Our results demonstrate that the association between HLA molecules conferring slow HIV-1 disease progression and CTL responses to HIV-1 Gag is due to an intrinsic preference of these HLA alleles for peptides from the p24 protein, and not merely a side-effect of the high immunogenicity of the Gag protein. Several recent studies have also established a link between low HIV viral loads and CTL responses against Gag or p24 
. Bailey et al. 
reported that CTL responses in elite suppressors of HIV infection focus on HLA-B57 restricted Gag epitopes. Moreover, a significant negative correlation between the magnitude of the CD8 T cell response and HIV-1 viral load was only found for responses against p24 and not for responses against other HIV-1 proteins 
. When we performed a partial correlation on data published by Frahm et al. 
, who determined CTL responses to a large panel of HIV-1 peptides spanning all HIV-1 proteins in 150 infected individuals, we found that the number of CTL responses directed against p24 correlated positively with the CD4+
T cell count (p
0.04) and negatively with viral load (p
0.01), while the number of CTL responses against Nef correlated positively
with viral load (p
0.03) and not with the CD4+
T cell count (p
0.96) (unpublished results). Interestingly, the main HIV-1 specific CTL responses in chimpanzees, which have a low viral load and do not develop AIDS, have been found to be directed against the very same peptides from p24 that are targeted by human individuals with HLA-B57 or B27, who tend to be long-term non-progressors 
. All these data suggest that preferential targeting of p24 delays disease progression. However, previous studies could never rule out the possibility that the apparent preference for p24 was a side-effect of the high immunogenicity of p24, causing immune responses to p24 to be better maintained than other responses in individuals with low viral load. Our analyses based on HLA-peptide binding predictions conclusively show that HLA alleles associated with slow HIV disease progression have an intrinsic preference for peptides from the HIV p24 protein. Our results thereby suggest that the class I restricted CTL immune response, particularly against p24, plays a key role in controlling HIV-1 infection.
Why then is preferential targeting of p24 beneficial? The first reason that comes to mind is that p24 is one of the most functionally and structurally constrained proteins of HIV. P24 contains a stretch of 20 amino acids which is conserved across retroviruses, and is essential for viral assembly, maturation, and infectivity 
. This region contains a B14 epitope (a low RH allele), and Wagner et al. 
found that all mutations abrogating the CTL response to this epitope drastically reduced the replication capacity of the virus. In line with this, we found several HIV epitopes that are presented by protective HLA molecules to be located at the dimer interface of p24, which is expected to be sensitive to mutations. The peptide binding motifs of HLA alleles with low and high relative hazards () also suggest that low RH alleles tend to present peptides that are more sensitive to mutations, because they prefer tryptophan (W) at position 9. Tryptophan is the only amino acid coded by a single triplet. Any mutation in a tryptophan triplet will thus lead to an amino acid substitution or a stop-codon. Due to the unusual side chain properties of tryptophan, such amino acid substitutions tend to affect the protein structure and function. As a consequence, tryptophan is the most conserved amino acid. HLA alleles with a high RH, on the other hand, prefer proline (P) at position 2, which is coded by 4 triplets, and is known to be less conserved than tryptophan. HLA alleles with a low RH thus seem to preferentially bind the parts of the HIV genome that are most sensitive to mutations. This may in part explain why they confer better protection against disease progression.
Since HIV-1 proteins other than p24 may also contain functionally and structurally constrained regions, we investigated if there is a general correlation between the relative hazard of HLA alleles and the tendency to target constrained parts of the HIV-1 proteome. We predicted the HIV-1 epitopes for a large number of HLA alleles and used the entropy of each predicted epitope as a measure of functional and structural constraint. The Shannon entropy 
of each residue in the HIV-1 proteome was calculated using the HIV-1 protein alignments for clade B available in the Los Alamos HIV database (September 2005). Surprisingly, the average entropies of the three predicted best-binding epitopes for HLA alleles with a low RH were not significantly different from the ones presented by HLA alleles with a high RH (Mann-Whitney, p
0.87, unpublished results). There was also no significant correlation between the entropy score of the best-binding predicted epitopes and the RH of the HLA presenting the epitope (p
0.79, unpublished results). Presentation of conserved HIV epitopes is thus not explaining the difference between HLA alleles associated with slow and rapid disease progression.
There are two non-mutually exclusive explanations for this surprising finding. The first is that targeting conserved epitopes is required but not sufficient to delay disease progression (see also 
). Apart from its conservedness, p24 may have other properties that explain why CTL responses against p24 are most beneficial. Possible factors include the fact that p24 is one of the most immunogenic and abundant HIV proteins. An immature HIV particle contains approximately 1500 copies of p24, while other HIV proteins are present at much lower copy numbers 
. P24 epitopes are therefore expected to induce stronger immune responses than other HIV epitopes. Additionally, it was recently shown that p24 can be detected within two hours after the infection of a target cell, which is well before other HIV-1 proteins are produced, and before Nef can down-regulate HLA expression 
. The origin of the early expressed p24 is probably the large amount of p24 packaged in viral particles 
. Preferential targeting of conserved epitopes from an early and abundantly expressed, highly immunogenic protein, may hence be the clue to slowing down disease progression.
A second explanation is that entropy is not the correct measure of functional and structural constraints. Indeed, peptides that are hardly constrained may nevertheless have a low entropy if there is no strong CTL pressure on the peptide, or if the HLA molecule by which the peptide is restricted is very rare in the human population. Conversely, peptides may have a high entropy despite functional or structural constraint if the CTL pressure on the peptide is so high that the peptide mutates despite a high viral fitness cost 
. The latter is exactly what has been described for p24 epitopes targeted by elite suppressors of HIV-1 
. Despite the lack of correlation between the relative hazard of HIV disease progression and the tendency to present peptides with low entropy, preferential targeting of functionally and structurally constrained regions of HIV-1 may thus be key to slowing down disease progression.
HLA-B27 was the first HLA that was described to be associated with slow HIV-1 disease progression 
. Our analyses reveal an important difference between HLA-B27 on the one hand and HLA-B57 and B58 on the other. While the latter two HLA alleles target at least 3 different p24 peptides with high affinity, HLA-B27 has only one good-binding p24 peptide (see ). It has been reported that CTL escape of this epitope caused a rapid increase of viremia in an HLA-B27-positive long-term non-progressing child 
. Our analyses suggest that this abrupt breakage of protection may be caused by the absence of other protective anti-p24 CTL responses.
The differences between HLA alleles associated with slow and rapid disease progression have also been sought in their frequencies in the human population. Trachtenberg et al. 
demonstrated a significant correlation between the population frequency of HLA supertypes 
and the HIV-1 viral load at set point in a large group of HIV-1 infected homosexual men. Scherer et al. 
extended these results by showing that common HLA alleles are associated with a lack of CTL responses to known HIV-1 epitopes, further supporting the idea that HIV-1 is adapting to the most common HLA molecules in the human population. Frahm et al. 
demonstrated that an HLA molecule associated with a low viral load in a population in which the HLA is rare, lacked this association in another population where the HLA is common. Data are conflicting, however, because no significant correlation could be found between the relative hazard of HIV disease progression and the population frequency of HLA molecules 
Whatever the effect of the population frequency of HLA molecules on the rate of HIV disease progression is, the current study shows that qualitative differences in the epitopes targeted by different HLA molecules contribute to the association between HLA molecules and the rate of HIV-1 disease progression.