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1.
PLoS Pathog ; 16(5): e1008393, 2020 05.
Article in English | MEDLINE | ID: mdl-32433711

ABSTRACT

Infection with (SAg)-producing bacteria may precede or follow infection with or vaccination against influenza A viruses (IAVs). However, how SAgs alter the breadth of IAV-specific CD8+ T cell (TCD8) responses is unknown. Moreover, whether recall responses mediating heterosubtypic immunity to IAVs are manipulated by SAgs remains unexplored. We employed wild-type (WT) and mutant bacterial SAgs, SAg-sufficient/deficient Staphylococcus aureus strains, and WT, mouse-adapted and reassortant IAV strains in multiple in vivo settings to address the above questions. Contrary to the popular view that SAgs delete or anergize T cells, systemic administration of staphylococcal enterotoxin B (SEB) or Mycoplasma arthritidis mitogen before intraperitoneal IAV immunization enlarged the clonal size of 'select' IAV-specific TCD8 and reshuffled the hierarchical pattern of primary TCD8 responses. This was mechanistically linked to the TCR Vß makeup of the impacted clones rather than their immunodominance status. Importantly, SAg-expanded TCD8 retained their IFN-γ production and cognate cytolytic capacities. The enhancing effect of SEB on immunodominant TCD8 was also evident in primary responses to vaccination with heat-inactivated and live attenuated IAV strains administered intramuscularly and intranasally, respectively. Interestingly, in prime-boost immunization settings, the outcome of SEB administration depended strictly upon the time point at which this SAg was introduced. Accordingly, SEB injection before priming raised CD127highKLRG1low memory precursor frequencies and augmented the anamnestic responses of SEB-binding TCD8. By comparison, introducing SEB before boosting diminished recall responses to IAV-derived epitopes drastically and indiscriminately. This was accompanied by lower Ki67 and higher Fas, LAG-3 and PD-1 levels consistent with a pro-apoptotic and/or exhausted phenotype. Therefore, SAgs can have contrasting impacts on anti-IAV immunity depending on the naïve/memory status and the TCR composition of exposed TCD8. Finally, local administration of SEB or infection with SEB-producing S. aureus enhanced pulmonary TCD8 responses to IAV. Our findings have clear implications for superinfections and prophylactic vaccination.


Subject(s)
Immunologic Memory/immunology , Influenza A virus/immunology , Superantigens/immunology , Animals , CD8-Positive T-Lymphocytes/immunology , Epitopes/immunology , Female , Humans , Immunologic Memory/physiology , Influenza A virus/metabolism , Influenza, Human/immunology , Influenza, Human/metabolism , Lymphocyte Activation/immunology , Mice , Mice, Inbred BALB C , Staphylococcus aureus/immunology , Superantigens/physiology , Superinfection/immunology , Vaccination
3.
J Immunol ; 202(12): 3370-3380, 2019 06 15.
Article in English | MEDLINE | ID: mdl-31092636

ABSTRACT

The importance of antiviral CD8+ T cell recognition of alternative reading frame (ARF)-derived peptides is uncertain. In this study, we describe an epitope (NS1-ARF21-8) present in a predicted 14-residue peptide encoded by the +1 register of NS1 mRNA in the influenza A virus (IAV). NS1-ARF21-8 elicits a robust, highly functional CD8+ T cell response in IAV-infected BALB/c mice. NS1-ARF21-8 is presented from unspliced NS mRNA, likely from downstream initiation on a Met residue that comprises the P1 position of NS1-ARF21-8 Derived from a 14-residue peptide with no apparent biological function and negligible impacts on IAV infection, infectivity, and pathogenicity, NS1-ARF21-8 provides a clear demonstration of how immunosurveillance exploits natural errors in protein translation to provide antiviral immunity. We further show that IAV infection enhances a model cellular ARF translation, which potentially has important implications for virus-induced autoimmunity.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Epitopes, T-Lymphocyte/metabolism , Influenza A virus/physiology , Influenza, Human/immunology , Orthomyxoviridae Infections/immunology , Viral Nonstructural Proteins/metabolism , Alternative Splicing , Animals , Disease Models, Animal , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/immunology , HEK293 Cells , Host-Pathogen Interactions , Humans , Immunologic Surveillance , Mice , Mice, Inbred BALB C , Open Reading Frames/genetics , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/immunology
4.
J Infect Dis ; 219(8): 1307-1317, 2019 04 08.
Article in English | MEDLINE | ID: mdl-30418594

