Host Responses to Viruses: Overview

Host Responses to Viruses: OverviewBackground Slides:Levels of immune protectionCytokines, chemokinesComplementPatterns of viral infection (Acute vs. Chronic)CD8+ CTL: mechanism of cytolytic actionWhat are neutralizing antibodies?RNA viruses and error catastrophe

2. Viral evasion mechanismsA. Viral mimicry of cytokines, chemokinesB.Complement evasionInhibition of Natural killer cellsAntigenic variationEscape from CD8+ CTLsEscape from Neutralizing antibodies (Influenza, HIV)Host susceptibility to virus infectionPersistence in immunologically privileged sites (e.g. neurons)

3. Immunological techniques to explore host responses to viruses

References

Pabio552 Lecture 17; NL Haigwood

Learning Objectives:

What are the major mechanisms used by viruses to evade innate and adaptive immunity?

What is the association between types of viral infection (acute and chronic infection) and modulation of immune responses?

How does HIV trick the various arms of the immune system?

What are the major methods used to measure adaptive immunity?


Levels of immune protectionSource: http://mil.citrus.cc.ca.us/cat2courses/bio104/ChapterNotes/Chapter39notesLewis.htm


Cytokine, ChemokinesCytokines are small soluble proteins secreted by one cell that can alter the behavior or properties of the cell itself or of another cell. They are released by many cells in addition to those of the immune system.

Cytokines, such as interferons (IFNs) and tumor-necrosis factor (TNF), induce intracellular pathways that activate an anti-viral state or apoptosis, and thereby limit viral replication.

Chemokines are chemoattractant cytokines that regulate trafficking and effector functions of leukocytes, and so have an important role in inflammation and immune surveillance

Chemokines: divided into 4 classes, CC-, CXC-, C-, CX3C-

Expression of specific chemokines, together with differential expression of respective chemokine receptors by leukocytes, determines which immune cells migrate during inflammationHIV utilizes chemokine receptors CCR5, CXCR4 for entry into target cellsSee Flint Table 14.6Blocking interferon action


Viral Mimicry of cytokines, chemokines and their receptorsAlacami A. Nat Rev Immunol. 2003 Jan;3(1):36-50. Properties of poxvirus-encoded soluble cytokine receptors or binding proteins.


ComplementInteracting set of enzymes (proteases) that upon activation give rise to cascade of reactions culminating in the destruction of pathogens and infected cells.

Three pathways:Classical: implicated in controlling HIV, HTLV, CMV infected cells

Mannan binding lectin (MBL) : similar to classical; lyses HBV, HCV and infuenza infected cells

Alternative pathway: default pathway; spontaneous and indiscriminate deposition of complement factor C3b on host cell surface or foreign particles; complement activation will proceed unless downregulated by complement regulators CD59, DAF


Complement EvasionRemove Ag:Ab complexes; express Fc receptors on the infected cellsHSV gE-gI glycoprotein complex binds non-immune IgG and sterically hindered access of virus specific effector cells or neutralizing antibodiesPRV gE cytoplasmic domain has two tyr residues that internalizes through clathrin mediated endocytosis, followed by degradation

Viral mimicry of complement regulatorsPoxvirus and -Herpesvirus infected cells secrete soluble viral encoded factors that are genetically similar to complement regulators; inhibit by inactivating C3b and C4b, C3 convertase-herpesvirus (HSV) gC glycoprotein acts as complement receptor for C3b, C5 and P factor

Incorporation or up-regulation of cellular complement control factorsEnveloped viruses like HIV, Ebola, influenza incorporate complement control proteins CD46, CD55, CD59 (enriched in lipid rafts) during virus releaseHCMV upregulates expression of CD59 in infected cells leading to suppression of alternate pathway.


Natural Killer CellsLymphocytes that do not undergo genetic recombination events to increase their affinity for particular ligands (effectors of the innate immune system)Capable of killing without previous stimulationCharacterized by the absence of conventional receptors for antigen (TCR); display CD3- CD16+ phenotypeCD16 (FcRIII) is a low affinity receptor for IgG and is involved in Antibody Dependent Cell mediated Cytotoxicity (ADCC)

NK cell activating receptors:Human natural cytotoxicity receptors NKp30, NKp44, NKp46LFA-1 and CD2 familyInhibitory receptors: receptors for MHC class I belonging to killer cell inhibitory receptor (KIR) family; KIR receptors recruit phosphatases (Src domain containing protein tyrosine phosphatase 1) to transduce the inhibitory signalImmunoglobulin-like inhibitory receptors (ILT)Lectin like heterodimer CD94-NKG2A

