HIV Pathogenesis

1
HIV Pathogenesis And Immune Reconstitution Savi
ta Pahwa, MD University of Miami, Miller School
of Medicine
2
Topics for Discussion

• Pathogenic events in acute and chronic HIV
  infection
• Protective antiviral immune responses against HIV
• HIV escape from the immune system and
  antiretroviral drugs
• Extent of damage to the immune system and
  potential for immune reconstitution

3
HIV infection of target cells is dependent on
CD4 and chemokine receptors
Transmission

• Main cells infected by HIV
• CD4 T cells, monocytes
• Two main receptors for HIV
• CCR5 and CXCR4
• Transmission occurs with R5
• virus through CCR5 and CD4

R5, M-tropic virus
CCR5
CCR5
CXCR4
CD4
CD4
CD4 T Lymphocyte
Macrophage
4

DC express dendritic cell-specific
ICAM-3-grabbing nonintegrin (DC-SIGN), a C-type
lectin that specifically recognizes fungal, viral
and bacterial pathogens. DC-SIGN binds HIV gp120
and transports HIV
Dendritic Cells
5
Nature Medicine  11, 469 - 470 (2005)
6
Summary Initial events in Acute HIV Infection

• Infection established at point of entry
  predominantly in CCR5 CD4 memory T cells, and
  spreads to regional lymph nodes via DC and to the
  mucosal associated lymphoid tissue (MALT)
• Gastrointestinal tract is the most prominent
  early site of virus replication (1-3 weeks post
  infection) with widespread infection and
  depletion of memory CD4 T cells that is only
  partially restored with later HAART
• A latent reservoir of HIV is established early in
  resting memory CD4 T cells (integrated virus not
  visible to the immune system) which is minimally
  affected by HAART

7
Course of Untreated HIV Infection
Chronic Phase
Acute Phase
Qualitative and Quantitative loss of CD4 T cells
CD4 counts
Plasma HIV RNA copies/ml
CD4 gt25
CD4 15-24
CD4 lt15
Limit of detection of plasma HIV
Latent HIV Reservoir
Time
years
months
8
Components of anti-viral responses

• Innate Immunity
• Antigen presenting cells Mf and Dendritic cells
• Other cell types NK, gd, NKT cells
• Soluble factors Chemokines
• Adaptive Immunity
• CD8 T cells (CTL)
• CD4 T helper cells
• Regulatory T cells
• B cells Neutralizing antibodies

9
HIV Immunity Innate and Adaptive Immune Systems
CELLULAR SECRETED FACTORS
ADAPTIVE
INNATE
Helper function
Cellular antiviral factors
CD4 T cells
Macrophages
Cytokines
(
Suppression
CD8 suppressor factor
Regulatory T cells

Chemokines
Cytotoxicity (CTL)
Dendritic cells

Complement System
CD8 T cells
Neutralizing Antibodies
NK Cells
Antibodies
B cells
NKT Cells
gd T Cells
10
Dendritic Cell T cell interaction and T Cell
Activation
CD4 or CD8 T Cell
MHC Class I / II
Adapted from, Abbas Sharpe, Nat Immunol, 2005
11
Host Virus Interaction Components of Adaptive
Immunity
Helper T Cells
HIV virion
CD4
CTL
CD8
NeutralizingAntibody
B cell
Y Y Y
Y Y
CD4
Helper T Cells
CD4 T cell
12
Evidence for Role of HIV-specific Cellular
Immune Responses in Controlling HIV

• Natural immune control of HIV
• Temporal relationship of establishment of viral
  set-point and HIV- specific cellular immune
  response
• Animal studies removal of CD8 T cells in vivo
  results in rapid increase in the steady-state
  level of viremia in SIV infected macaques
• Identification of HIV-infected individuals who
  dont develop disease long term non-progressors

Goulder and Watkins, Nature Reviews Immunology 4
631-640, 2004
13
T cell receptor antigen recognition
CTL

• CTL play a dominant role in control of HIV
• Some CTL are more effective than others quality
  of CTL more important than frequency or magnitude
• HLA plays a dominant role in CTL response
• HLA B restricted responses are the most dominant
  and B allele variation makes an impact on Viral
  Load
• Dominant CTL responses include those directed
  against regulatory viral genes (tat, nef) and
  structural genes (gag)