ABSTRACT

Superantigens (SAgs) released by common Gram-positive bacterial pathogens have been reported to delete, anergize, or activate mouse T cells. However, little is known about their effects on preexisting memory CD8+ T cell (TCD8) pools. Furthermore, whether SAgs manipulate human memory TCD8 responses to cognate antigens is unknown. We used a human peripheral blood mononuclear cell culture system and a nontransgenic mouse model in which the impact of stimulation by two fundamentally distinct SAgs, staphylococcal enterotoxin B and Mycoplasma arthritidis mitogen, on influenza virus- and/or cytomegalovirus-specific memory TCD8 could be monitored. Bacterial SAgs surprisingly expanded antiviral memory TCD8 generated naturally through infection or artificially through vaccination. Mechanistically, this was a T cell-intrinsic and T cell receptor ß-chain variable-dependent phenomenon. Importantly, SAg-expanded TCD8 displayed an effector memory phenotype and were capable of producing interferon-γ and destroying target cells ex vivo or in vivo. These findings have clear implications for antimicrobial defense and rational vaccine design.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Superantigens/immunology , Adult , Animals , Antigens, Bacterial/immunology , Enterotoxins/immunology , Female , Humans , Immunologic Memory/immunology , Influenza Vaccines/immunology , Lymphocyte Activation/immunology , Male , Mice , Mice, Inbred BALB C , Orthomyxoviridae/immunology , Young Adult
5.
J Immunol ; 199(9): 3348-3359, 2017 11 01.
Article in English | MEDLINE | ID: mdl-28939757

ABSTRACT

The interactions between programmed death-1 (PD-1) and its ligands hamper tumor-specific CD8+ T cell (TCD8) responses, and PD-1-based "checkpoint inhibitors" have shown promise in certain cancers, thus revitalizing interest in immunotherapy. PD-1-targeted therapies reverse TCD8 exhaustion/anergy. However, whether they alter the epitope breadth of TCD8 responses remains unclear. This is an important question because subdominant TCD8 are more likely than immunodominant clones to escape tolerance mechanisms and may contribute to protective anticancer immunity. We have addressed this question in an in vivo model of TCD8 responses to well-defined epitopes of a clinically relevant oncoprotein, large T Ag. We found that unlike other coinhibitory molecules (CTLA-4, LAG-3, TIM-3), PD-1 was highly expressed by subdominant TCD8, which correlated with their propensity to favorably respond to PD-1/PD-1 ligand-1 (PD-L1)-blocking Abs. PD-1 blockade increased the size of subdominant TCD8 clones at the peak of their primary response, and it also sustained their presence, thus giving rise to an enlarged memory pool. The expanded population was fully functional as judged by IFN-γ production and MHC class I-restricted cytotoxicity. The selective increase in subdominant TCD8 clonal size was due to their enhanced survival, not proliferation. Further mechanistic studies utilizing peptide-pulsed dendritic cells, recombinant vaccinia viruses encoding full-length T Ag or epitope mingenes, and tumor cells expressing T Ag variants revealed that anti-PD-1 invigorates subdominant TCD8 responses by relieving their lysis-dependent suppression by immunodominant TCD8 To our knowledge, our work constitutes the first report that interfering with PD-1 signaling potentiates epitope spreading in tumor-specific responses, a finding with clear implications for cancer immunotherapy and vaccination.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Epitopes/immunology , Immunity, Cellular , Neoplasm Proteins/immunology , Neoplasms, Experimental/immunology , Programmed Cell Death 1 Receptor/immunology , Signal Transduction/immunology , Animals , CD8-Positive T-Lymphocytes/pathology , Cell Death/genetics , Cell Death/immunology , Cell Line, Tumor , Epitopes/genetics , Female , Interferon-gamma/genetics , Interferon-gamma/immunology , Mice , Neoplasm Proteins/genetics , Neoplasms, Experimental/genetics , Programmed Cell Death 1 Receptor/genetics , Signal Transduction/genetics
6.
J Immunol ; 196(9): 3608-17, 2016 05 01.
Article in English | MEDLINE | ID: mdl-27016602

