Virologic and Immunologic findings from an Indian NeuroAIDS Cohort Anita Desai, Anupa Kamat, V' Rav

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  Virologic and Immunologic findings
from an Indian NeuroAIDS Cohort Anita
Desai, Anupa Kamat, V. Ravi, P. Satishchandra,
KS Satish, DK Subbukrishna, I Borodowsky and
Mahendra KumarDept of Neurovirology,
NIMHANS, Bangalore, INDIASewa Clinic,
Bangalore, IndiaUniversity of Miami, USA
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Global distribution of HIV subtypes is non-uniform
B
B/C
C
B/C
C
B
D
C
B
C
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What were the challenges ?

• (i) HIV-1 Clade C virus
  predominates in India
• (ii) Genetic heterogeneity of
  HIV-1 is an important
• factor that could
  jeopardize reliability of viral load
• measurements (Swanson et
  al., 2005).
• (iii) There was no single
  commercial plasma
• viral load assay suitable
  for monitoring all HIV-1
• infected patients at a
  reasonable price
• (v) Opportunistic infections
  more common
• (vi) AIDS cohort for longitudinal
  study of markers

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Subtype C specific PCR
1 2 3 4 5 6 7
8 9 10 11 12 13
232bp
138bp

• Nested PCR
• First round 975bp product (LTR-gag)
• Second round
• 138bp product LTR region specific to HIV-1
  Subtype C
• 232bp product LTR leader-gag region common to
  all HIV-1
• subtypes

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Real Time PCRPrimer and probe selection

• The primers and probe were selected in the gag
  region of HIV-1 genome using primer express
  software and synthesized commercially by Applied
  Biosystems (USA).
• Forward primers ACC CAT GTT TAC AGC ATT ATC AGA
  AG and reverse primer GCT TGA TGT CCC CCT ACT GTA
  TTT at positions 498 and 578 on the reference
  Indian subtype C HIV-1 strain (Accession number
  AF533121, Los Alamos database) strain generate a
  80bp product in the gag region.
• A highly specific probe was also selected at
  position 525 of the above HIV-1 subtype C strain.
  The HIV-1 gag oligonucleotide probe (5 AGC CAC
  CCC ACA AGA TTT AAA CAC CAT GT 3) with a
  reporter fluorescein dye (FAM) attached to the 5
  end and no quencher (Minor groove binding probe)
  linked to the 3 end was used for detection.

• Logic
• Gag is a relatively more conserved gene as
  compared to other HIV structural genes
• LTR region though conserved had failed to
  amplify virus in the hands of other Indian
  investigators

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Amplification plots obtained in the TaqMan Real
time PCR assay with all the major HIV-1 group M
plasmid subtypes
Subtype C
Subtypes A, B, D, F, G H
Standard curve generated in Real Time PCR using a
HIV 1 viral load reference standard
The sensitivity of this assay was determined to
be 180 copies/ml
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Range of Plasma and CSF viral loads estimated in
HIV-1 infected patients by Real Time PCR
In these 46 patients, plasma viral loads were
comparable (within the same log) to CSF viral
loads in 7/46 patients (15.21 ), plasma viral
loads were higher than (by 1 log) than CSF in
27/46 patients (58.69) while in the remaining
12/46 patients (26.08 ) CSF viral loads were
greater (by 1 log or more) than plasma viral
loads.
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Reproducibility of the TaqMan Real time PCR
assay Intra-assay variation
Inter-assay variation
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Scatter plot depicting the correlation of viral
loads obtained in the TaqMan Real time PCR assay
and Amplicor HIV-1 monitor assay (Version 1.5).
(n36)
All samples positive by both assays. 30/36
samples showed concordant values in both assays.

• The X-axis depicts the HIV-1 RNA log copies /ml
  of plasma obtained using the Amplicor HIV-1
  monitor assay while the Y-axis depicts represents
  values obtained with the TaqMan Real Time PCR
  assay.