CDR3 region of TCR
7/98
14
Generation of Cellular Immune Response against HIV
Death of HIV-infected target cells
Helper T cell
APC
CD4
CTL
TCR
CD8
CD8
CD8
CD8
MHC Class I
TCR
Effector
CM
EM
CD27 /- -- CD45RA -- --
CD62L --
--
Lysis of infected cells perforin, granzyme,
Fas L Soluble Factors Cytokines IFN?,
TNF? Chemokines MIP1?/?,
RANTES CD8 suppressor factor,
CAF
Development of CTL Activation Proliferation Matu
ration/Differentiation
15
Study of CD8 T Cells

• Phenotypic assays to determine maturation stages
  of CD8 T cells
• Tetramer binding to enumerate percentage of
  antigen-specfic cells
• Antigen-stimulated cytokine production in Elispot
  or by flow cytometry to evaluate CTL
• Antigen-stimulated proliferation
• CTL effector function cytolytic granules
  perforin /granzyme and lysosomal
  degranulation/cytotoxicity assays
• Evaluation of viral escape mutations that impair
  generation of CTL

16
Effector Memory CD8 T Cells Accumulate in HIV
Infection Skewed Maturation
Pahwa et al, JID, in press
17
APC Gag and Pol Tetramers
APC
Recombinant MHC-1 heavy chain
biotin
streptavidin
Peptide
18
Representative histogram demonstrating the high
granzymeperforin ratio in HIV-specific CD8 T
cells
Lymphocyte gate
CD8 T cell gate
Tetramer cell gate
CD8 36.9
Lymphs 93
Gag8.2
ECD CD8
SSC
SSC
FSC
FITC perforin
APC gag tetramer
Analysis of perforin and granzyme A
4 color staining in whole blood ECD-CD8
APC-Tetramer Lyse RBC, Fix, Permeabilize, FITC-Pe
rforin PE-Granzyme A
50.9
3.0
86.2 1.9
GrA 88.1 Pf 3.2
GrA 53.9 Pf 3.1
PE Granzyme A
PE Granzyme A
0.1
46.0
11.6 1.3
Haridas et al, AIDS, 2003
FITC Perforin
FITC Perforin
19
Summary of CD8 T cell defects

• Maturation of HIV specific CD8 T cells is
  impaired, with an expansion of effector memory T
  cells and a decrease of naïve cells
• Qualitative deficiency of HIV-specific CD8 T
  cells include lack of a polyfunctional response
  IFN-g production may occur without IL-2
  synthesis, and proliferative responses are
  impaired similar defects in CD4 T cells leads to
  lack of help for CD8 T cells
• CD8 effector T cells and HIV tetramer positive
  cells exhibit increased granzyme perforin ratio
  with relative perforin deficiency
• Expression of CD127 (memory marker) is
  diminished expansion of CD127 negative,
  activated CD8 T cells is associated with disease
  progression

20
Viral evolution and immune escape
APC
Brander C and Walker B Nature Medicine
9, 1359 - 1362 (2003) 
21
HLA Leaves Its Footprints on HIV
                                               
                   
McMichael and Klenerman, Science 2002 296
1410-1411
22
HLA and HIV Infection

• Protective
• Heterozygote advantage
• A32
• B14
• B27
• B44
• B51
• B57
• C8
• Homozygosity for Bw4
• Bw4-80Ile alleles/KIR3DS1

• Susceptible
• A24
• B3
• B8
• B35-Px
• C4
• C16

Buchbinder, et al., 1992 Klein, et al., 1994
Goulder, et al., 1996 Kaslow, et al., 1996 Keet
et al., 1999 Carrington, et al., 1999 Hendel,
et al., 1999 Flores-Villanueva, et al., 2001
Martin, et al., 2002
23
Escape mutations Barriers to Effective CTL

• CTL escape mutations associated with progression
  to AIDS
• Immunodominance of ineffective CTL causes
  interference with effective CTL responses
• However,
• Escape may come at a price and reduce viral
  replication capacity
• virus reverts mutation if transmitted to MHC
  mismatched subject, including MTCT

24
Summary mechanisms by which HIV eludes the
immune system

• HIV establishes a latent reservoir early in
  infection
• HIV develops mutations to escape immune
  responses, CTL escape best studied but escape
  from neutralizing antibody also documented
• HIV depletes functional cells of innate immune
  system
• HIV depletes CD4 T cells the conductor of the
  immune orchestra and blunts immune responses
• HIV causes deleterious effects on the entire
  immune system resulting in aberrant immune
  activation

25
Excessive T cell activation Can it be
Deleterious?
26
Cell Surface Marker Changes in HIV Disease
CD45 RA CD45 RO
CD 28 CD95 (Fas) DR CD38 CD57
changes seen in both, CD4 and CD8 subsets
Expansion of CD8 DR CD38 T cells
Loss of CD45RA naive T cells
predominantly in CD8 T cells
6/6/1995
27
Correlation between plasma viremia and levels of
CD8 T cells expressing CD38
.                                             
                      