ABSTRACT

Influenza A virus gene segment 7 encodes two proteins: the M1 protein translated from unspliced mRNA and the M2 protein produced by mRNA splicing and largely encoded by the M1 +1 reading frame. To better understand the generation of defective ribosomal products relevant to MHC class I Ag presentation, we engineered influenza A virus gene segment 7 to encode the model H-2 K(b) class I peptide ligand SIINFEKL at the M2 protein C terminus. Remarkably, after treating virus-infected cells with the RNA splicing inhibitor spliceostatin A to prevent M2 mRNA generation, K(b)-SIINFEKL complexes were still presented on the cell surface at levels ≤60% of untreated cells. Three key findings indicate that SIINFEKL is produced by cytoplasmic translation of unspliced M1 mRNA initiating at CUG codons within the +1 reading frame: 1) synonymous mutation of CUG codons in the M2-reading frame reduced K(b)-SIINFEKL generation; 2) K(b)-SIINFEKL generation was not affected by drug-mediated inhibition of AUG-initiated M1 synthesis; and 3) K(b)-SIINFEKL was generated in vitro and in vivo from mRNA synthesized in the cytoplasm by vaccinia virus, and hence cannot be spliced. These findings define a viral defective ribosomal product generated by cytoplasmic noncanonical translation and demonstrate the participation of CUG-codon-based translation initiation in pathogen immunosurveillance.


Subject(s)
Defective Viruses/genetics , Influenza A virus/genetics , Peptides/genetics , Ribosomes/metabolism , Viral Matrix Proteins/genetics , Animals , Antigen Presentation/drug effects , Cell Line , Defective Viruses/chemistry , Defective Viruses/drug effects , Defective Viruses/metabolism , Genes, MHC Class I , HeLa Cells , Humans , Influenza A virus/chemistry , Influenza A virus/drug effects , Influenza A virus/metabolism , Mice , Mice, Inbred C57BL , Peptides/chemistry , Protein Biosynthesis , Pyrans/pharmacology , RNA Splicing/drug effects , Spiro Compounds/pharmacology
7.
PLoS Pathog ; 12(3): e1005493, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26991092

ABSTRACT

Skin infection with the poxvirus vaccinia (VV) elicits a powerful, inflammatory cellular response that clears virus infection in a coordinated, spatially organized manner. Given the high concentration of pro-inflammatory effectors at areas of viral infection, it is unclear how tissue pathology is limited while virus-infected cells are being eliminated. To better understand the spatial dynamics of the anti-inflammatory response to a cutaneous viral infection, we first screened cytokine mRNA expression levels after epicutaneous (ec.) VV infection and found a large increase the anti-inflammatory cytokine IL-10. Ex vivo analyses revealed that T cells in the skin were the primary IL-10-producing cells. To understand the distribution of IL-10-producing T cells in vivo, we performed multiphoton intravital microscopy (MPM) of VV-infected mice, assessing the location and dynamic behavior of IL-10 producing cells. Although virus-specific T cells were distributed throughout areas of the inflamed skin lacking overt virus-infection, IL-10+ cells closely associated with large keratinocytic foci of virus replication where they exhibited similar motility patterns to bulk antigen-specific CD8+ T cells. Paradoxically, neutralizing secreted IL-10 in vivo with an anti-IL-10 antibody increased viral lesion size and viral replication. Additional analyses demonstrated that IL-10 antibody administration decreased recruitment of CCR2+ inflammatory monocytes, which were important for reducing viral burden in the infected skin. Based upon these findings, we conclude that spatially concentrated IL-10 production limits cutaneous viral replication and dissemination, likely through modulation of the innate immune repertoire at the site of viral growth.


Subject(s)
Interleukin-10/metabolism , Vaccinia virus/immunology , Vaccinia/immunology , Animals , Cytokines/genetics , Cytokines/metabolism , Female , Interleukin-10/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Monocytes/immunology , Skin/immunology , Specific Pathogen-Free Organisms , T-Lymphocytes/immunology , Vaccinia/virology , Virus Replication
8.
mBio ; 6(4): e01156, 2015 Aug 04.
Article in English | MEDLINE | ID: mdl-26242629

