxin (LpxL1) and/or by the use of mutant FHbp antigens with low factor H (FH) binding increased serum bactericidal antibody (SBA) responses. In this study, we immunized 13 infant macaques with 2 doses of NOMV with overexpressed mutant (R41S) FHbp with low binding to macaque FH (NOMV-FHbp). Control macaques received MenB-4C (n = 13) or aluminum hydroxide adjuvant alone (n = 4). NOMV-FHbp elicited a 2-fold higher IgG anti-FHbp geometric mean titer (GMT) than MenB-4C (P = 0.003), and the anti-FHbp repertoire inhibited binding of FH to FHbp, whereas anti-FHbp antibodies to MenB-4C enhanced FH binding. MenB-4C elicited a 10-fold higher GMT against strain NZ98/254, which was used to prepare the dOMV component, whereas NOMV-FHbp elicited an 8-fold higher GMT against strain H44/76, which was the parent of the mutant NOMV-FHbp vaccine strain. Against four strains with PorA mismatched to both of the vaccines and different FHbp sequence variants, NOMV-FHbp elicited 6- to 14-fold higher SBA GMTs than MenB-4C (P ≤ 0.0002). Two of 13 macaques immunized with MenB-4C but 0 of 17 macaques immunized with NOMV-FHbp or adjuvant developed serum anti-FH autoantibodies (P = 0.18). Thus, the mutant NOMV-FHbp approach has the potential to elicit higher and broader SBA responses than a licensed group B vaccine that contains wild-type FHbp that binds FH. The mutant NOMV-FHbp also might pose less of a risk of eliciting anti-FH autoantibodies.IMPORTANCE There are two licensed meningococcal capsular B vaccines. Both contain recombinant factor H binding protein (FHbp), which can bind to host complement factor H (FH). The limitations of these vaccines include a lack of protection against some meningococcal strains and the potential to elicit autoantibodies to FH. We immunized infant macaques with a native outer membrane vesicle (NOMV) vaccine with genetically attenuated endotoxin and overproduced mutant FHbp with low binding to FH. The NOMV-FHbp vaccine stimulated higher levels of protective serum antibodies than a licensed meningococcal group B vaccine against five of six genetically diverse meningococcal strains tested. Two of 13 macaques immunized with the licensed vaccine, which contains FHbp that binds macaque FH, but 0 of 17 macaques given NOMV-FHbp or the negative control developed serum anti-FH autoantibodies Thus, in a relevant nonhuman primate model, the NOMV-FHbp vaccine elicited greater protective antibodies than the licensed vaccine and may pose less of a risk of anti-FH autoantibody.
Subject(s)
Antibodies, Bacterial/blood , Antigens, Bacterial/immunology , Bacterial Outer Membrane Proteins/immunology , Bacterial Proteins/immunology , Complement Factor H/immunology , Meningococcal Vaccines/immunology , Animals , Antigens, Bacterial/genetics , Autoantibodies/immunology , Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/genetics , Carrier Proteins/genetics , Carrier Proteins/immunology , Macaca mulatta , Meningitis, Meningococcal/immunology , Meningitis, Meningococcal/prevention & control , Mutant Proteins/immunology , Neisseria meningitidis/genetics , Neisseria meningitidis/immunology , Neisseria meningitidis, Serogroup B/genetics , Neisseria meningitidis, Serogroup B/immunology , Serum Bactericidal Antibody AssayABSTRACT
We previously showed that single amino acid substitutions at seven positions in haemagglutinin determined major antigenic change of influenza H3N2 virus. Here, the impact of two such substitutions was tested in 11 representative H3 haemagglutinins to investigate context-dependence effects. The antigenic effect of substitutions introduced at haemagglutinin position 145 was fully independent of the amino acid context of the representative haemagglutinins. Antigenic change caused by substitutions introduced at haemagglutinin position 155 was variable and context-dependent. Our results suggest that epistatic interactions with contextual amino acids in the haemagglutinin can moderate the magnitude of antigenic change.
Subject(s)
Amino Acid Substitution , Antigens, Viral/immunology , Epistasis, Genetic , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza A Virus, H3N2 Subtype/classification , Influenza A Virus, H3N2 Subtype/immunology , Mutant Proteins/immunology , Antigens, Viral/genetics , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Humans , Influenza A Virus, H3N2 Subtype/genetics , Influenza, Human/virology , Mutant Proteins/geneticsABSTRACT
HCV core is an attractive HCV vaccine target, however, clinical or preclinical trials of core-based vaccines showed little success. We aimed to delineate what restricts its immunogenicity and improve immunogenic performance in mice. We designed plasmids encoding full-length HCV 1b core and its variants truncated after amino acids (aa) 60, 98, 152, 173, or up to aa 36 using virus-derived or synthetic polynucleotides (core191/60/98/152/173/36_191v or core152s DNA, respectively). We assessed their level of expression, route of degradation, ability to trigger the production of reactive oxygen species/ROS, and to activate the components of the Nrf2/ARE antioxidant defense pathway heme oxygenase 1/HO-1 and NAD(P)H: quinone oxidoreductase/Nqo-1. All core variants with the intact N-terminus induced production of ROS, and up-regulated expression of HO-1 and Nqo-1. The capacity of core variants to induce ROS and up-regulate HO-1 and Nqo-1 expression predetermined their immunogenicity in DNA-immunized BALB/c and C57BL/6 mice. The most immunogenic was core 152s, expressed at a modest level and inducing moderate oxidative stress and oxidative stress response. Thus, immunogenicity of HCV core is shaped by its ability to induce ROS and oxidative stress response. These considerations are important in understanding the mechanisms of viral suppression of cellular immune response and in HCV vaccine design.