Chun et al PNAS 20041012464-9.
28
CD8 Cell Activation Correlates with CD4 Cell
Loss
CD4gt500 cells/µl 500gtCD4gt200 cells/µl CD4lt200
cells/µl
CD8 T Cells
Sousa AE et al J Immunol 1693400, 2002
29
Excessive T-cell apoptosis correlates with
plasma virus load and inversely with CD4 counts
20
Virus Load
CD4 T cells
10
0
-10
Slope of T cell apoptosis
-20
-30
-40
-50
rs0.82 Plt0.00001
-60
-70
-1.5
-1.0
-0.5
0.0
Slope of log HIV RNA copies
Slope of CD4 counts
Chavan et al 2000
30
Proposed Mechanisms of Aberrant Immune Activation

• HIV specific T cells produce inflammatory
  cytokines
• HIV gene products, e.g. envelope gp120 activate
  uninfected CD4 T cells
• Potential role of macrophages/innate immune cells
  
• Deficiency/depletion of regulatory T cells

31
HIV induced immune dysregulation
Co-receptor
HIV
Cytokines (IFN-g, TNF-a, GM-CSF IL-1, IL-6
) NF-kB induction Fas, Fas-L CD40L (gp160)
gp-120
CD4
Anti-gp120
Anergy
CD4 crosslinking
PI hydrolysis IL-2 proliferation B7 on APC
Aberrant Activation
Sensitization
Oyaizu Chirmule Tamma Than McCloskey Chavan
Apoptosis
Bcl-2
CD4 T Cell
32
Summary T cell depletion in the HIV infected host
Lymphoid progenitor Cell
Thymus
Peripheral lymphoid compartment (including GALT)

• Restricted T cell Repertoire
• Loss of HIV specific Immune T cells

Naïve T cells
?

• Immune activation and increased
• T cell turnover CD8gtCD4
• Increased T cell apoptosis
• Infection and depletion of
• HIV-specific memory CD4 T cells

HIV
10/98
33
Different course of SIV in two primate models

Rhesus macaque Macaca Mulatta
Sooty mangabey (Cercocebus atys)
Location Northern India and southern China
Location Subsaharan western Africa Sierra Leone
to Ghana
Slow or non-progression
Rapid Progression
34
Immunity, Vol 18, 441-452, March
2003Nonpathogenic SIV Infection of Sooty
Mangabeys Is Characterized by Limited Bystander
Immunopathology Despite Chronic High-Level
Viremia Guido Silvestri 1,2, Donald L. Sodora 3,
Richard A. Koup 3,4, Mirko Paiardini 1,2, Shawn
P. O'Neil 5, Harold M. McClure 5, Silvija I.
Staprans 1,2, and Mark B. Feinberg 1,2
1Departments of Medicine and Microbiology and
Immunology and, Emory University School of
Medicine, Atlanta, GA USA 2Emory Vaccine
Research Center, Emory University School of
Medicine, Atlanta, GA USA 3University of
Texas-Southwestern Medical Center, Dallas, TX USA
4NIH Vaccine Research Center, Bethesda, MD USA
5Yerkes National Primate Research Center, Emory
University, Atlanta, GA USA
35
HIV-Specific CD8 T-cells From Patients with Poor
Restriction of Viral Replication Have Low
Proliferative Capacity
LTNP
Progressor
Day 6 after stimulation with HIV-infected
autologous CD4 T cells
B57-KAF11 tetramer
Gated on CD8 T cells
Gated on tetramer CD8 T cells
Cells
CFSE
Courtesy, M Connor
36
Correlates of delayed disease progression in
HIV-1-infected Kenyan children Functional
HIV-specific CD4 T cells and higher naïve CD4 T
cells
LTS Long term survivor Age gt8 years, no
therapy NP Non-progressor LTSP Progressors Age
lt 6 years, no therapy ENP Early
non-progressors TP Typical progressors HAART
on potent ART
Chakraborty R J Immunol. 2005 Jun
15174(12)8191-9
37
Estimated probability of developing AIDS within
12 months at selected ages in HIV-infected
children receiving no therapy or zidovudine
monotherapy
by CD4
From Guidelines for the Use of Antiretroviral
Agents in Pediatric HIV Infection, March
2005 (Adapted From HPPMS publication in Lancet
2003 362, 1605-11)
by virus load
38
Effect of HAART on the course of HIV infection
? Immune Restoration
CD4 counts
Plasma HIV RNA copies/ml
HAART
Limit of detection of plasma HIV
Latent HIV Reservoir
months
years
Time
39
Effect of HAART How effective is immune
reconstitution ?