ABSTRACT

UNLABELLED: Antibody (Ab) affinity maturation enables an individual to maintain immunity to an increasing number of pathogens within the limits of a total Ig production threshold. A better understanding of this process is critical for designing vaccines that generate optimal Ab responses to pathogens. Our study describes a simple flow-cytometric method that enumerates virus-specific germinal center (GC) B cells as well as their AC50, a measure of Ab avidity, defined as the antigen concentration required to detect 50% of specific B cells. Using a model of mouse Ab responses to the influenza A virus hemagglutinin (IAV HA), we obtained data indicating that AC50 decreases with time postinfection in an affinity maturation-dependent process. As proof of principle of the utility of the method, our data clearly show that relative to intranasal IAV infection, intramuscular immunization against inactivated IAV in adjuvant results in a diminished GC HA B cell response, with increased AC50 correlating with an increased serum Ab off-rate. Enabling simultaneous interrogation of both GC HA B cell quantity and quality, this technique should facilitate study of affinity maturation and rational vaccine design. IMPORTANCE: Though it was first described 50 years ago, little is known about how antibody affinity maturation contributes to immunity. This question is particularly relevant to developing more effective vaccines for influenza A virus (IAV) and other viruses that are difficult vaccine targets. Limitations in methods for characterizing antigen-specific B cells have impeded progress in characterizing the quality of immune responses to vaccine and natural immunogens. In this work, we describe a simple flow cytometry-based approach that measures both the number and affinity of IAV-binding germinal center B cells specific for the IAV HA, the major target of IAV-neutralizing antibodies. Using this method, we showed that the route and form of immunization significantly impacts the quality and quantity of B cell antibody responses. This method provides a relatively simple yet powerful tool for better understanding the contribution of affinity maturation to viral immunity.


Subject(s)
Antibody Affinity , B-Lymphocytes/chemistry , B-Lymphocytes/immunology , Cell Differentiation , Flow Cytometry/methods , Influenza A virus/immunology , Receptors, Antigen, B-Cell/analysis , Animals , B-Lymphocytes/physiology , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Injections, Intramuscular , Mice
9.
Elife ; 42015 Aug 07.
Article in English | MEDLINE | ID: mdl-26252514

ABSTRACT

Immunoglobulins (Igs) are a crown jewel of jawed vertebrate evolution. Through recombination and mutation of small numbers of genes, Igs can specifically recognize a vast variety of natural and man-made organic molecules. Jawless vertebrates evolved a parallel system of humoral immunity, which recognizes antigens not with Ig, but with a structurally unrelated receptor called the variable lymphocyte receptor B (VLRB). We exploited the convergent evolution of Ig and VLRB antibodies (Abs) to investigate if intrinsic chemical features of foreign proteins determine their antigenicity and immunogenicity. Surprisingly, we find lamprey VLRB and mouse Ig responses to influenza A virus are extremely similar. Each focuses ~80% of the response on hemagglutinin (HA), mainly through recognition of the major antigenic sites in the HA globular head domain. Our findings predict basic conservation of Ab responses to protein antigens, strongly supporting the use of animal models for understanding human Ab responses to viruses and protein immunogens.


Subject(s)
Adaptive Immunity , Fish Diseases/immunology , Influenza A virus/immunology , Lampreys/immunology , Orthomyxoviridae Infections/veterinary , Animals , Antigens, Viral/immunology , Fish Proteins/genetics , Fish Proteins/immunology , Lymphocytes/immunology , Mice , Orthomyxoviridae Infections/immunology , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology
10.
J Biol Chem ; 290(26): 16431-9, 2015 Jun 26.
Article in English | MEDLINE | ID: mdl-25971973

ABSTRACT

Green fluorescent protein (GFP) and other fluorescent proteins are essential tools for biological research. When fused to peptides or proteins as a reporter, GFP enables localization and quantitation of gene products in otherwise unmanipulated live cells or organisms. We previously reported that a sizable fraction of nascent GFP is post-translationally converted into a 20-kDa Triton X-100-insoluble proteasome substrate (Qian, S. B., Princiotta, M. F., Bennink, J. R., and Yewdell, J. W. (2006) J. Biol. Chem. 281, 392-400; Dolan, B. P., Li, L., Veltri, C. A., Ireland, C. M., Bennink, J. R., and Yewdell, J. W. (2011) J. Immunol. 186, 2065-2072). Here, we show that a similarly sized fragment is generated by all GFP and red fluorescent protein family members we examined. We demonstrate that fragmentation is a by-product of GFP chromophore rearrangement. A non-rearranging GFP mutant fails to fragment and generates diminished levels of K(b)-SIINFEKL complexes when SIINFEKL is genetically fused to either the C- or N-terminal domains of GFP fusion proteins. Instructively, another fragmenting GFP mutant that cannot create the functional chromophore but still generates fragments also demonstrates diminished K(b)-SIINFEKL generation. However, the mutant and wild-type fragments differ fundamentally in that wild-type fragments are rapidly liberated from the intact molecule and degraded quickly, accounting for increased K(b)-SIINFEKL generation. In the fragmenting mutant, the fragments are generated slowly and remain associated, likely in a native conformation based on their original structural description (Barondeau, D. P., Kassmann, C. J., Tainer, J. A., and Getzoff, E. D. (2006) J. Am. Chem. Soc. 128, 4685-4693). The wild-type GFP fragments represent the first biochemically defined natural defective ribosomal products to contribute peptides for immunosurveillance, enabling quantitation of peptide generation efficiency from this source of defective ribosomal products. More broadly, given the wide use of fluorescent proteins, their ubiquitous and abundant fragmentation must be considered when interpreting experiments using these extremely useful probes.


