Vaccines and immunomonitoring

Transcription

1 Vaccines and immunomonitoring Anna-Karin Hermansson Corso di Microbiologia cellulare e vaccinologia,

2 What is immunomonitoring? Evaluation of immune responses induced by a vaccine?

3 Why immunomonitoring? Understand how a vaccine works Evaluation of vaccine candidates Predict protection

4 What we ll discuss during this lesson Which immune responses do pathogens and vaccines induce? How can they protect? How can we evaluate them? Can we use this knowledge to predict protection?

5 Understand how a vaccine works Which immune responses do pathogens and vaccines induce?

6 1 PAMPS/ DAMPS 2 Pro-inflammatory cytokines 3 DC activation 4 Presentation 5 Cytotoxic T cells 6 Antibodies 5 Th responses Desmet & Ishii (2012) Nature Reviews Immunology

7 Immune responses induced by a vaccine 1. Innate immune responses (a) Dendritic cell activation (b) Inflammatory response (c) Complement activation 2. Adaptive immune responses (i) Antibody and B cell immune responses (ii) T cell immune responses

8 Understand how a vaccine works How can they protect? Depends on the pathogen! Entry mechanism? Target organs? Mechanisms involved in survival of pathogen? Damage mechanism? Wound? Mucosa? Lungs? Nervous system? Liver? Intestinal lumen? Intra/extracellular? Toxin? Cell death?

9 How to protect against virus? Cytopathic virus kills the cell neutralizing antibodies Pictures: Stopenterics/HSeT

10 How to protect against virus? Cytopathic virus kills the cell neutralizing antibodies Non cytopathic virus do not kill the cell Cytotoxic T lymphocytes (CTL) Pictures: Stopenterics/HSeT

11 How to protect against bacteria? Neutralizing antibodies siga block bacterial entry, neutralize toxin IgG neutralize bacteria and toxin in tissue Activation of complement Phagocytosis by neutrophils and macrophages Complement receptor bind C3b Fc receptor binds antibodies Pictures: Stopenterics/HSeT

12 How can we evaluate immune responses? Mucosal or systemic Humoral or cell mediated Acute or memory Type and function - antibody isotype - neutralizing capacity of antibodies - bactericidal activity of antibodies - avidity of antibody binding - T cell effector mechanisms (cytotoxicity, cytokine production etc)

13 Mucosal or systemic Systemic - Serum - Blood cells Mucosal - Faeces - Lavage - Saliva - Urine - IgG - siga

14 B-cell responses How strong (Fold rises): Immune responses before and after vaccination or infection How many (Responder rate): Proportion (%) of subjects mounting a response to infection/immunization above a certain cut-off level (often 2 or 4 fold rise) ELISA ELISpot ALS

15 ELISA What to measure? Antibodies quantity isotype Antigen Cytokines Where to measure? Serum Feces Saliva Urine Cell culture supernatants

16 ELISA Enzyme-Linked ImmunoSorbent Assay For detection of antibodies in a sample Antigen W A S H Antigen specific antibody W A S H Enzyme linked antibody W A S H Substrate catalyzed by enzyme - color change Pictures: Stopenterics/HSeT

17 Vaccine DCs pick up antigen Lymphocyts enters site of induction DCs present antigen Clonal expansion of lymphocytes Lymphocytes enters circulation Time point for collection important! Lambrecht & Hammad 2003 (Nat Rev Immunol.)

18 ELISpot Detect antibody secreeting cells (B cells or plasma cells) for a specific antigen Add cells to antigencoated wells Enzyme-linked secondary antibody Extract blood cells Add substrate enzyme color change Red spot = antibodies for the specific antigen Pictures: Stopenterics/HSeT

19 ALS Antibodies in lymphocyte supernatant Combination of Elispot + ELISA Requires less sample Faster Extract blood cells Culture cells Collect supernatant E L I S A

20 T cell responses T cells recognize antigen bound to MHC molecules on antigen presenting cells. Therefore, the specificity of T cells is more difficult to asses than the specificity of antibody responses. Restimulation of T cells with antigen in vitro is often required to assess the specificity and function of T cells.

