Supplemental Data
Table 1. dN/dS analysis.
Fig. 1. Predicted amino acid variation in the 5' exon of Tat of Mamu-A*01
negative macaques. Individual clones spanning the Mamu-A*01-restricted SL8
epitope (highlighted in yellow) were sequenced and aligned to the Genbank
sequence for SIVmac239 (accession number M33262). Viral clones derived from
Mamu-A*01 negative animals 8 weeks post-infection. Few amino acid
replacements accumulate within the Mamu-A*01-restricted CD8 positive T
lymphocyte epitope. However, tightly clustered substitutions elsewhere in Tat
may represent selection against other non-A*01-restricted CTL responses (shown
in gray scale).
Fig. 2. Sequence of entire virus at four weeks post-infection by both bulk and
clonal analysis (only 96114) in two Mamu-A*01 positive animals (96114 and
96118).
A. Strategy used to amplify entire virus.
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B. Primers used for virus amplification.
C. Sequencing of virus at 4 weeks post-infection reveals few changes from the
SIVmac239nef open inoculum. RT-PCR products from animals 96114 and 96118
that spanned the entire viral genome at four weeks post-infection were directly
sequenced (bulk). In addition, 6-10 cDNA clones were isolated and sequenced
from each RT-PCR product from animal 96114. The amino acid sequences were
aligned against the consensus sequence for SIVmac239nef open. Genes that
spanned more than 1 template had different sets of clones for each RT-PCR
product. The Mamu-A*01-restricted CD8 positive T lymphocyte epitope in Tat2835
STPESANL is noted in yellow shading.
Fig. 3 ELISPOT reveals CD8 positive T lymphocyte responses to Tat in three
Mamu-A*01 negative animals, suggesting that Tat is an important target for early
CD8 positive T lymphocyte responses. ELISPOT responses were assessed against
overlapping 15-mer or 20-mer peptides spanning regions of the Tat protein.
Freshly isolated or frozen PBMC were plated in 96-well plates at 2.0 x 105
cells/well and stimulated with either 15-mers or 20-mer peptides from Tat, left
unstimulated (negative control) or stimulated with 10µg/ml concanavalin A
(positive control). Samples were tested in triplicate and at peptide
concentrations of 10µM. Fresh cells were incubated for 16 hours in the assay,
frozen cells were incubated for 48 hours. Background responses (as indicated by
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the dashed line) were calculated by adding five to twice the number of Spot
Forming Cells (SFCs) from the control wells containing cells. Mean values and
standard deviations from triplicate wells were calculated. Yellow bars indicate
those responses where a minimum of 5 spots were detected above background
and the responses was significant according to Student’s T-test ( p < 0.05).
Fig. 4. Sliding window analysis of dN and dS in all animals. Sliding window
analysis of vaccinated Mamu-A*01 positive animals (a), naïve Mamu-A*01
animals (b and c) and naïve non-Mamu-A*01 positive animals (d-k). dS (number
of synonymous substitutions per synonymous site) and dN (number of
nonsynonymous substitutions per nonsynonymous site) in a sliding window of
eight codons were plotted against they codon position at the start of the window.
Fig. 5. Naïve animals were divided into two groups dependent on evidence for
high dN in Tat (Fig. 4). Viral loads were then averaged at 2, 4 and 8 weeks postpeak viremia (peak viremia occurred at 2 weeks in animals infected with
SIVmac239 Nef open and 4 weeks in animals infected with SIVmac239 Nef stop).
Animals with higher dN in Tat had lower plasma viral concentration after peak
viremia. There were no statistically significant differences between groups in
terms of peak levels regardless of how the data were analyzed (p > .70). The
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group difference in post-peak viremia levels was clearly significant (p=.008). At 2
weeks post-peak viremia levels, animals with high dN had lower viral loads than
those with low dN (2-tailed p=0.011 by t-test; p=0.019 by non-parametric MannWhitney U test). At 4 weeks post-peak viremia , the high dN animals also had
lower plasma virus concentrations than the low dN animals (2-tailed p=0.001 by
t-test; p=0.011 by non-parametric Mann-Whitney U test). Finally, at 8 weeks postpeak viremia, the high dN animals had lower plasma virus concentrations than
the low dN animals, but the p-values were of marginal significance (2-tailed
p=.077 by t-test (.180 with Welch correction); p=.088 by non-parametric MannWhitney U test).
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Table 1. Mean number of synonymous (dS) and nonsynonymous (dN) nucleotide substitutions
per 100 sites in comparisons of Tat/Vpr protein regions demonstrate evidence for selection.
Epitope
Remainder
dS (±s.e.m)
dN(±s.e.m)
dS(±s.e.m)
dN(±s.e.m)
0.2 ± 0.2
1.1 ± 0.5
0.5 ± 0.2
0.3 ± 0.1
0.5 ± 0.5
0.9 ± 0.3
0.6 ± 0.2
0.4 ± 0.1
0.4 ± 0.4
5.7 ±
0.4 ± 0.1
0.1 ± 0.0
0.4 ± 0.1
0.2 ± 0.1
Tat reading
frame
Mamu- A*01-
versus
negative
inoculum
within
sample
Mamu-A*01-
versus
positive
inoculum
within
0.4***†††
0.7 ± 0.7
sample
7.3 ±
1.0***†††
In the Tat reading frame, the epitope region encompasses the 8 codons aligned with the Tat28-35 STPESANL
epitope.
Paired sample t-tests of the hypothesis that mean dS = mean dN: *** P < 0.001.
T-tests of the hypothesis that mean dS or mean dN in Mamu-A*01-positive animals equals the corresponding
value in Mamu-A*01-negative animals: ††† P < 0.001.