Subject(s)
Green Fluorescent Proteins/chemistry , Luminescent Proteins/chemistry , Peptide Fragments/immunology , Antigen Presentation , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/immunology , HeLa Cells , Humans , Luminescent Proteins/genetics , Luminescent Proteins/immunology , Monitoring, Immunologic , Peptide Fragments/chemistry , Peptide Fragments/genetics , Red Fluorescent Protein
11.
Immunity ; 42(3): 524-37, 2015 Mar 17.
Article in English | MEDLINE | ID: mdl-25769612

ABSTRACT

CD8(+) T cells play a critical role in limiting peripheral virus replication, yet how they locate virus-infected cells within tissues is unknown. Here, we have examined the environmental signals that CD8(+) T cells use to localize and eliminate virus-infected skin cells. Epicutaneous vaccinia virus (VV) infection, mimicking human smallpox vaccination, greatly increased expression of the CXCR3 chemokine receptor ligands CXCL9 and CXCL10 in VV-infected skin. Despite normal T cell numbers in the skin, Cxcr3(-/-) mice exhibited dramatically impaired CD8(+)-T-cell-dependent virus clearance. Intravital microscopy revealed that Cxcr3(-/-) T cells were markedly deficient in locating, engaging, and killing virus-infected cells. Further, transfer of wild-type CD8(+) T cells restored viral clearance in Cxcr3(-/-) animals. These findings demonstrate a function for CXCR3 in enhancing the ability of tissue-localized CD8(+) T cells to locate virus-infected cells and thereby exert anti-viral effector functions.


Subject(s)
Keratinocytes/immunology , Poxviridae Infections/immunology , Receptors, CXCR3/immunology , Skin/immunology , T-Lymphocytes, Cytotoxic/immunology , Vaccinia virus/immunology , Adoptive Transfer , Animals , Cell Movement , Chemokine CXCL10/genetics , Chemokine CXCL10/immunology , Chemokine CXCL9/genetics , Chemokine CXCL9/immunology , Female , Gene Expression Regulation , Humans , Keratinocytes/pathology , Keratinocytes/virology , Mice, Transgenic , Poxviridae Infections/genetics , Poxviridae Infections/pathology , Poxviridae Infections/virology , Receptors, CXCR3/deficiency , Receptors, CXCR3/genetics , Signal Transduction , Skin/pathology , Skin/virology , T-Lymphocytes, Cytotoxic/pathology , T-Lymphocytes, Cytotoxic/transplantation , Viral Load
12.
Proc Natl Acad Sci U S A ; 111(47): 16854-9, 2014 Nov 25.
Article in English | MEDLINE | ID: mdl-25385602

ABSTRACT

The influenza A virus (IAV) genome is divided into eight distinct RNA segments believed to be copackaged into virions with nearly perfect efficiency. Here, we describe a mutation in IAV nucleoprotein (NP) that enhances replication and transmission in guinea pigs while selectively reducing neuraminidase (NA) gene segment packaging into virions. We show that incomplete IAV particles lacking gene segments contribute to the propagation of the viral population through multiplicity reactivation under conditions of widespread coinfection, which we demonstrate commonly occurs in the upper respiratory tract of guinea pigs. NP also dramatically altered the functional balance of the viral glycoproteins on particles by selectively decreasing NA expression. Our findings reveal novel functions for NP in selective control of IAV gene packaging and balancing glycoprotein expression and suggest a role for incomplete gene packaging during host adaptation and transmission.


