The Chaos of Vaccine Responses: The Importance of Tissue-Level Activity - Dr. David Hurley, Department of Large Animal Medicine, University of Georgia, from the 2016 Ceva Swine U.S. Launch & Scientific Symposium, February 26, New Orleans, LA, USA. More presentations at http://www.swinecast.com/2016-ceva-symposium-aasv

1. The Chaos of Vaccine Responses:
The Importance of
Tissue Level Activity
David J Hurley, PhD
Professor, Food Animal Health and Management
University of Georgia

2. How to Understand Vaccine Responses
• The traditional approach to understanding the vaccine response is through a
classical reductionist approach
• Isolate individual factors and components
• Try to understand the role of each
• Show an association between the individual component and an outcome
(i.e. SN titer to virus)
• My approach – use Chaos Theory (or Complexity Theory if you prefer)

3. Chaos Theory
• Chaos is a branch of statistical practice that deals with predictive models of
complex systems
• Complex systems have several important properties:
• They have multiple “inputs” from different sources and of different types
• They generate multiple “output products” that impact the overall “outcome” we
observe
• The many inputs and output products combine (most often by summation) to
generate critical conditions called attractors (or strange attractors by some) that
can be use to predict the overall outcome

4. Complex System Properties
• Complex systems are dynamic – we do not get to chose a starting set of
conditions and must monitor them as they happen (the weather is the most
common example)
• Outcomes in complex systems are governed by initial conditions, summation
of critical attractors, and a set of operators that translate output products into
functional outcomes

5. ChaosMathematical expression
Graphic iterations
Social Context

6. How is the Immune System Chaotic?
• Initial conditions in the tissues determine the outcome products and actions of
immunity
• Inputs at the tissue level generate output products that provide the information
as “orders” presented to the adaptive immune system through a summation
process.
• The adaptive immune system acts as a network of factories to produce the
outcome effectors that protect the tissues from damage and invasion

7. The Tissue Environment

8. “Information” Flows to the Adaptive Factory from the Tissues
• “Information” (primarily antigen and danger
signals) flow to the node
• Clonal expansion is followed by effector
differentiation and memory cell development
• The organized lymphoid tissues are divided
into B cell and T cell areas that are connected
and after expansion the effector cells (and
eventually the memory cells) move to the
blood and lymph.

9. • This micrograph shows the organization of the B cell
area (center of the ring) abutting the T cell area (to the
right side)
• B cells divide forming an expanding ring of cells
specific for the antigen involved. T cells divide 2-4
times then begin to provide helper support or become
effector cells.
• Differentiated B cells produce antibody and become
plasma cells. Antibody leaves the factory. Effector T
cells also leave to return to the tissues and find the
damaged areas presenting evidence of danger.
• Memory cells for both B and T cells follow
“Information” Flows to the Adaptive Factory from the
Tissues

10. Infection, Immunity and How Vaccines are Intended
to Model the Ecology of Disease Management
• Tissue level interaction between infectious invaders and the host are the factors that
guide development of lasting immunity by transfer of target antigen to the structured
lymphoid centers and providing the context for the response.
• Vaccines are designed to mimic the production of the necessary factors, provide
sufficient and lasting antigen in tissue, and to set the context of response.
• Most vaccines are designed to induce lasting immunity to pathogens in a tissue context
that does not reflect natural exposure. Thus, they most often function to prevent
disease and not block or stop colonization of the host. However, they provide immunity
that seeks areas of damage and danger, so find their way to the tissue that is infected.

11. The Classical View of How a Vaccine Functions
• Vaccines are most often viewed as
the placement of antigen (live or
with adjuvant) in a black box and
allowing the magic in the tissues to
send antigen information to the
secondary immune centers where
“all the action” happens

12. How We Think About Vaccine
Response
• Antigen is delivered to the organized lymphoid tissue by dendritic cells (DC) coming
from the tissues.
• Antigen peptides and DC surface proteins activate T cells, and whole 3D antigen
activates B cells in the lymphoid tissues.
• Matching T and B cells undergo “clonal expansion” by dividing.
• Somehow, B cells become antibody producing cells (first IgM then IgG) and the
antibody enters the circulation with IgG entering the tissues. T cells differentiate
into effector cells (T cells that make cytokines on meeting antigen again, and T cells
that kill infected targets that express antigen in MHC protein).
• If we are lucky, memory cells develop among the expanded T and B cells so that
future response is faster and stronger.

