HIV-1 conserved-element vaccines: relationship between sequence conservation and replicative capacity

Abstract

To overcome the problem of HIV-1 variability, candidate vaccine antigens have been designed to be composed of conserved elements of the HIV-1 proteome. Such candidate vaccines could be improved with a better understanding of both HIV-1 evolutionary constraints and the fitness cost of specific mutations. We evaluated the in vitro fitness cost of 23 mutations engineered in the HIV-1 subtype B Gag-p24 Center-of-Tree (COT) protein through fitness competition assays. While some mutations at conserved sites exacted a high fitness cost, as expected under the assumption that the most conserved residue confers the highest fitness, there was no overall strong relationship between sequence conservation and replicative capacity. By comparing sites that have evolved since the beginning of the epidemic to those that have remain unchanged, we found that sites that have evolved over time were more likely to correspond to HLA-associated sites and that their mutation had limited fitness costs. Our data showed no transcendent link between high conservation and high fitness cost, indicating that merely focusing on conserved segments of HIV-1 would not be sufficient for a successful vaccine strategy. Nonetheless, a subset of sites exacted a high fitness cost upon mutation--these sites have been under selective pressure to change since the beginning of the epidemic but have proved virtually nonmutable and could constitute preferred targets for vaccine design.

Fig 1

Alignment of HIV-1 p24-COT (Center-of-Tree) sequences. Conserved elements (CE) used in initial vaccine studies (33) are in boldface. Mutated sites evaluated in fitness assays are boxed, and the frequency of the consensus subtype B residue is indicated. Sites shown in red were associated with a fitness cost and those in blue with a higher relative fitness. Sites shown in black showed no difference in relative fitness, and mutations at sites in gray were regarded as lethal as they yielded no productive infection.

Fig 2

Replicative fitness in PBMC and CEM cells as a function of HIV-1 subtype B sequence conservation (database [Db] amino acid [AA] frequency of the consensus residue). Mean relative fitness values are reported for mutants of Gag-p24-COT-B: 22 mutants in PBMC and 20 mutants in CEM cells. (Two mutant viruses that were noninfectious are not depicted.) The lines represent a least-squares fit to the data. Filled symbols correspond to mutations at signature sites: i.e., sites that showed a change in amino acid frequency between the 1980s and the 2000s.

Fig 3

Sequence conservation over time and HLA-associated sites. The change in frequency between the consensus residue in the 1980s and 2000s is shown for all Gag-p24 sites (left panel) and for the 23 mutants of Gag-p24-COT-B assayed (right panel). Sites that have been previously identified as HLA associated and sites where no HLA association has been identified are compared.

Fig 4

Signature sites and replicative fitness. The relative fitness measured for 23 mutants of Gag-p24-COT-B is shown for signature sites (i.e., sites with a change in frequency between the 1980s and the 2000s) and nonsignature (nonsig.) sites. For the PBMC comparison, the P value in parentheses corresponds to all the data, including the zero value.