HIV-1 Conserved-Element Vaccines: Relationship between Sequence Conservation and Replicative Capacity

Abstract

bTo 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.

DNA Prime-Boost Vaccine Regimen To Increase Breadth, Magnitude, and Cytotoxicity of the Cellular Immune Responses to Subdominant Gag Epitopes of Simian Immunodeficiency Virus and HIV

“…To address the problem of viral variability, many approaches have been tested including strategies that use consensus, center-of-tree or ancestral sequences, multiple strains or mosaic immunogens, immunogens consisting of known epitopes, and chimeric molecules expressing a selection of the most conserved epitopes from different clades of HIV . In our approach (20)(21)(22), we designed a plasmid DNA (pDNA) vaccine to focus the immune response to conserved elements (CE) of the HIV-1 proteome based on stringent conservation, broad HLA-coverage, and association with HIV control (19,(23)(24)(25). The p24…”

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

“…To study the fitness impact of single amino acid changes in HIV-1 Gag-p24, we used oligonucleotide directed mutagenesis to introduce mutations into a pNL4-3 plasmid containing the HIV-1 COTB-p24 gene 23,32,33 . Viral stocks were generated by transfection of 293T cells and harvested after 48 hr.…”

“…Another alternative is the use of cell lines. The protocol presented here was used successfully with the T-cell line CEMx174 23,36 . However, it is important that the numbers of cells seeded are re-optimized to achieve consistent cell growth.…”

Gag and env conserved element CE DNA vaccines elicit broad cytotoxic T cell responses targeting subdominant epitopes of HIV and SIV Able to recognize virus-infected cells in macaques

“…During primary infection in humans the reduction of the original viral burst is associated with the emergence of HIV-specific cytotoxic CD8 + T lymphocytes (CTL). 19 , 33 , 34 , 39–53 Their role in controlling viremia was confirmed by systemic CD8 depletion in SIV/SHIV infected macaques, which resulted in a rapid increase in viremia, 54–57 demonstrating a CD8-dependent immunological mechanisms of viral control. 58–67 Failure to contain viral replication in macaques correlated with the emergence of viral mutants that escaped from CTL immune surveillance.…”

“…20 , 21 , 73 Seven CE, spanning 12 to 24 AA in length, were selected based on their stringent conservation across all known HIV sequences, broad HLA-coverage, and association with HIV control. 19 , 53 , 74 , 75 By positional analogy and sequence homology to the HIV p24CE, an SIV p27 Gag (p27CE) derived molecule was generated. 76 Using a similar approach based on sequence conservation, association with virologic control, and CTL escape resulting in a loss in viral fitness, we generated HIV Env CE molecules 77 comprising 12 highly conserved regions of Env, spanning 11 to 43 AA in length.…”