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Neutralizing antibody response against V3 peptide

• HIV envelope major target for humoral immune
  response - antibody neutralization-localized in
• gp120 and external portion of gp41.
• V3 region of gp120.central of this
  sequence-Gly-Pro-Gly (conserved between different
  HIV-1, flanked by amino acid that differ between
  isolates.
• V3 peptide with neutralizing domain to estimate
  neutralizing antibody levels (Guido van marle et
  al 2002, Cao et al 1995.
• Neutralizing antibody response in LTNPs is
  greater in magnitude than in other HIV-1 infected
  persons (Lifson et al 1991, Cecilia et al 1999,
  Pilgrim et al 1997)

V3 loop
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Neutralizing antibodies V3 Peptide ELISA

• HIV-1MN, 33 mer (1mg/ml)
• TRPNYNKRKRIHIGPGRAFYTTKNIIGTIRQAH
  NH2
• V3 peptide coating (1ug/ml)
• Serum (1200), CSF (110)-diluted in PBS-T with
  1 milk powder and 5 NGS along with positive and
  negative control
• Pooled HIV positive serum positive control
• CSF samples obtained from HIV negative
  individuals undergoing spinal anaesthesia were
  tested to calculate the cutoff value
• Cutoff Mean absorbance 3SD (standard
  deviation)
• Serum cutoff 0.6, CSF cutoff 0.2
• ELISA ratio (E Ratio) Test OD/Cutoff
• E Ratio gt 1 was considered as positive.

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Molecular Epidemiology C2-V3 sequencing
Transmembrane glycoprotein (TM)
Surface glycoprotein (SU)
gp41
gp120
Variable domains (gp120)
1 100 200 300 400 500
600 700 800 AA


transmembrane
ED5
ES7
ES8
ED12
anchor
RNA was extracted from serum and CSF samples.
cDNA was made with commercially available cDNA
archive kit (Applied Biosystems). HIV-1 env
C2-V3 region amplified and sequenced using
commercial services ES7 and ES8 were used for
sequencing of env C2-V3 region
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SYMPTOMATICS Vs ASYMPTOMATICS

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A VIROLOGICAL AND IMMUNOLOGICAL MARKERS

• HIV positive symptomatic
  (n20)
• HIV positive asymptomatic individuals
  (n20).
• Virological - Viral load,
• - p24 antigen
• Immunological - CD4 cell count,
• - Cytokines (TNFa,
  IL-6, IL-1a),
• - ß-2 microglobulin,
• - Neopterin,
• - Neutralizing
  antibodies
• Correlation of these parameters with
  neurologic disease occurrence and evolution of
  determinants in serum and CSF of the same
  individual in the two groups.

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Clinical data

• Asymptomatic HIV seropositive patients had no
  neurological illness at the time of recruitment.
• Symptomatic patients were HIV seropositive with
  neurological illness.
• 9/20- TBM, 3/20-CM, 3/20-PML, 1/20-HIV
  myelopathy,
• 3/20-Dementia and 1/20-Toxoplasmosis
• 1/20 died after 8 months of
  recruitment due to PCP,
• two developed peripheral neuropathy
  and 1 CMV
• retinitis

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CD4, CD8 cell counts and CD4/CD8 ratio in HIV
infected asymptomatic and symptomatic patient
group
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Comparison of mean viral loads obtained in plasma
and CSF samples of symptomatic and asymptomatic
group of cases
(p lt 0.001)
(p 0.011)
(n 20 / group)
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IL-1 alpha, IL-6 and TNF alpha levels in serum
and CSF of HIV infected asymptomatic and
symptomatic subjects as well as control subjects.
Serum
CSF
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ß 2 microglobulin and Neopterin levels in serum
and CSF of HIV infected asymptomatic symptomatic
as well as control subjects.
Serum
CSF
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p24 Antigen levels within two groups
4/10 (CSF), 1/10 (serum)- Symptomatic group 1/20
(CSF), 4/20 (serum)- Asymptomatic group
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Comparison of Neutralizing Antibody levels in
symptomatic and asymptomatic group
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Neutralizing Antibody levels in symptomatic and
asymptomatic subjects
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HIV-1 envelope sequences in serum and CSF

• Envelope sequences (C2 V3 region) in CSF and
  serum clustered in host dependent fashion rather
  than tissue specific compartmentalization.
• B/C recombinant was identified in the CSF and
  serum of one the sequence analysed.
• Amino acid alignments obtained from plasma and
  CSF did not reveal any significant differences