13. Common Vaccine Constructs
• Modified Live or Live-Attenuated vaccines
• Designed for subcutaneous delivery
• Designed for intramuscular delivery
• Designed for mucosal (often intranasal) delivery (less
common)
• Killed and Subunit Vaccines with Adjuvant
• Designed for subcutaneous delivery
• Designed for intramuscular delivery
• Proposed for mucosal delivery

14. IM Vaccine with Live Agent

16. Adjuvanted Vaccine

17. Adaptive Response to Vaccines
Core of Adaptive Response What happens here
• Antigen presenting cells from tissue drive
proliferation of lymphocytes that recognize
antigen targets
• The clones that arise differentiate to produce
antibody (at the edge of the ring) or
cytokine/kill T cell activity over time
• The antibody is released to the circulation
and some of the T cells leave to function in
tissue

18. Trafficking of Effector Cells
• As effector T cells are
differentiated, they leave the
organized lymphoid tissues and
enter the lymph (also a route to
circulation via the thoracic duct)
and circulation
• From there they have effect in the
body by entering tissues showing
signs of damage and danger
Treg
Tc
T
B
B
Antibody
Efferent
vessel

19. How Do We Measure Vaccine Effect?
• In general, we look for antibody that either
binds or neutralizes cellular infection or toxicity
in the serum, or
• We look for the activation of circulating (or
splenic in rodent) lymphocytes by exposure to
the vaccine antigen in culture. We measure
cell division, cytokine production, expression
of activation antigens, or ability to kill antigen
presenting targets.
• Unfortunately, there is only moderate
concordance between these measures and
protection of animals from disease

20. How Do We Measure Vaccine Effect?
• In general, we look for antibody that either
binds or neutralizes cellular infection or toxicity
in the serum, or
• We look for the activation of circulating (or
splenic in rodent) lymphocytes by exposure to
the vaccine antigen in culture. We measure
cell division, cytokine production, expression
of activation antigens, or ability to kill antigen
presenting targets.
• Unfortunately, there is only moderate
concordance between these measures and
protection of animals from disease

21. How Can We Measure
Protection From Disease?
• For many agents, we can not measure protection directly as we have no
reliable model to induce disease at will.
• In systems with a disease induction model, we challenge animals and
determine the reduction in expected development of symptoms and the
duration of shedding of the challenge agent.
• The models we use are based on single pathogen challenge in a world of
polymicrobial disease.

22. What is the Current Model for
Understanding the Function of Vaccines?
• The clonal expansion of adaptive cells in organized lymphoid
tissue is the sine quo non of vaccine response.
• Effector cells and molecules generated by some members of
those clones mediate clearance of infectious agents
• The duration of these effectors in circulation and/or the
presence of memory cells in the organized lymphoid tissues
are the focus of effective vaccination.

23. • The Chaotic model is focused on tissue level actions that
“place orders” to the adaptive immune system.
• The orders are placed democratically (by summation of the
“reports” about damage, danger and target antigen from
multiple tissue microenvironments), and they are updated
dynamically – the reports change with time.
A Different Way to Look At
Vaccine Induced Immunity

24. • The tissue has a large number of somatic cells (epithelial,
muscle and endothelial) that interact with foreign invaders
• Leukocytes enter and reside in the tissues and some enter
then traffic with the lymphatic system
• All these cells respond to inputs and produce outputs that
impact the level of damage, danger and target antigen in the
tissue
Many Players Leading to Three Focused
Messages (The Attractors)

25. Somatic Cells and Bacteria

26. Somatic Cells and Bacteria
Drive signals of
Damage
Differentiate iDC
Changes in
movement of cells,
platelets and
factors from
circulation
Local cell gene
expression and
activation

27. Somatic Cells and Bacteria
Drive signals of
Damage
Differentiate iDC
Changes in
movement of cells,
platelets and
factors from
circulation
Local cell gene
expression and
activation

28. Somatic Cells and Bacteria
Drive signals of
Damage
Differentiate iDC
Changes in
movement of cells,
platelets and
factors from
circulation
Local cell gene
expression and
activation

29. Somatic Cells and Bacteria
Drive signals of
Damage
Differentiate iDC
Changes in
movement of cells,
platelets and
factors from
circulation
Local cell gene
expression and
activation

30. Somatic Cells and Virus

31. Somatic Cells and Virus
Drive signals of
Damage
Differentiate iDC
Changes in
movement of cells,
platelets and
factors from
circulation
Local cell gene
expression and
activation