Subject(s)
Influenza A virus/physiology , Influenza, Human/transmission , Neuraminidase/genetics , Nucleoproteins/physiology , Viral Proteins/physiology , Genes, Viral , Humans , Influenza A virus/genetics , Influenza, Human/virology , Virus Assembly , Virus Replication
14.
PLoS Pathog ; 10(6): e1004204, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24945804

ABSTRACT

Antigenic variation in the globular domain of influenza A virus (IAV) hemagglutinin (HA) precludes effective immunity to this major human pathogen. Although the HA stem is highly conserved between influenza virus strains, HA stem-reactive antibodies (StRAbs) were long considered biologically inert. It is now clear, however, that StRAbs reduce viral replication in animal models and protect against pathogenicity and death, supporting the potential of HA stem-based immunogens as drift-resistant vaccines. Optimally designing StRAb-inducing immunogens and understanding StRAb effector functions require thorough comprehension of HA stem structure and antigenicity. Here, we study the biogenesis of HA stem epitopes recognized in cells infected with various drifted IAV H1N1 strains using mouse and human StRAbs. Using a novel immunofluorescence (IF)-based assay, we find that human StRAbs bind monomeric HA in the endoplasmic reticulum (ER) and trimerized HA in the Golgi complex (GC) with similar high avidity, potentially good news for producing effective monomeric HA stem immunogens. Though HA stem epitopes are nestled among several N-linked oligosaccharides, glycosylation is not required for full antigenicity. Rather, as N-linked glycans increase in size during intracellular transport of HA through the GC, StRAb binding becomes temperature-sensitive, binding poorly to HA at 4°C and well at 37°C. A de novo designed, 65-residue protein binds the mature HA stem independently of temperature, consistent with a lack of N-linked oligosaccharide steric hindrance due to its small size. Likewise, StRAbs bind recombinant HA carrying simple N-linked glycans in a temperature-independent manner. Chemical cross-linking experiments show that N-linked oligosaccharides likely influence StRAb binding by direct local effects rather than by globally modifying the conformational flexibility of HA. Our findings indicate that StRAb binding to HA is precarious, raising the possibility that sufficient immune pressure on the HA stem region could select for viral escape mutants with increased steric hindrance from N-linked glycans.


Subject(s)
Antibodies, Monoclonal/immunology , Epitopes/immunology , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza A Virus, H1N1 Subtype/immunology , Animals , Antibodies, Monoclonal/therapeutic use , Antibodies, Viral/immunology , Antibody Affinity , Antigenic Variation/genetics , Antigenic Variation/immunology , Cell Line , Dogs , Glycosylation , Golgi Apparatus/immunology , Humans , Influenza Vaccines/immunology , Madin Darby Canine Kidney Cells , Mice , Protein Structure, Tertiary , Recombinant Proteins/immunology , Vaccination
15.
J Virol ; 87(17): 9742-53, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23824811

ABSTRACT

Influenza A virus (IAV) remains an important human pathogen largely because of antigenic drift, the rapid emergence of antibody escape mutants that precludes durable vaccination. The most potent neutralizing antibodies interact with cognate epitopes in the globular "head" domain of hemagglutinin (HA), a homotrimeric glycoprotein. The H1 HA possesses five distinct regions defined by a large number of mouse monoclonal antibodies (MAbs), i.e., Ca1, Ca2, Cb, Sa, and Sb. Ca1-Ca2 sites require HA trimerization to attain full antigenicity, consistent with their locations on opposite sides of the trimer interface. Here, we show that full antigenicity of Cb and Sa sites also requires HA trimerization, as revealed by immunofluorescence microscopy of IAV-infected cells and biochemically by pulse-chase radiolabeling experiments. Surprisingly, epitope antigenicity acquired by HA trimerization persists following acid triggering of the globular domains dissociation and even after proteolytic release of monomeric heads from acid-treated HA. Thus, the requirement for HA trimerization by trimer-specific MAbs mapping to the Ca, Cb, and Sa sites is not dependent upon the bridging of adjacent monomers in the native HA trimer. Rather, complete antigenicity of HA (and, by inference, immunogenicity) requires a final folding step that accompanies its trimerization. Once this conformational change occurs, HA trimers themselves would not necessarily be required to induce a highly diverse neutralizing response to epitopes in the globular domain.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza A virus/chemistry , Influenza A virus/immunology , Animals , Antibodies, Monoclonal , Antibodies, Viral , Antigens, Viral/chemistry , Cell Line , Dogs , Epitopes/chemistry , Humans , Mice , Models, Molecular , Protein Folding , Protein Structure, Quaternary
16.
J Clin Invest ; 123(5): 1976-87, 2013 May.
Article in English | MEDLINE | ID: mdl-23543059