21 T cell assays ELISA ELISpot Intracellular staining Polyclonal cell expansion Tetramer staining

22 ELISA Enzyme-Linked ImmunoSorbent Assay 1 = procedure to detect antibodies in sample 1 2 Antigen W A S H Antigen specific antibody W A S H Enzyme linked antibody W A S H Substrate catalyzed by enzyme - color change Cytokine specific antibody Cytokine 2 = procedure to detect cytokines in sample Enzyme linked antibody Substrate catalyzed by enzyme - color change Pictures: Stopenterics/HSeT

23 ELISpot Detect cytokine secreeting cells (mainly T cells) for a specific antigen Add cells + peptide to antibody coated wells Enzyme-linked secondary antibody Extract blood mononuclear cells Add substrate enzyme color change Red spot = cytokine secreated by peptide specific T cells Pictures: Stopenterics/HSeT

24 Intracellular staining T cells, APCs and antigen T cells start producing cytokines Block of cytokine release Add antibodies to select cells of interest, eg CD8 conjugated to fluorofor Permeabilize cells, add antibody to cytokine of interest, eg INFγ conjugated to fluorofor Evaluate with flow cytometry Pictures: Stopenterics/HSeT

25 CFSE - Carboxyfluorescein diacetate succinimidyl ester CFSE proliferation assay PBMC + CFSE CFSE enters cells binds proteins Antigen added Specific T cells proliferate Intensity of CFSE decreases with each division Pictures: Stopenterics/HSeT

26 Pictures: Stopenterics/HSeT Tetramer staining Detection of antigen specific CD8 T cells Tetramer of four MHC I and peptide antigen Peptide specific T cells bind to fluorofor conjugated tetramere Evaluation by flow cytometry Davis et al., 2011, Nature Reviews Immunology MHC I Peptide

27 Functional assays antibody isotype neutralizing capacity of antibodies bactericidal activity of antibodies avidity of antibody binding T cell effector mechanisms (cytotoxicity, cytokine production etc)

28 CFU/ml (*10^3) Neutralizing effect Immunization vaccine candidate serum control serum without serum Rabbit serum Bacteria Bacteria added to cells Plating of bacteria Cell lysis Gentamycin kills extracellular bacteria

29 Neutralizing antibodies assay Virus added Virus enter, express luciferase Addition of luciferase substrate Virus with luciferase gene Virus added Entry inhibited, luciferase not expressed Addition of luciferase substrate

30 T cell cytotoxicity Cells exposing antigen on MHC I killed by cytotoxic T cells Label target cells with Na 2 51 CrO 4 Add T cells Killed cells release radioactive Cr Target cells: eg virus infected or tumor cells

31 Why immunomonitoring? Evaluation of new vaccines Proceed from animal studies to clinical trials? Proceed to next step of clinical trials? Adjuvant needed? Immunization route?

32 Two examples: evaluation of immune responses induced by vaccines in clinical trials - Methods used - Data representation

33 Example: Ebola vaccine Phase I clinical trial Adenovirus type 26 vector encoding Ebola glycoprotein and a modified vaccinia ankara vector encoding glycoproteins from Ebola virus, Sudan virus, Marburg virus and Tai Forest virus nucleoprotein Two injections Safety and Immunogenicity of Novel Adenovirus Type 26 and Modified Vaccinia Ankara Vectored Ebola Vaccines A Randomized Clinical Trial Milligan et al., 2016, JAMA

34 Example: Ebola vaccine Serum IgG against Ebola glycoprotein ELISA PBMC: T cells specific for Ebola glycoprotein ELISpot T cells specific for Ebola glycoprotein intracellular cytokine staining Antibodies to virus vector (Ad26) neutralizing antibodies assay Safety and Immunogenicity of Novel Adenovirus Type 26 and Modified Vaccinia Ankara Vectored Ebola Vaccines A Randomized Clinical Trial Milligan et al., 2016, JAMA

35 % Safety and Immunogenicity of Novel Adenovirus Type 26 and Modified Vaccinia Ankara Vectored Ebola Vaccines A Randomized Clinical Trial Milligan et al., 2016, JAMA Example: Ebola vaccine ELISA Serum IgG to EBOV GP ELISpot IFNγ secreting EBOV GP specific T cells Intracellular staining IFNγ/TNFα/IL2+ EBOV GP specific T cells Neutralizing antibodies assay Preexisting antibodies to Ad26 3.4% of the population before immunization

36 Example: HIV vaccine Recombinant envelope gp140 protein Test of different immunization routes: intramuscular (IM), intranasal (IN) and intravaginal (IVAG) 3 immunizations: week 0, 4 and 8 For the IVAG group first immunization was IM and the two others IVAG Comparative Immunogenicity of HIV-1 gp140 Vaccine Delivered by Parenteral, and Mucosal Routes in Female Volunteers; MUCOVAC2, A Randomized Two Centre Study Cosgrove et al., 2016, PloS One