32. Somatic Cells and Vaccine Adjuvant

33. Somatic Cells and Vaccine Adjuvant
Drive signals of
Damage
Differentiate iDC
Changes in
movement of cells,
platelets and
factors from
circulation
Local cell gene
expression and
activation

34. Tissue Players Among Leukocytes

35. Tissue Players Among Leukocytes
Opens vascular
epithelial spaces
Recruits cells
and factors
Make cytokines
for iDC
development
Enhance
inflammatory
environment

36. Tissue Players Among Leukocytes

37. Recruit
neutrophils and
monocytes
Activate
macrophage, DC
and neutrophil
activation
Arm local
defenses kill
invaders directly
Release target
antigen and
danger signals
Mediate damage
Tissue Players Among Leukocytes

38. Tissue Players Among Leukocytes

39. Tissue Players Among Leukocytes
Mediate tissue
damage
Enhance
processing and
traffic of target
antigen
Direct killing of
invaders
Enhance local
inflammation
Traffic target
antigen to instruct
the “order” of
support

40. Actions of the Neutrophil

41. Actions of the Neutrophil
Phagocytosis
Radical damage – invader
and host
Proteolytic, lipolytic and
nucleic acid enzyme attack
Chemokine and cytokine
production augment
inflammatory cells
Netosis leading to trapping
and killing of invader and
damage signals

42. Tissue Players Among Leukocytes

43. Tissue Players Among Leukocytes
Source for iDC and
target antigen
processing,
trafficking and
presentation activity
Enhance inflammation
Source material for
development of
macrophages (killer
M1 macrophages and
director M2
macrophages) in
tissue
Carry proof of danger
to adaptive tissues

44. Fates of the Monocyte

45. Tissue Players Among Leukocytes:
Dendritic Cells
Family of dendritic cells arising
from the CD34+ stem cell
parents
Monocyte derived dendritic
cells developed in tissues

46. Dendritic Cell Interactions with Agents
in Infection or Vaccine

47. Dendritic Cell Interactions with Agents
in Infection or Vaccine
Dendritic cells
(iDC and their bone marrow
derived cousins)
are the core signal (antigen
and danger) carrier from
tissues to
Adaptive Immune
Factories
(lymph nodes, spleen, and other
lymphoid tissues)

48. Tissue Players Among Leukocytes:
The Macrophage Family

49. Tissue Players Among Leukocytes:
The Macrophage Family
M1 macrophages are phagocytes and
aggressive promoters of the
inflammatory response with tissue
damage, release of danger signals, and
destruction of invaders to release target
antigen
M2b macrophages are the tissue
macrophage that manage the tissue
level responses of all immune
components
M2c macrophages are the sensors of
danger and activators of monocytes
M2a macrophage are the drivers of
tissue repair

50. Macrophage Cells in Action

51. Macrophage Cells in Action
React to invaders and vaccines
Clear damaged cells in tissue
Respond to physiological
imbalance
Cause tissue level damage in
exuberant inflammatory response
Lead restoration of tissue

52. Tissue Players:
How this comes together in tissues
• Tissue Macrophages lead the response and drive recruitment of
monocytes
• NK and gamma-delta cells recruit monocytes and neutrophils, somatic
tissue cells
• Mast cells lead iDC differentiation
• That cascade leads to a multi-pronged attack in the tissues of the
invader or response to the vaccine. This is the basis for organizing the
order to the adaptive response.

53. Tissue Players:
How this comes together in tissues

54. Tissue Players:
How this comes together in tissues

55. Transition to the Adaptive Tissues –
Making and Filling the Orders
• Tissue pressure increases with activation in the tissues
• DC traffic in the lymph carrying signals of danger and target antigen,
neutrophils bring whole antigen context (for B cells) and factors
indicating damage also traffic.
• The composite of damage, danger and target antigen are the
necessary and sufficient components to launch the order.
• What is made and delivered is dependent on the absolute and relative
levels of each of these three.

56. Tissue Response and the Signals the Travel to
Organized Lymphoid Tissue

57. The T cell Response

58. The T cell Response
In the organized lymphoid tissues,
tissue DC, factors and other
leukocytes interact with local DC to
present antigen to T cells in the
context of damage and danger
Low levels of damage, danger and
antigen allows for initial clonal
expansion
As the levels from tissues rise with
time, effector cells are differentiated
(DTH, killer cells), and if the balance
changes (less danger and damage,
but lots of antigen) memory is
developed, or excessive damage
Treg develop and traffic.
When all drop, excess clones are
deleted

59. The Cascade of Orders
• Initial stimulation in the context of damage, danger and target antigen leads to
expansion of clones (fuel and raw materials)
• After 2-5 days, increase levels of all signals (as factors with the migrating DC) allow
the expanded cells from clones to differentiate effector cells and make them return to
the tissues
• After a period (6-20 days), excessive damage may occur, leading to differentiation of
Treg cells to dampen the level of response and control tissue level killing
• Damage and danger may drop off (30-60 days), but target antigen may remain high
from tissue leading to memory cell development. Memory cells form two pools, local
to lymphoid tissue and circulating in blood and tissues.