NK cell responses are also coordinated and regulated by cytokines such as IFN-, IFN-, IL-2, IL-12, IL-15 and IL-18Spontaneously active, unless they are inhibited by self-MHC molecules. Well-suited to perform immunosurveillance for nonself MHC-bearing targets (eg, transplanted cells, tumors, virally-modified cells)


Viral mechanisms for evading NK cells. Viral homologs of MHC class I Binds to inhibitory NK cell class I receptors

Selective modulation of MHC class I allele expressionIncrease relative expression of HLA-C, HLA-E by down regulating HLA-A, HLA-B; NK cells inhibited through class I CD94-NKG2A and KIRHIV Nef causes endocytosis of Class I, except HLA-C, HLA-EHCMV UL40 enhances expression of HLA-E

Inhibition of activating receptor functionViral cytokine binding proteins (ii) viral homologs (iii) induce host cell secretion of NK cell inhibitory cytokinesHIV Tat blocks NK cell activation by specifically binding and blocking Ca2+ influx channel important for NK Cell cytotoxicityKSHV K5 protein ubiquitinates, and decreases surface expression of ICAM-1, B7-2 inhibits NK cell cytotoxicity

Secrete antagonists of NK cell receptor-infected cell ligand interactionsKSHV vMIP-I, vMIP-II acts as a chemokine antagonist and intereferes with NK cell traffickingHPV E6, E7 binds IL-18 receptor and inhibits IFN- production by NK cells

Directly infects NK cells or ligates NK cell inhibitory receptors through envelope glycoproteinsHIV in vitro infects NK cells through an unknown receptorHCV E2 glycoprotein binds CD81 on NK cells and sends inhibitory signal to NK cells


General patterns of viral infectionAcute InfectionRhinovirusRotavirusInfluenza virusPersistent InfectionLymphocytic choriomeningitis virusLatent, Reactivating infectionHSVSlow virus infectionLentivirus (HIV)

Virus is clearedMemory responses establishedSource: Flint, Principles of Virology 4th Ed Fig: 16.1


High rate of viral evolutionEarly escape from CTLEscape from neutralizing antibodiesDown regulation of MHC CD4+ T helper cell destructionIntegration and reactivation

HIV: drives the host defenses into exhaustion, and ultimately failure


Immune responses to HIVControl of primary HIV-1 infection coincides with the appearance of virus-specific Cytotoxic T lymphocytes (CTLs)

2. Antibodies form early; neutralizing antibodies arise later in infection

Strong immune responses to HIV infection control it for many years but ultimately host diesafter erosion of T cells


HIV: Antigenic Escape, Quasispecies, Error ThresholdE. coli: Genome size 4.6 x 106 bp or ~107 bpError rate in DNA replication is 10-101 error for every 1000 genome replications

RNA Viruses: Genome size ~104 nucleotidesError rate of RNA replicases is 10-41 error with every replication


HIV intrapatient viral evolution


Viral Escape from CD8+ Cytotoxic T lymphocytes (CTLs)

Flint Table 15.4 and Figure 15.7*RSV--less MHC class I transcription*HIV--Tat, Vpu interfer with MHC class I synthesis*Adenovirus--E3 and E1a reduce MHC in ER and transcription*CMV, HSV, EBV--Tap transporter inhibition, retention of peptides in ER


CD8+ CTLs: Mechanism of cytolytic actionPotent defenders against virus infection and intracellular pathogens

Mediates apoptotic death through Fas-FasL interactions

Upon interaction with an infected cell, an immunological synapse between the CTL and the target cell is formed

Targeted release of effector molecules Perforin: forms pore in the target cell (release of intracellular contents from infected cell); translocation channel for granzymes

Granzyme B: belongs to family of serine proteases that mediate apoptotic cell death through the (i) direct cleavage of pro-caspase-3 or, (ii) indirectly, through caspase-8, (iii) cleavage of BID resulting in its translocation, with other members of the pro-apoptotic BCL2-family such as BAX, to the mitochondria, (iv) direct activation of DFF40/CAD (DNA fragmentation 40/caspase-activated deoxynuclease) which damages DNA and leads to cell death by granzyme-B-mediated proteolysis of the inhibitor ICAD.