ABSTRACT

CD8+ T cells (TCD8) confer protective immunity against many infectious diseases, suggesting that microbial TCD8 determinants are promising vaccine targets. Nevertheless, current T cell antigen identification approaches do not discern which epitopes drive protective immunity during active infection - information that is critical for the rational design of TCD8-targeted vaccines. We employed a proteomics-based approach for large-scale discovery of naturally processed determinants derived from a complex pathogen, vaccinia virus (VACV), that are presented by the most frequent representatives of four major HLA class I supertypes. Immunologic characterization revealed that many previously unidentified VACV determinants were recognized by smallpox-vaccinated human peripheral blood cells in a variegated manner. Many such determinants were recognized by HLA class I-transgenic mouse immune TCD8 too and elicited protective TCD8 immunity against lethal intranasal VACV infection. Notably, efficient processing and stable presentation of immune determinants as well as the availability of naive TCD8 precursors were sufficient to drive a multifunctional, protective TCD8 response. Our approach uses fundamental insights into T cell epitope processing and presentation to define targets of protective TCD8 immunity within human pathogens that have complex proteomes, suggesting that this approach has general applicability in vaccine sciences.


Subject(s)
Antigens/metabolism , CD8-Positive T-Lymphocytes/cytology , T-Lymphocytes/cytology , Vaccinia virus/metabolism , Animals , Antigen Presentation/immunology , Epitopes/immunology , Epitopes, T-Lymphocyte/immunology , HeLa Cells , Histocompatibility Antigens Class I/metabolism , Humans , Immunodominant Epitopes/immunology , Mass Spectrometry , Mice , Mice, Transgenic , Peptides/immunology , Phenotype
17.
Cell Host Microbe ; 13(3): 314-23, 2013 Mar 13.
Article in English | MEDLINE | ID: mdl-23498956

ABSTRACT

Human influenza A virus (IAV) vaccination is limited by "antigenic drift," rapid antibody-driven escape reflecting amino acid substitutions in the globular domain of hemagglutinin (HA), the viral attachment protein. To better understand drift, we used anti-hemagglutinin monoclonal Abs (mAbs) to sequentially select IAV escape mutants. Twelve selection steps, each resulting in a single amino acid substitution in the hemagglutinin globular domain, were required to eliminate antigenicity defined by monoclonal or polyclonal Abs. Sequential mutants grow robustly, showing the structural plasticity of HA, although several hemagglutinin substitutions required an epistatic substitution in the neuraminidase glycoprotein to maximize growth. Selecting escape mutants from parental versus sequential variants with the same mAb revealed distinct escape repertoires, attributed to contextual changes in antigenicity and the mutation landscape. Since each hemagglutinin mutation potentially sculpts future mutation space, drift can follow many stochastic paths, undermining its unpredictability and underscoring the need for drift-insensitive vaccines.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Antigenic Variation , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza A virus/immunology , Influenza, Human/virology , Amino Acid Sequence , Amino Acid Substitution , Animals , Antibodies, Monoclonal/analysis , Antibodies, Viral/analysis , Humans , Immune Evasion , Influenza A virus/genetics , Influenza, Human/immunology , Mice , Molecular Sequence Data , Mutation
18.
J Virol ; 87(8): 4330-8, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23365443