60. T and B cells Return to the Tissue

61. T and B cells Return to the Tissue
In the matured
response, T and B
cells return in
relatively small
numbers (compared
to macrophage and
neutrophils) to induce
and regulate tissue
level activities to
tissues showing signs
of damage and
danger.
These cells provide
specificity to the
response to invaders.
This may be the result
of an infection or prior
vaccine exposure

62. The B cell Response

63. The B cell Response
Whole antigen, tissue B cells and
factors traffic from the tissues to the
organized lymphoid tissues.
B cells are directly activated by whole
antigens and the clones are expanded
Under the influence of T cells, the
orders from the tissues are enhanced
with time and expanded B cell clones
are differentiated to produce antibody
and class switch occurs to optimize the
impact systemically and in tissue
With reduced damage and danger, but
sustained antigen, memory develops
Extra clones are deleted

64. B cells Form Germinal Centers to
Produce and Export Antibody
Germinal centers are the product of the collection of expanded clones of B cells
enhanced by T cell help (to the right on the first image) that then differentiate to
produce antibody for export. The right hand image shows the pathway to traffic
antibody to rest of the body. Further, B memory cells reside both in the lymphoid
tissue and circulate to the other tissues of the body to provide rapid response to
invasion.

65. The Cascade of Orders
• Initial stimulation (D0-2) in the context of damage, danger and target antigen leads to
expansion of B cell clones (fuel and raw materials)
• After 2-5 days, increase levels of all provide signals (as factors with the migrating DC)
to differentiate antibody producing cells, then cells with the proper antibody isotype
(IgM, IgG, IgA), and make them traffic to the tissues
• After a period (6-20 days), excessive tissue damage may occur, leading to
differentiation of Treg cells to dampen the level of response and control tissue level
killing. However, antibody does not change its targeting function.
• Damage and danger may drop off (30-60 days), but target antigen may remain high
from tissue leading to B cell memory cell development. Memory cells form two pools,
local to lymphoid tissue and circulating in blood and tissues.

66. Adaptive Immune Tissue is a Factory
Filling Orders for Use by Tissue Environments
• The tissue signals of damage, danger and target antigen control the
activation and specific differentiation of adaptive cells.
• This process makes “tools” for use in activating, targeting, dampening,
or effecting molecularly specific responses in the tissues when the
products traffic to those sites.
• The adaptive response is the servant of the tissues and the “products”
from the immune factories return to the tissues with preference for
those showing damage and danger.

67. A Typical Vaccine Response

68. A focused initial response leads to
a primary production of antibody
and trafficking of T cells to the
tissues (as seen by circulation in
serum).
A later exposure takes advantage
of the memory pools of B and T
cells to make a larger and more
rapid response to the antigen
Both exposures are influenced by
the tissue “volume” exposed,
duration of damage, danger and
antigen signal, and match to prior
exposure
A Typical Vaccine Response

69. Secondary Response
 If priming vaccine or exposure is effective, the factory has more copies of the
“right” lymphocytes available in stock, and they are ready to respond
 This shortens the number of clonal expansion divisions required to provide
effectors
 Effectors are released sooner and more antibody enters the circulation sooner
on subsequent responses

70. Desired Vaccine Outcome

71. Optimized and Suboptimal
Use of Vaccines
• Vaccine response is based on the duration and quantity of damage, danger and
target antigen in tissues.
• The sequence of activities in the organized lymphoid tissues is driven by the
progressive “reporting” of these three attractors from multiple tissue micro-
environments and the quantity, quality, persistence and distribution of the
products of the adaptive response is a direct result of this sequence
• Optimal vaccine usage provides the greatest quality and quantity of protective
immunity and capacity to respond to invading pathogens, but suboptimal use of
vaccines provides a limited response that may lack quantity of immune effectors
and a lack of memory capacity delivered to the tissues.