CD8+ CTLs have non-cytolytic action too (todays discussion paper)


CD8+ CTL control of HIV infectionEvidenceControl of high viral loads associated with the acute phase of HIV infection is coincident with the appearance of anti-HIV CTLs

CTLs can be found at sites of HIV replication in vivo

The quantity of anti-HIV CTLs, as measured by MHC-I tetrameric complexes, correlates inversely with viral load (data conflicting)

When SIV infected macaques were immunodepleted for CD8+ T cells, a dramatic increase in SIV replication was observed

HIV vaccine strategies targeting CTL responses show decrease in viral load in SIV/ SHIV infected macaques

CTLs from Long-term nonprogressors secrete higher levels of perforin, granzyme B

BUT, vaccines that elicit only CTL cannot protect from infection, only control, and patients can be superinfected with another HIV-1 isolate, even if CTL are present


Impairment of the cellular immune responseInterference with proteosomal degradationPeptide transport associated with Ag presentationRetention of MHC I molecules in subcellular compartmentsDestruction of MHC class I heavy chainsInduction of functional paralysis of DCsDownregulation of MHC Class II expressionCircumvent NK-cell mediated killingAntigenic variation of T cell epitopes


Viral load is critical to the control of disease


Viral Escape from Neutralizing Antibodies


CD4CCR5/ CXCR4What are Neutralizing antibodies?Target cell


CD4 binding induces a conformational change in envelope leading to exposure of the coreceptor binding siteWhat are Neutralizing antibodies?Target cell


Binding to CCR5 exposes the fusion domain leading to subsequent viral core entryWhat are Neutralizing antibodies?Target cell


Most antibodies however, bind the virus but do not neutralize

Neutralizing antibodies: Block virus infection in the target cells by directly binding to virion and prevent their interaction with receptor on the target cells

What are Neutralizing antibodies?Target cell


NAbs act at different levels


Envelope modifications enable NAb escapeInfluenza Virus

No cross-protective immunityEscape from neutralizing antibodies

Original antigenic sin: Abs made only to epitopes present on the initial viral variant when infected with a second variant

Source: http://www.gak.co.jp/FCCA/glycoword/GD-A06/GD-A06_E.html


HIV: Escape from Antibody-mediated NeutralizationReasonsTransient exposure of neutralizing epitopes only at critical moments during the entry process

CCR5 binding site is highly protected and is exposed only after conformation changes has occurred upon binding CD4

CD4 independent viruses are highly neutralization sensitive

Occlusion of conserved domains within the oligomer

Wyatt R., et al., Nature 393: 705 - 711 (1998)CD4 binding siteCCR5 binding siteSchematic of envelope trimer


HIV: Escape from Antibody mediated NeutralizationReasonsExtrusion of variable domains from the exposed surface of the oligomeric complex

Serves as an antigenically variable shield and covers the more conserved envelope core

Removal of V2 loop opened up the CD4 binding site rendered the virus susceptible to neutralization from other clades, and also resulted in high titer neutralizing antibodies in a SHIV infected macaque and reduced viral burden (Stamatatos et al; J Virol. 1998. 72:7840)


HIV: Escape from Antibody mediated NeutralizationReasonsExtensive glycosylation of the envelope proteinsHIV/ SIV envelope contains 28-30 potential N-linked glycosylation sites that account for 50% of the mass of gp120

Position of the glycans modulate antibody neutralization specificity

Removal of N-linked glycan in V3 loop increased HIVs sensitivity to neutralizing antibodies

Longitudinal analyses indicate that the number of glycans remain fairly constant, while positions vary, with indels, and most commonly a shift by a couple of aas

Evolving Glycan shield


HIV: Escape from Antibody mediated NeutralizationEvidenceViral variants are resistant to plasma (NAbs) from the same time point

During chronic HIV infection, there are high levels of NAbs, however they are not directed towards autologous virus from the patient at the same time point

NAbs that recognize heterologous isolates arise late in infection (Broad)

The development of heterologous NAb hinders the development of autologous NAb responses that may control infection

Richman DD., et al. PNAS. 100(7): 4144. (2003)Despite these changes, Env is by necessity relatively conserved in CD4, co-receptor binding regions


Broad NAbs do arise in some patients


Passive immunization studies Combination of broad human monoclonal NAbs act synergistically to confer protection against SHIV and SIV

