In vitro evolution of a T cell receptor with high affinity for peptide/MHC

T cell receptors (TCRs) exhibit genetic and structural diversity similar to antibodies, but they have binding affinities that are several orders of magnitude lower. It has been suggested that TCRs undergo selection in vivo to maintain lower affinities. Here, we show that there is not an inherent genetic or structural limitation on higher affinity. Higher-affinity TCR variants were generated in the absence of in vivo selective pressures by using yeast display and selection from a library of Vα CDR3 mutants. Selected mutants had greater than 100-fold higher affinity (KD ≈ 9 nM) for the peptide/MHC ligand while retaining a high degree of peptide specificity. Among the high-affinity TCR mutants, a strong preference was found for CDR3α that contained Pro or Gly residues. Finally, unlike the wild-type TCR, a soluble monomeric form of a high-affinity TCR was capable of directly detecting peptide/MHC complexes on antigen-presenting cells. These findings prove that affinity maturation of TCRs is possible and suggest a strategy for engineering TCRs that can be used in targeting specific peptide/MHC complexes for diagnostic and therapeutic purposes.

In vitro evolution of a highly replicating, doxycycline-dependent HIV for applications in vaccine studies

A major concern associated with the use of vaccines based on live-attenuated viruses is the possible and well documented reversion to pathogenic phenotypes. In the case of HIV, genomic deletions or mutations introduced to attenuate viral pathogenicity can be repaired by selection of compensating mutations. These events lead to increased virus replication rates and, eventually, disease progression. Because replication competence and degree of protection appear to be directly correlated, further attenuation of a vaccine virus may compromise the ability to elicit a protective immune response. Here, we describe an approach toward a safe attenuated HIV vaccine. The system is not based on permanent reduction of infectivity by alteration of important viral genomic sequences, but on strict control of replication through the insertion of the tetracycline (Tet) system in the HIV genome. Furthermore, extensive in vitro evolution was applied to the prototype Tet-controlled HIV to select for variants with optimized rather than diminished replication capacity. The final product of evolution has properties uniquely suited for use as a vaccine strain. The evolved virus is highly infectious, as opposed to a canonically attenuated virus. It replicates efficiently in T cell lines and in activated and unstimulated peripheral blood mononuclear cells. Most importantly, replication is strictly dependent on the nontoxic Tetanalogue doxycycline and can be turned on and off. These results suggest that this in vitro evolved, doxycycline-dependent HIV might represent a useful tool toward the development of a safer, live-attenuated HIV vaccine.

Long term trends in the evolution of H(3) HA1 human influenza type A

We have studied the HA1 domain of 254 human influenza A(H3N2) virus genes for clues that might help identify characteristics of hemagglutinins (HAs) of circulating strains that are predictive of that strain’s epidemic potential. Our preliminary findings include the following. (i) The most parsimonious tree found requires 1,260 substitutions of which 712 are silent and 548 are replacement substitutions. (ii) The HA1 portion of the HA gene is evolving at a rate of 5.7 nucleotide substitutions/year or 5.7 × 10−3 substitutions/site per year. (iii) The replacement substitutions are distributed randomly across the three positions of the codon when allowance is made for the number of ways each codon can change the encoded amino acid. (iv) The replacement substitutions are not distributed randomly over the branches of the tree, there being 2.2 times more changes per tip branch than for non-tip branches. This result is independent of how the virus was amplified (egg grown or kidney cell grown) prior to sequencing or if sequencing was carried out directly on the original clinical specimen by PCR. (v) These excess changes on the tip branches are probably the result of a bias in the choice of strains to sequence and the detection of deleterious mutations that had not yet been removed by negative selection. (vi) There are six hypervariable codons accumulating replacement substitutions at an average rate that is 7.2 times that of the other varied codons. (vii) The number of variable codons in the trunk branches (the winners of the competitive race against the immune system) is 47 ± 5, significantly fewer than in the twigs (90 ± 7), which in turn is significantly fewer variable codons than in tip branches (175 ± 8). (viii) A minimum of one of every 12 branches has nodes at opposite ends representing viruses that reside on different continents. This is, however, no more than would be expected if one were to randomly reassign the continent of origin of the isolates. (ix) Of 99 codons with at least four mutations, 31 have ratios of non-silent to silent changes with probabilities less than 0.05 of occurring by chance, and 14 of those have probabilities <0.005. These observations strongly support positive Darwinian selection. We suggest that the small number of variable positions along the successful trunk lineage, together with knowledge of the codons that have shown positive selection, may provide clues that permit an improved prediction of which strains will cause epidemics and therefore should be used for vaccine production.