72. 0
5
10
15
20
25
0 1 3 7 10 14 21 28 35 60
Responselevel
Days after Delivery
Optimized Vaccine Delivery
Damage
Danger
Antigen
Time Related Damage, Danger and Target Antigen Profile:
Optimized Vaccine

73. 0
5
10
15
20
25
0 1 3 7 10 14 21 28 35 60
Responselevel
Days after Delivery
Optimized Vaccine Delivery
Damage
Danger
Antigen
A balance of
damage, danger and
target antigen that
leads to productive
and lasting adaptive
immunity
Time Related Damage, Danger and Target Antigen Profile:
Optimized Vaccine

74. 0
10
20
30
40
50
60
70
80
0 1 3 7 10 14 21 28 35 60
Magnitude
Time related damage, danger and antigen profile
Response to Vaccine: Optimized
clonal expansion
effector differentiation
Treg differentation
memory cell development
repair activators
apoptosis
Time Related Damage, Danger and Target Antigen Profile:
Optimized Vaccine

75. 0
10
20
30
40
50
60
70
80
0 1 3 7 10 14 21 28 35 60
Magnitude
Time related damage, danger and antigen profile
Response to Vaccine: Optimized
clonal expansion
effector differentiation
Treg differentation
memory cell development
repair activators
apoptosis
Production of:
• Strong clonal
expansion
• Effector activity
• Memory
• Appropriate Treg
activity and
tissue repair
Time Related Damage, Danger and Target Antigen Profile:
Optimized Vaccine

76. Optimized Vaccines Generate a Broad
Response that Returns to Tissues
• Optimized vaccines provide balanced damage, danger and target antigen to the factory to drive clonal
expansion.
• With time, the level of danger and target antigen rise in the context of continued damage, and effector
differentiation is favored
• When antigen rises relative to danger and damage, memory is generated
• If damage and antigen are high relative to danger, Treg activity is generated.
• When damage is higher than danger and antigen, restoration factor producing lymphocytes are sent to the
tissue.
• The cells that leave circulate to tissues showing damage and danger and often become resident waiting for
the next attack.

77. 0
5
10
15
20
25
0 1 3 7 10 14 21 28 35
Responselevel
Days after Delivery
Low Dose Vaccine Delivery
Damage
Danger
Target Antigen
Time Related Damage, Danger and Target Antigen Profile:
Suboptimal Vaccine

78. 0
5
10
15
20
25
0 1 3 7 10 14 21 28 35
Responselevel
Days after Delivery
Low Dose Vaccine Delivery
Damage
Danger
Target Antigen
• Short duration of
damage and danger
that limits the response
to target antigen
• Too few tissue sites
responding due to
reduced loading dose
• Fewer cycles of
“instructions and
orders” to the adaptive
tissue
Time Related Damage, Danger and Target Antigen Profile:
Suboptimal Vaccine

79. 0
10
20
30
40
50
60
70
80
0 1 3 7 10 14 21 28 35 60
Magnitude
Time related damage, danger and antigen profile
Response to Vaccine: Low Vaccine Dose
clonal expansion
effector differentiation
Treg differentation
memory cell development
repair activators
apoptosis
Time Related Damage, Danger and Target Antigen Profile:
Suboptimal Vaccine

80. 0
10
20
30
40
50
60
70
80
0 1 3 7 10 14 21 28 35 60
Magnitude
Time related damage, danger and antigen profile
Response to Vaccine: Low Vaccine Dose
clonal expansion
effector differentiation
Treg differentation
memory cell development
repair activators
apoptosis
• Reduced clonal
expansion and
effector
differentiation
• Minimal or no
memory, weak Treg
differentiation
• Failure to reach
vaccine potential for
protective immunity
Time Related Damage, Danger and Target Antigen Profile:
Suboptimal Vaccine

81. • Shorter and less effective period of antigen transferred to the adaptive
immune factory
• Less robust response in clonal expansion and delivery of effectors to
the tissue during the vaccine response period
• Little development of memory or Treg activity that could impact the
response to infection in tissues, fewer circulating memory cells to home
to tissues with damage and danger driven vascular access.
Suboptimal Vaccination Outcomes

82. Summary
• Tissue level events – their duration and strength plus their diversity
(involvement of multiple microenvironments) are the drivers of a complex
and effective vaccine response
• The dose, timing and delivery of vaccine is critical to having an effective
response that meets the goals of the vaccine
• Vaccines are able to protect target tissues other than the vaccine site
because once ordered and produced in effective quantity and for the
right challenge, the products will enter all body tissues expressing
damage and danger.