In vitro assessment of vaccines measures of immune response and attempts to predict protection

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In vitro assessment of vaccines - measures of
immune response and attempts to predict protection

• David J. Hurley, PhD
• Associate Professor and Molecular Microbiologist
• Food Animal Health and Management Program
• College of Veterinary Medicine
• University of Georgia
• Athens, GA

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Definition of vaccines

• Ideal vaccines - those that provide complete
  blockage of infection or symptoms in individual
  animals after vaccination, and provide herd
  immunity when a significant fraction (say 60) of
  individuals are vaccinated.

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Definition of vaccines

• Real world vaccines - those that provide
  modulation of infection or disease that reduces
  the social and economic impact of the presence of
  the pathogen in the environment.

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Primary vaccine model
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Vaccines and herd immunity
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Assessment of vaccine protection

• Gold standard - challenge trials using balanced
  populations of animals. Success is defined by
  problem.

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Protection - how to chose an endpoint?

• Protection based on many factors including
• The ecology of the pathogen in the external
  environment.
• The life-cycle of the pathogen in the host.
• Rate of replication within host.
• Pathogen factors involved in managing host
  inflammatory and immune function.

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Protection means?

• Reduced clinical signs in vaccinated animals?
• The attainment of a specific circulating antibody
  titer?
• Elimination of clinical disease in vaccinated
  animals?
• Prevention of infection of vaccinated animals?
• YES! - but not to everyone and not at the same
  time.

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In vitro measures of vaccine response

• Antibody titers
• Cellular responses based on endogenous or recall
  antigen stimulation
• Cytotoxicity
• Proliferation
• Activation antigens
• Cytokines
• Antibody release

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Antibody titers

• A snap shot of history.
• Not generally very useful as single measurement,
  but can offer insight when done as serial
  samples.

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Herd serology assessment
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Antibodies and protection

• SN titers against viral antigens associated with
  the extracellular portion of viral life cycle can
  indicate protection (like polio).
• Antibody that neutrilizes toxins or binds
  bacterial components for clearance can be
  predictive in some cases.
• Mucosal titers can indicate protection.

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Paired Serology
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Isotype assessment of response context

• IgG1 and IgG2 are often used to assess Th2 and
  Th1 response context ratios, respectively.
• Can offer indication of vaccine efficacy in
  systems defined as strongly Th1 or Th2 dependent,
  like herpes or polio.

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Antibody pitfalls

• How long do titers last?
• What is their relationship to memory?
• Is this agentregulated by antibody?

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Cellular responses

• Endogenous vs recall stimulation.
• When to sample for endogenous responses?
• What samples offer insight?
• Blood and secondary tissues a question for
  rodents and other mammals.

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Assessments of cell mediated immunity
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Cytotoxic cell responses

• Total cytotoxic responses measure what is
  happening in the animal.
• MHC restricted cytotoxic responses measured to
  show the expansion of pTc memory cells in the
  population.

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Total cytotoxic response

• Measures activation of monocyte derived cells
  (and neutrophils if included), as well as NK,
  NK-T, gamma-delta T, and MHC restricted T cells.
  No selection or in vitro activation of cells.

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Total cytotoxic response

• Advantage is that it is a quick assay, direct
  from blood or secondary lymphoid tissues and can
  be easily run with MHC matched cells plus or
  minus antigen. Tools to determine role of NK
  and MHC restricted cells available for most
  species.

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Total cytotoxicity induced by Vaccines
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MHC restricted cytotoxicity

• Requires purified lymphocytes and APC.
• In vitro priming of pTc, addition of cytokines to
  promote differentiation and activation of clones,
  and long priming periods.

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MHC restricted cytotoxicity

• Assay conducted using MHC matched cells.
• Antigen expression by infection or transfection.
• Responder dilution assay generally required.

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Cytotoxicity and protection

• Total cytotoxicity appears to be associated with
  protection against respiratory viruses when done
  at the time of challenge in cattle.
• MHC restricted toxicity appears to correlate with
  protection against many intracellular agents in
  mice.

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Examples

• West K, Petrie L, Konoby C, Haines DM, Cortese V,
  Ellis JA. The efficacy of modified-live bovine
  respiratory syncytial virus vaccines in
  experimentally infected calves. Vaccine 1999 Dec
  1018(9-10)907-19
• Ellis JA, Steeves E, Wright AK, Bogdan JR, Davis
  WC, Kanara EW, Haines DM. Cell-mediated cytolysis
  of equine herpesvirus-infected cells by
  leukocytes from young vaccinated horses. Vet
  Immunol Immunopathol 1997 Jul57(3-4)201-14

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Cytotoxicity and protection

• Total cytotoxicity measurement offers view of
  current status, MHC restricted cytotoxicity
  measurement (as currently measured) offers a
  window on memory.
• Both are expensive and difficult to do.

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Proliferation responses

• Most widely used with in vitro recall antigen.
• Many ways to measure based on DNA synthesis,
  physiological activity or total cell number.
• Measured in bulk or on an individual cell basis.

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Bulk DNA synthesis assays

• 3H-TdR and BrdU are commonly used after
  stimulation.
• Assays involve stimulation of a fixed number
  (50K-5000K per sample) for 2-10 days.
• Measured using radio-incorporation or by ELISA.