37 Example: HIV vaccine Serum and mucosal IgG against gp140 specific IgG ELISA Antibody funcion neutralizing antibody assay PBMC: T cells specific for HIV gp140 Intracellular cytokine staining Comparative Immunogenicity of HIV-1 gp140 Vaccine Delivered by Parenteral, and Mucosal Routes in Female Volunteers; MUCOVAC2, A Randomized Two Centre Study Cosgrove et al., 2016, PloS One

38 Example: HIV vaccine Serum IgG Comparative Immunogenicity of HIV-1 gp140 Vaccine Delivered by Parenteral, and Mucosal Routes in Female Volunteers; MUCOVAC2, A Randomized Two Centre Study Cosgrove et al., 2016, PloS One

39 Example: HIV vaccine Neutralizing antibodies against various HIV types Week 12 Comparative Immunogenicity of HIV-1 gp140 Vaccine Delivered by Parenteral, and Mucosal Routes in Female Volunteers; MUCOVAC2, A Randomized Two Centre Study Cosgrove et al., 2016, PloS One

40 IM20 IM100 IN IVAG IM20 IM100 IN IVAG IM20 IM100 IN IVAG IM20 IM100 IN IVAG Example: HIV vaccine IL-2 CD4+ T-cells No response Response 0 weeks 9 weeks 12 weeks 24 weeks Comparative Immunogenicity of HIV-1 gp140 Vaccine Delivered by Parenteral, and Mucosal Routes in Female Volunteers; MUCOVAC2, A Randomized Two Centre Study Cosgrove et al., 2016, PloS One

41 Why immunomonitoring? Understand how a vaccine works Evaluation of new vaccines Predict protection Are new lots as efficient as the previous? Is a new vaccine comparable to an old one? Is a certain population or individual more susceptible? What happens when vaccines are combined?

42 Correlates of protection Is there an immune parameter that corresponds to protection? E. g.: - Antibody level - Antibody funcion - Cellular responses

43 Correlate of Protection Nomenclature for Immune Correlates of Protection After Vaccination Plotkin and Gilbert, 2012, Clin Infect Dis.

44 Immunological correlation Correlates of vaccine-induced protection: methods and implications Innitiative for Vaccine Research (IVR) of the Department of Immunization, Vaccines and Biologicals WHO, 2013

48 What do we mean by protection? Protection against carriage interrupt transmission Protection against disease Protection against severe disease

49 What do we mean by protection? Protection against carriage Protection against disease E g vaccine against morbilli

50 What do we mean by protection? Protection against carriage Protection against disease Protection against severe disease Eg BCG protects mainly against the severe forms of TB such as TB meningitis and miliary TB

51 Correlates of protection Responses induced after natural infection Passive immunization Responses induced by vaccines Specific correlates needed for each vaccine, specific tests often developed

52 Examples for specific vaccines

53 Haemophilus influenzae b Hib declined at the age when most of the population showed antibody concentrations of > 0.15 μg/ml Studies on gamma globulin prophylaxis and of the decline of maternal antibodies: concentrations between 0.03 and 0.1 μg/ml are protective Prevention of nasopharyngeal carriage: diffusion of H. influenzae antibodies from serum, correlated with postimmunization levels of >5 μg/ml.

54 Clostridium tetani Antibodies against tetanus toxin protective 0,01 μg/ml some protection 0,1 μg/ml full protection

55 Bordetella pertussis Antibodies against toxin and adhesion factors protective: Pertussis toxin Pertactin Fimbrial hemagglutinins

56 Variola virus Neutralizing antibodies directed against various viral proteins: lifelong protection against variola induced death Mild disease may occur when CD8 + cytotoxic T cell responses fade

57 Varicella Zoster Virus Infection leads to immunity Passive transfer of antibodies protects Antibody responses induced by vaccine correlate with protection Children protected despite low antibody titers after vaccination T cell responses important: decrease in older age while antibodies persist Herpes Zoster can occur

58 Hepatitis B Memory B cells important 10 miu/ml antibodies after vaccination correlates with induction Th cells that mediate memory B cells Protection lasts much longer than detectable antibodies Long incubation period enough time to activate memory

59 Summary Many ways to evaluate immune responses Useful to understand how a vaccine works Predict protectivity complicated!

60 Further reading Correlates of vaccine-induced protection: methods and implications Innitiative for Vaccine Research (IVR) of the Department of Immunization, Vaccines and Biologicals WHO, 2013 Correlates of Protection Induced by Vaccination Plotkin, 2010, Clinical and Vaccine Immunology