Prevention of SHIV transmission to macaque monkeys when IgG1 b12 was mucosally applied Post-exposure prophylaxis with human monoclonal antibodies prevented SHIV89.6P infection or disease in neonatal macaques

Potent cross-group neutralization (Clades A, C, D, E, and F) of primary HIV isolates with monoclonal antibodies - IgG1b12 (b12), F105, 2G12, 2F5, 4E10, F424, and Clone 3 (CL3)


Host susceptibility to viral infectionHLA haplotypes influences HIV disease progressionAssociated with slower disease progression: A*11, B*27, B*57, B858, Cw*02, Cw*14Associated with rapid disease: B*35, B*53, Cw*04Maternally acquired protective alleles were no longer protective against disease progression in vertically infected infants

Mutant forms/ Polymorphisms of coreceptor modulates progression to AIDSIndividuals homozygous for a 32-bp deletion (32) in CCR5 are resistant to infection by HIV-1 HIV-1-infected individuals heterozygous for CCR5 32 (-/+) show a slightly slower disease progression than CCR5+/+ homozygotes Common polymorphism in the 3'-untranslated region of the stromal derived factor gene (SDF-1; natural ligand for CXCR4) associated with delayed disease progression from some cohorts. In others, the same homozygous genotype has been associated with rapid progression to AIDS, but with prolonged survival after diagnosis of AIDSPolymorphism in 3 UTR SDF-1 is associated with increased risk of vertical transmission


Immunological techniquesNot an exhaustive list!Humoral Immunity (B cells)ELISA; Radioimmunoassay : checks for the development of virus-specific antibodies in plasma, other body fluidsAb competitive inhibition assayAntibody-dependent cellular cytotoxicity (ADCC)How are monoclonal antibodies generated?What is Biacore? What does it measure?Neutralization assay viral infectivity on target cells in the presence or absence of Ab

Cellular immunity (CD4, CD8 T cells)How do you check for the presence of cytokine secreted by a T cell?Antigen proliferation assay (also called lymphocyte proliferation assay, thymidine uptake assay)MHC Tetramer Assay: Ag specific T cell staining reagent

Functional AssaysCytokine production: ELIspot, intracellular cytokine stainingB- cell activation: What is an ELIspot?Target Cell Killing: Chromium release assay: what does it measure?Antibody dependent cell-mediated cytotoxicity (ADCC)Chemotaxis assay


ReferencesReviewsAlcami A. Viral mimicry of cytokines, chemokines and their receptors. Nat Rev Immunol. 3: 36-50 (2003).Favoreel HW., et al. Virus Complement evasion strategies. J. Gen. Virol. 84: 1-15 (2003).Orange JS., et al. Viral evasion of natural killer cells. Nat. Immunol. 3: 1006 (2002).Vossen MTM., et al. Viral Immune evasion: am masterpiece of evolution. Immunogenetics. 54: 527 (2002).Domingo E. Viruses at the edge of adaptation. Virology. 270: 251 (2000) Barry M., et al. Cytotoxic T lymphocytes: all roads lead to death. Nat. Rev. Immunol. 2: 401 (2002).Ferrantelli F, et al., Neutralizing antibodies against HIV -- back in the major leagues? Curr Opin Immunol. 14: 495 (2002).Land A., et al., Folding of the human immunodeficiency virus type 1 envelope glycoprotein in the endoplasmic reticulum. Biochimie. 83: 783 (2001). Burton DR. Antibodies, viruses and vaccines. Nat Rev Immunol. 2: 706-13 (2002). Wyatt R. et al., The antigenic structure of the HIV gp120 envelope glycoprotein. Nature. 393: 705-11 (1998).

Primary papersSchlender et al. Inhibition of toll-like receptor 7- and 9-mediated alpha/beta interferon production in human plasmacytoid dendritic cells by respiratory syncytial virus and measles virus. J Virol. 2005 May;79(9):5507-15.Richman DD., et al. Rapid evolution of the neutralizing antibody response to HIV type 1 infection. PNAS. 100: 4144 (2003).Ferrantelli F., et al., Potent cross-group neutralization of primary human immunodeficiency virus isolates with monoclonal antibodies--implications for acquired immunodeficiency syndrome vaccine. J Infect Dis. 189: 71-4 (2004). Mascola JR., et al., Protection of macaques against vaginal transmission of pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nat Medicine 6: 207-210 (2000). Chackerian et al., Specific N-linked and O-linked glycosylation modifications in the envelope V1 domain of simian immunodeficiency virus variants that evolve in the host alter recognition by neutralizing antibodies. J Virol. 1997 Oct;71(10):7719-27.Wei X et al. Antibody neutralization and escape by HIV-1. Nature. 2003 Mar 20;422(6929):307-12.