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Typical proliferative response to vaccine antigen
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Examples

• Ellis JA, Hassard LE, Morley PS. Bovine
  respiratory syncytial virus-specific immune
  responses in calves after inoculation with
  commercially available vaccines. J Am Vet Med
  Assoc 1995 Feb 1206(3)354-61
• Ellis JA, Bogdan JR, Kanara EW, Morley PS, Haines
  DM. Cellular and antibody responses to equine
  herpesviruses 1 and 4 following vaccination of
  horses with modified-live and inactivated
  viruses. J Am Vet Med Assoc 1995 Mar
  15206(6)823-32

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Physiological activation assays

• Use of MTT, MTS or other enzyme indicator dyes.
• Made as bulk measurements, can be confounded by
  other physiological activation, such as
  secretion.
• Limited headroom, uses OD, new fluorescent probes
  available - improves from 2 to 4 logs of signal.

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Single cell measurements

• BrdU can be used to monitor the fraction of
  responsive cells by flow cytomerty.
• Total DNA dyes can be used in flow cytometry to
  assess cell number and fraction of dividing cells
  (n vs 2n).
• Coulter counters have been used to assess cell
  number and fraction in size class after recall
  stimulation.

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Proliferation and protection

• Has been used to assess vaccines for many years,
  and several studies show correlation to
  protection.
• The success at predicting protection based on
  recall proliferation has not been observed in
  most studies.
• May be part of total story.

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Activation antigens

• CD25 and CD26 antigens most widely used, but the
  Act antigen family (Bill Davis at WSU) and MHC
  class II also employed.
• Recall stimulation most commonly employed, can be
  combined with phenotype staining.

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Sample of activation analysis
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Examples

• Endsley JJ, Quade MJ, Terhaar B, Roth JA.
  BHV-1-Specific CD4, CD8, and gammadelta T cells
  in calves vaccinated with one dose of a modified
  live BHV-1 vaccine. Viral Immunol
  200215(2)385-93
• Sandbulte MR, Roth JA. T-cell populations
  responsive to bovine respiratory syncytial virus
  in seronegative calves. Vet Immunol Immunopathol
  2002 Jan 184(1-2)111-23

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Memory markers

• CD45R isoforms (CD45R, CD45RO, CD45RA) have been
  monitored to assess the development of memory.
• Many markers available in human, mouse.
• Fewer for cattle, sheep, goats and swine.
• Correlation to protection has been reported in
  some studies.

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Activation antigens and protection

• In cattle championed by Roth et al.
• Correlation to protection with BRSV vaccination.
• Can be followed during vaccine and challenge
  period after the work of Davis et al. in MAP
  infection of cattle.

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Vaccines and cytokine

• Broad interest in cytokine relative to vaccines.
• Measurement of proteins and message both widely
  done.
• Diverse methods and little known about the
  relationship of each measurement to the others.

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Cytokine protein measuements

• Secreted protein ELISA measurement common in
  human, rodent and swine. Some for cattle, but
  few for horses.
• Recall production by ELISPOT advancing in swine
  (Merial, Univ. Minnesota) and cattle (Compton,
  UK). Combine ELISA and single cell analysis.

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ELISPOT assays
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Cytokine protein measurements

• Intracellular cytokines measured by flow
  cytometery.
• Generally gives estimates of cytokine positive
  cells at least twice expected.
• Difficult to do well, cells become fragile and
  dirty.

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Cytokine message

• Has been measured endogenously after vaccine and
  challenge.
• Most often measured after recall antigen
  stimulation in vitro.
• Real-time and competitive RT-PCR (Zarlenga) most
  widely used.

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Examples

• Estes DM, Brown WC Type 1 and type 2 responses in
  regulation of Ig isotype expression in cattle.
  Vet Immunol Immunopathol 2002 Nov90(1-2)1-10
• Werling D, Collins RA, Taylor G, Howard CJ.
  Cytokine responses of bovine dendritic cells and
  T cells following exposure to live or inactivated
  bovine respiratory syncytial virus. J Leukoc Biol
  2002 Aug72(2)297-304

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Cytokine message and microarray

• In humans, mice and cattle microarrays available
  for vaccine studies.
• Coussens (MSU) has established a microarray
  screening and real-time evaluation of affected
  genes for MAP that may be the model for
  microarray analysis in vaccines.

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Cytokines and protection

• Well established in assessing the context of
  response (Th1 vs Th2).
• Predicts protection in HIV.
• Other applications await further proof.
• May be most useful combined with other measures.

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Recall antigen driven antibody production

• Clonal frequency of Ab producing cells can be
  monitored after in vivo or in vitro recall
  antigen.
• ELISPOT provides an outstanding tool to monitor
  antibody production following recall antigen
  stimulation in vitro.

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Base on clasical plaque assays
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B cell memory

• Methods to measure B cell memory in vitro
  evolving.
• Offer promise for prediction of protection.

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A summary

• No single, reliable in vitro assessment is
  predictive of protection for all vaccines.
• In diseases where antibody is protective,
  workable paired serology protocols can predict
  vaccine protection.

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More summary

• Herd protection in these cases can be more
  confidently predicted than individual protection.
• CMI induction by vaccines has been correlated
  with protection in some cases.

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Still more summary

• CMI is defined in the eyes of the lab doing the
  assays, and is not always reproduced easily in
  other labs.
• As yet, proliferation, cytotoxicity, activation
  antigen and cytokine assessments tell us more
  about immune response to vaccine than protection.