ABSTRACT

Influenza A virus (IAV) infects a remarkably wide variety of avian and mammalian hosts. Evolution finely hones IAV genes to optimally infect and be transmitted in a particular host species. Sporadically, IAV manages to jump between species, introducing novel antigenic strains into the new host population that wreak havoc until herd immunity develops. IAV adaptation to new hosts typically involves reassortment of IAV gene segments from coinfecting virus strains adapted to different hosts in conjunction with multiple adaptive mutations in the various IAV genes. To better understand host adaptation between mammalian species in real time, we passaged mouse-adapted A/PR8/34 (PR8) in guinea pigs. Guinea pigs, unlike mice, support spontaneous and robust IAV transmission. For some IAV strains, including PR8, adaptation is required for a virus to attain transmissibility, providing an opportunity to understand the evolution of transmissibility in guinea pigs. Multiple guinea pig-adapted PR8 mutants generated by serial nasal wash passaging in independent lines replicated more efficiently and were transmitted by cocaging. All transmissible variants possessed one of two nonsynonymous mutations in M1, either alone or in combination with mutations in PB2, HA, NP, or NA. Rapid reassortment between independently selected variants combined beneficial mutations in NP and M1 to form the fittest virus capable of being transmitted. These findings provide further insight into genetic determinants in NP and M1 involved in PR8 IAV adaptation to be transmitted in a new host and clearly show the benefit of a segmented genome in rapidly generating optimal combinations of mutations in IAV evolution.


Subject(s)
Adaptation, Biological , Influenza A virus/genetics , Mutation, Missense , RNA-Binding Proteins/genetics , Recombination, Genetic , Viral Core Proteins/genetics , Viral Matrix Proteins/genetics , Animals , Disease Models, Animal , Female , Guinea Pigs , Mice , Nasal Cavity/virology , Nucleocapsid Proteins , Orthomyxoviridae Infections/transmission , Orthomyxoviridae Infections/virology , Serial Passage
19.
Cell Host Microbe ; 13(2): 155-68, 2013 Feb 13.
Article in English | MEDLINE | ID: mdl-23414756

ABSTRACT

Despite extensive ex vivo investigation, the spatiotemporal organization of immune cells interacting with virus-infected cells in tissues remains uncertain. To address this, we used intravital multiphoton microscopy to visualize immune cell interactions with virus-infected cells following epicutaneous vaccinia virus (VV) infection of mice. VV infects keratinocytes in epidermal foci and numerous migratory dermal inflammatory monocytes that outlie the foci. We observed Ly6G(+) innate immune cells infiltrating and controlling foci, while CD8(+) T cells remained on the periphery killing infected monocytes. Most antigen-specific CD8(+) T cells in the skin did not interact with virus-infected cells. Blocking the generation of reactive nitrogen species relocated CD8(+) T cells into foci, modestly reducing viral titers. Depletion of Ly6G(+) and CD8(+) cells dramatically increased viral titers, consistent with their synergistic but spatially segregated viral clearance activities. These findings highlight previously unappreciated differences in the anatomic specialization of antiviral immune cell subsets.


Subject(s)
Adaptive Immunity , CD8-Positive T-Lymphocytes/virology , Epidermis/pathology , Immunity, Innate , Vaccinia virus/immunology , Administration, Cutaneous , Animals , Antigens, Ly/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Movement , Chemokines/immunology , Epidermis/immunology , Epidermis/virology , Inflammation/immunology , Inflammation/pathology , Keratinocytes/virology , Mice , Microscopy, Fluorescence, Multiphoton , Monocytes/immunology , Monocytes/pathology , Monocytes/virology , Peroxynitrous Acid/metabolism , Reactive Nitrogen Species/metabolism , Skin Diseases, Viral/immunology , Skin Diseases, Viral/virology , Vaccinia/immunology , Vaccinia/virology , Viral Load
20.
J Virol ; 87(6): 3155-62, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23283949

ABSTRACT

Segmentation of the influenza A virus (IAV) genome enables rapid gene reassortment at the cost of complicating the task of assembling the full viral genome. By simultaneously probing for the expression of multiple viral proteins in MDCK cells infected at a low multiplicity with IAV, we observe that the majority of infected cells lack detectable expression of one or more essential viral proteins. Consistent with this observation, up to 90% of IAV-infected cells fail to release infectious progeny, indicating that many IAV virions scored as noninfectious by traditional infectivity assays are capable of single-round infection. This fraction was not significantly affected by target or producer cell type but varied widely between different IAV strains. These data indicate that IAV exists primarily as a swarm of complementation-dependent semi-infectious virions, and thus traditional, propagation-dependent assays of infectivity may drastically misrepresent the true infectious potential of a virus population.


Subject(s)
Gene Expression Regulation, Viral , Influenza A virus/genetics , Viral Proteins/biosynthesis , Virion/genetics , Animals , Cell Line , Defective Viruses/genetics , Defective Viruses/growth & development , Dogs , Genes, Essential , Influenza A virus/growth & development , Viral Proteins/genetics , Virion/growth & development
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