Translocation events in the evolution of aminoacyl-tRNA synthetases.

We have characterized hisS, the gene encoding the histidyl-tRNA synthetase (HisRS) from the tetraodontoid fish Fugu rubripes. The hisS gene is about 3.5 kbp long and contains 13 exons and 12 introns of 172 bp, on average. The Fugu hisS gene encodes a putative protein of 519 amino acids with the three motifs identified as signatures of class 2 aminoacyl-tRNA synthetases. A model for the shifting of intron 8 between Fugu and hamster is proposed based on the successive appearance of a cryptic splicing site followed by an insertion mutation that created a new acceptor site. In addition, sequence comparisons suggest that the hisS gene has undergone a translocation through the first intron. As a result, the Fugu HisRS has an N-terminal sequence markedly different from that in the human and hamster enzymes. We propose that similar events have been responsible for variations at the N-terminal end of other aminoacyl-tRNA synthetases. Our analysis suggests that this involves exchanges through introns of two exons encoding an ancestral 32-amino acid motif.

Slip complexity in dynamic models of earthquake faults.

We summarize recent evidence that models of earthquake faults with dynamically unstable friction laws but no externally imposed heterogeneities can exhibit slip complexity. Two models are described here. The first is a one-dimensional model with velocity-weakening stick-slip friction; the second is a two-dimensional elastodynamic model with slip-weakening friction. Both exhibit small-event complexity and chaotic sequences of large characteristic events. The large events in both models are composed of Heaton pulses. We argue that the key ingredients of these models are reasonably accurate representations of the properties of real faults.

Chloroplast DNA evidence of colonization, adaptive radiation, and hybridization in the evolution of the Macaronesian flora.

Most evolutionary studies of oceanic islands have focused on the Pacific Ocean. There are very few examples from the Atlantic archipelagos, especially Macaronesia, despite their unusual combination of features, including a close proximity to the continent, a broad range of geological ages, and a biota linked to a source area that existed in the Mediterranean basin before the late Tertiary. A chloroplast DNA (cpDNA) restriction site analysis of Argyranthemum (Asteraceae: Anthemideae), the largest endemic genus of plants of any volcanic archipelago in the Atlantic Ocean, was performed to examine patterns of plant evolution in Macaronesia. cpDNA data indicated that Argyranthemum is a monophyletic group that has speciated recently. The cpDNA tree showed a weak correlation with the current sectional classification and insular distribution. Two major cpDNA lineages were identified. One was restricted to northern archipelagos--e.g., Madeira, Desertas, and Selvagens--and the second comprised taxa endemic to the southern archipelago--e.g., the Canary Islands. The two major radiations identified in the Canaries are correlated with distinct ecological habitats; one is restricted to ecological zones under the influence of the northeastern trade winds and the other to regions that are not affected by these winds. The patterns of phylogenetic relationships in Argyranthemum indicate that interisland colonization between similar ecological zones is the main mechanism for establishing founder populations. This phenomenon, combined with rapid radiation into distinct ecological zones and interspecific hybridization, is the primary explanation for species diversification.

The hypocone as a key innovation in mammalian evolution.

The hypocone, a cusp added to the primitively triangular upper molar teeth of therian mammals, has evolved convergently > 20 times among mammals during the Cenozoic. Acquisition of the hypocone itself involves little phenotypic change, but subsequent diversification of groups possessing the hypocone may be greatly enhanced. Our analysis of the Cenozoic mammalian radiations, including the Recent fauna, shows that high species diversity of mammals with hypocones and association of the hypocone with herbivory strongly support recognition of the hypocone as a key innovation that has allowed invasion of, and diversification within, herbivorous adaptive zones. In contrast, mammals lacking hypocones show no marked increase in species diversity during the Cenozoic.

Twenty-eight national elections for a Parliament in constant evolution. European Policy Brief No. 26, March 2014

In the City, the citizen is king. At least theoretically. In the European City currently being built around twenty-eight national democracies, the citizen will soon be called upon, in May, to democratically elect his or her representative in the European Parliament for the next five years. Since the very first election of Members of the European Parliament by direct universal suffrage in 1979, spectacular progress has been made by the “European Economic Community” that we now all know as the European Union. And the powers vested in citizen representatives are equally impressive. But there is a real possibility that European citizens will turn their backs on the upcoming European elections like never before. Why?

Toward 2000: opportunities in transportation evolution.

Mode of access: Internet.