Neutralization AssayTarget cells (Cell line/ PBMC) expressing viral receptors for entry (For ex, HIV-1 CD4, CCR5) and a reporter gene (-gal, luciferase)Mechanism

Only infected cells produce Gal Easy visualization through X-gal stainingNeutralization assay: pretreatment of virus with AbReduction in Gal positive cells indicates virus neutralizationCalculate % neutralization


Generation of monoclonal antibodiesJaneway, Immunobiology 5th Edition; Fig A.15


Antigen proliferation assayAntigen: gp120, peptide pools (Gag, Env)AT-2 inactivated SHIV89.6 Contols: PHASEBa-CD3 mAbirrelavant peptidePlain media4. Harvest the cells onto filters using cell harvester5. Read filters on a scintillation counterCalculate Stimulation index = Samplecpm

Irr cpm8-16 hours incubation


ELIspotSource: www.bdbiosciences.com


Tetramer stainingStain antigen-specific T cells

MHC:peptide tetramers are formed from recombinant refolded MHC:peptide complexes containing a single defined peptide epitope

Recombinant MHC heavy chain is linked to a bacterial biotinylation sequence, a target for the E. coli enzyme BirA, which is used to add a single biotin group to the MHC molecule

Streptavidin is a tetramer, each subunit having a single binding site for biotin, thus the streptavidin/MHC:peptide complex is a tetramer of MHC:peptide complexes

While the affinity between the T-cell receptor and its MHC:peptide ligand is too low for a single complex to bind stably to a T cell, the tetramer, by being able to make a more avid interaction with multiple MHC:peptide complexes binding simultaneously, is able to bind to T cells whose receptors are specific for the particular MHC:peptide complex

Streptavidin is coupled to a fluorochrome, so that the binding to T cells can be monitored by flow cytometry. Janeway, Immunobiology 5th Edition; Fig A.32


BiacoreBased on Surface plasmon Resonance (SPR) technology

SPR arises when light is reflected under certain conditions from a conducting film at the interface between two media of different refractive index

SPR causes a reduction in the intensity of reflected light at a specific angle of reflection

Angle varies with the refractive index close to the surface on the side opposite from the reflected light (the sample side in Biacore)

When molecules in the sample bind to the sensor surface, the concentration and therefore the refractive index at the surface changes and an SPR response is detected

Plotting the response against time (called sensorgram) during the course of an interaction provides a quantitative measure of the progress of the interaction

Source: www.biacore.com


Chromium Release assayMeasures Cytotoxic T-cell activity Target cells are labeled with radioactive chromium as Na251CrO4, washed to remove excess radioactivity and exposed to cytotoxic T cellsCell destruction is measured by the release of radioactive chromium into the medium, detectable within 4 hours of mixing target cells with T cells.


The strategies by which viruses evade NK cells fall into five categories and are depicted in the interaction between a virus infected target cell (left) and an NK cell (right). (a) NK cells can be inhibited by a viral MHC class I homolog with structural similarity to endogenous host class I that binds to inhibitory class I receptors on NK cells. (b) Viruses can inhibit expression of HLA-A and HLA-B, resulting in a relative increase in HLA-C and HLA-E on the surface of the target cell; these inhibit NK cells through the class I inhibitory receptors CD94-NKG2A and KIR, respectively. Alternatively, viral gene expression can result in selectively increased expression of HLA-E, which inhibits NK cells through CD94-NKG2A. (c) Virus-encoded proteins can function as cytokine binding proteins that block the action of NK cell activating cytokines. In addition, viruses can produce homologs, or increase host production of cytokines that inhibit NK cells. (d) NK cell activities can also be avoided by decreased expression of NK cellactivating ligands in virus-infected target cells, which prevent signal transduction via NK cellactivating receptors. To achieve the same end, viruses can encode antagonists of the activating receptorligand interaction. (e) Viruses can also directly inhibit NK cells by infecting them or using envelope proteins to ligate NK cell inhibitory receptors. Proteins outlined in red are virally encoded. Each mechanism corresponds to the similarly numbered section of the text where additional details and examples are provided.

Documents

Host Responses to Viruses: Overview