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Phylogenetics Analysis
H.BE.93.VI991 AF190127
K.CD.97.EQTB11C AJ249235
79
F1.FI.93.FIN9363 AF075703
358
372
D.TZ.01.A280 AY253311
722
B.FR.83.HXB2-LAI-IIIB-BRU K034
999
B.NL.00.671 00T36 AY423387
A2.CY.94.94CY017 41 AF286237
HIV-1 env C2-V3 sequences clustered in host
dependent fashion.
905
A1.SE.94.SE7253 AF069670
TRICHOTOMY
998
S6C NIMHANS
O.SN.99.SEMP1300 AJ302647
C.ZA.04.SK164B1 AY772699
C.BR.92.BR025-d U52953
525
C.ET.86.ETH2220 U46016
982
S10C NIMHANS
124
C.IN.95.95IN21068 AF067155
540
921
07 BC.CN.97.CN54 AX149771
492
997
08 BC.CN.97.97CNGX 6F AY008715
880
879
142
S11C NIMHANS
1000
S11P NIMHANS
S3P NIMHANS
1000
S3C NIMHANS
G.FI.93.HH8793 12 1 AF061641
953
J.SE.93.SE7887 AF082394
0.1
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Summary of Symptomatics Vs Asymptomatics

• (i) High viral loads in plasma and CSF, High
  p24 antigen
• levels, low CD4 counts and higher
  levels of IL-1?, IL-6
• and TNF? as well as higher levels of
  ß 2 micro globulin
• and neopterin were noted in HIV
  infected subjects with
• neurological disease as compared to
  asymptomatic subjects.
• (ii) These results were found to be
  statistically significant,
• thereby indicating that these markers
  can indeed serve as
• excellent predictors of neurological
  involvement in HIV
• infection.
• (iii) Neutralizing antibody levels were
  found to be low in
• symptomatics than in asymptomatics

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LONGITUDINAL STUDY ON ASYMPTOMATIC INDIVIDUALS

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INDO-USA collaboration

• NIMHANS-University of Miami collaborative project
  (NIH, RO1 grant) on Neurological progression in
  HIV 12 infections
• HIV infected individuals clinically asymptomatic
  for neurological disease to be recruited and
  followed up for three years
• Clinical, radiological, psychological,
  neuroendocrine, virological and immunological
  parameters to be studied at recruitment and every
  six months

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Study carried with plasma samples - 128
casesParameters studied CD4 cell
count, Viral load, Neutralizing
antibodies against V3 peptide,
Cytokine IL-1, IL-6, TNF Surrogate
markers ß 2 microglobulin
and Neopterin
Longitudinal study (At recruitment Two years,
every 6 months)
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Serial CD4 and viral load measurements (mean) in
plasma
No significant increase/decrease in viral loads
and/or CD4 Counts noted in patients over a two
year period
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Plasma cytokine levels in asymptomatic HIV
patients at recruitment and subsequent follow up
visits

• Threefold increase in IL-6 levels noted in plasma
  over a two year period
• Gradual increase in plasma IL-1 alpha levels
  noted in two years
• Sevenfold increase in plasma TNF-a levels noted
  in two years

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Mean Plasma protein levels in asymptomatic HIV
patients at recruitment and subsequent follow up
visits

• Twofold increase in plasma Neopterin levels noted
  in two years
• Gradual increase in plasma ß2 micro globulin
  levels noted in two years

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Declining levels of neutralizing antibodies to
HIV-1 envelope V3 peptide in the serum of
asymptomatic patient over 2 years

E Ratio OD value of sample / Cut-off OD value
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Summary of Longitudinal studies

• CD4 counts and plasma viral loads did not
  significantly differ over a two year period
• Levels of pro-inflammatory cytokines (IL-1?, IL-6
  and TNF?) and surrogate markers (ß 2 micro
  globulin and Neopterin) were found to steadily
  increase in the plasma over a two year period.
• This steady increase was statistically
  significant over the two year period thereby
  suggesting that these markers can indeed serve as
  good prognosticators of neurological involvement
  in HIV infection.
• Neutralizing antibody levels progressively
  declined over the two year period of follow up.

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Acknowledgements

• The National Institute of Health, Bethesda,
  Maryland, USA for the RO-1 grant.

JNCASR, Bangalore Dr Udaykumar Ranga
NARI, Pune Dr Srikanth Tripathy

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THANK YOU