• Immunological Response (IR) and
• Virological Response (VR)
• 4 patterns
• Responders (IRVR) CD4 increase, virus
  suppressed
• Non responders (IR-VR-) CD4 low, and virus not
  suppressed
• Discordant type 1 (IRVR-) CD4 increase persists
  even after virus rebounds
• Discordant type 2 (IR-VR) CD4 remains low even
  though virus is suppressed

40
CD4 gains persist despite virus rebound (PACTG
366)
Kovacs A et al, J Infect Dis. 2005 Jul
15192(2)296-302
41
HIV Virus Evolution Due to Selection Pressure
Host
Antiretroviral Drugs
Immune response Genetic makeup
Virus
Viral fitness
Immune escape mutations Drug resistance mutations
Complete resistance to ART is uncommon Even in
advanced disease, immune pressure persists
42
TREC responses in HIV-infected children
Responder Pt. ID 675-1192
Discordant Pt. ID 675-786
50
40
30
CD4 T cells (-?-)
20
10
0
-2
0
2
4
6
8
10
-2
0
2
4
6
8
10
Time in Months
Chavan et al JID, 2001
43
Pediatric HIV patients CD4 T cells and CD127
CD8 T cells correlate with each other
50
40
30
percentage of CD4 T cells
20
10
p0.02
0
0
10
20
30
40
50
60
70
80
Percent IL-7Ra (CD127) positive CD8 T cells
Pahwa et al, JID in press
44
T cell apoptosis at baseline and after 12 months
of antiretroviral therapy
80
n23
60
T cell apoptosis
P0.006
40
20
0
Baseline
12 Months
Chavan et al, 2000
45
Innate Immunity pDC and mDC interaction
Lymph node
imDC
IFN a/b
46
CCR7 expression on plasmacytoid dendritic cells
correlates with CD4 reconstitution following ART
Desai et al, unpublished
47
Summary Immune Reconstitution following HAART
HAART

• Increases the no. of CD4 T lymphocytes and naïve
  T cells
• Increases TRECs and CD127 CD8 T cells
• Decreases activated CD8 T cells and reduces
  lymphocyte apoptosis
• Improves overall T and B cell function responses
  to recall antigens are enhanced by re-exposure/
  revaccination
• NK, monocytes, and dendritic cell function are
  under investigation
• HIV-specific CTL decrease on effective HAART
• Discontinuation of therapy, even after
  prolonged virus suppression usually results in
  rapid viral rebound

48
Immune reconstitution and HIV/TB co-infection

• Danger of Immune Reconstitution Inflammatory
  Syndrome (IRIS)
• Believed to represent exaggerated immune response
  to TB antigens following rapid recovery of CD4 T
  cells
• More common if CD4 count is very low at start of
  HAART
• If not on therapy, advisable to start TB
  treatment first and HAART later

49
Can we boost immunity to HIV in Infected
people???

• Therapeutic Vaccines
• Passive Immunity (HIV mAb)
• Immunomodulators (IL-2, IL-7, IL-15)
• Auto vaccination (STI)

50
Model of Memory T cell Differentiation
IL7 Ra (CD127)
long term memory precursors
Effectors
Naïve CD8 T cells
IL-21
Effector memory
IL-7
IL-2,IL-7,IL-15
Antigen-stimulation APC T helper cells
Central memory
51
CD8 T cells of patients proliferate in response
to cytokines
A
Percent proliferating CD8 T cells
Pahwa et al, JID in press
52
HIV- specific tetramer positive cells are
preserved in presence of IL-15
Cyr
Pahwa et al, JID in press
53
Enhancement of HIV-gag specific CD8 T cell IFN-g
producing spot forming cells with IL-15
Fig 10.
Chitnis et al, 2003
54
PACTG 402 s/c IL-2 in children with low CD4
Median Change from Baseline for CD4 In absolute
counts in percentage
4miu
2miu
3miu
Pahwa et al, unpublished
55
Approaches for More Effective Virologic Control
and Immune Reconstitution in HIV Disease
New Antiretroviral Therapies (entry inhibitors)
Host Immune Strategies to Attack HIV
Immune Based Therapies, Cytokines (IL-2)
IL-15, IL-7
Therapeutic Vaccines to boost HIV immunity
Virus Strategies for life-long persistence
Blunt Host Immunity
56

• The End
• Thank you

57
(No Transcript)
58
Excessive TCR Vb repertoire perturbations in a
HIV infected adolescent
Pahwa et al JAIDS 2004