A single amino acid substitution in the envelope protein of chimeric yellow fever-dengue 1 vaccine virus reduces neurovirulence for suckling mice and viremia/viscerotropism for monkeys.

A chimeric yellow fever-dengue 1 (ChimeriVax-DEN1) virus was produced by the transfection of Vero cells with chimeric in vitro RNA transcripts. The cell culture supernatant was subjected to plaque purification for the identification of a vaccine candidate without mutations. Of 10 plaque-purified clones, 1 containing no mutation (clone J) was selected for production of the vaccine virus. During subsequent cell culture passaging of this clone for vaccine production, a single amino acid substitution (K to R) occurred in the envelope (E) protein at residue 204 (E204) (F. Guirakhoo, K. Pugachev, Z. Zhang, G. Myers, I. Levenbook, K. Draper, J. Lang, S. Ocran, F. Mitchell, M. Parsons, N. Brown, S. Brandler, C. Fournier, B. Barrere, F. Rizvi, A. Travassos, R. Nichols, D. Trent, and T. Monath, J. Virol. 78:4761-4775, 2004). The same mutation was observed in another clone (clone E). This mutation attenuated the virus in 4-day-old suckling mice inoculated by the intracerebral (i.c.) route and led to reduced viremia in monkeys inoculated by the subcutaneous or i.c. route. The histopathology scores of lesions in the brain tissue of monkeys inoculated with either the E204K or E204R virus were reduced compared to those for monkeys inoculated with the reference virus, a commercial yellow fever 17D vaccine (YF-VAX). Both viruses grew to significantly lower titers than YF-VAX in HepG2, a human hepatoma cell line. After intrathoracic inoculation into mosquitoes, both viruses grew to a similar level as YF-VAX, which was significantly lower than that of their wild-type DEN1 parent virus. A comparison of the E-protein structures of nonmutant and mutant viruses suggested the appearance of new intramolecular bonds between residues 204R, 261H, and 257E in the mutant virus. These changes may be responsible for virus attenuation through a change in the pH threshold for virus envelope fusion with the host cell membrane.

Application of a very high-throughput digital imaging screen to evolve the enzyme galactose oxidase.

Directed evolution has become an important enabling technology for the development of new enzymes in the chemical and pharmaceutical industries. Some of the most interesting substrates for these enzymes, such as polymers, have poor solubility or form highly viscous solutions and are therefore refractory to traditional high-throughput screens used in directed evolution. We combined digital imaging spectroscopy and a new solid-phase screening method to screen enzyme variants on problematic substrates highly efficiently and show here that the specific activity of the enzyme galactose oxidase can be improved using this technology. One of the variants we isolated, containing the mutation C383S, showed a 16-fold increase in activity, due in part to a 3-fold improvement in K(m). The present methodology should be applicable to the evolution of numerous other enzymes, including polysaccharide-modifying enzymes that could be used for the large-scale synthesis of modified polymers with novel chemical properties.

Novel approaches for discovering industrial enzymes.

New technologies for enzyme discovery are changing the rules of the game for industrial biocatalysis. More kinds of enzymes are available, their hardiness is increasing, and their costs are coming down. These changes are the key drivers for a rebirth of interest in industrial applications of enzymes. The major enabling discovery approaches include screening of biodiversity, genomic sequencing, directed evolution and phage display.

Immunogenicity, genetic stability, and protective efficacy of a recombinant, chimeric yellow fever-Japanese encephalitis virus (ChimeriVax-JE) as a live, attenuated vaccine candidate against Japanese encephalitis.

Yellow fever (YF) 17D vaccine virus, having a 60-year history of safe and effective use, is an ideal vector to deliver heterologous genes from other medically important flaviviruses. A chimeric YF/Japanese encephalitis (JE) virus (ChimeriVax-JE virus) was constructed by insertion of the premembrane and envelope (prME) genes of an attenuated human vaccine strain (SA14-14-2) of Japanese encephalitis (JE) virus between core and nonstructural (NS) genes of a YF 17D infectious clone. The virus grew to high titers in cell cultures and was not neurovirulent for 3- to 4-week-old mice at doses /=10(3) pfu of ChimeriVax-JE virus were solidly protected against intraperitoneal challenge with a virulent JE virus. Genetic stability of the chimera was assessed by sequential passages in cell cultures or in mouse brain. All attenuating residues and the avirulent phenotype were preserved after 18 passages in cell cultures or 6 passages in mouse brains.

Effects of humanization by variable domain resurfacing on the antiviral activity of a single-chain antibody against respiratory syncytial virus.

HNK20 is a mouse monoclonal IgA that binds to the F glycoprotein of respiratory syncytial virus (RSV) and neutralizes the virus, both in vitro and in vivo. The single-chain antibody fragment (scFv) derived from HNK20 is equally active and has allowed us to assess rapidly the effect of mutations on affinity and antiviral activity. Humanization by variable domain resurfacing requires that surface residues not normally found in a human Fv be mutated to the expected human amino acid, thereby eliminating potentially immunogenic sites. We describe the construction and characterization of two humanized scFvs, hu7 and hu10, bearing 7 and 10 mutations, respectively. Both molecules show unaltered binding affinities to the RSV antigen (purified F protein) as determined by ELISA and surface plasmon resonance measurements of binding kinetics (Ka approximately 1x10(9) M-1). A competition ELISA using captured whole virus confirmed that the binding affinities of the parental scFv and also of hu7 and hu10 scFvs were identical. However, when compared with the original scFv, hu10 scFv was shown to have significantly decreased antiviral activity both in vitro and in a mouse model. Our observations suggest that binding of the scFv to the viral antigen is not sufficient for neutralization. We speculate that neutralization may involve the inhibition or induction of conformational changes in the bound antigen, thereby interfering with the F protein-mediated fusion of virus and cell membranes in the initial steps of infection.

Recombinant, chimaeric live, attenuated vaccine (ChimeriVax) incorporating the envelope genes of Japanese encephalitis (SA14-14-2) virus and the capsid and nonstructural genes of yellow fever (17D) virus is safe, immunogenic and protective in non-human primates.

Yellow fever 17D virus, a safe and effective live, attenuated vaccine, was used as a vector for genes encoding the protective antigenic determinants of a heterologous member of the genus Flavivirus, Japanese encephalitis (JE) virus, the leading cause of acute viral central nervous system infection and death throughout Asia. The viral envelope (prM and E) genes of a full-length cDNA clone of YF 17D virus were replaced with the corresponding genes of JE SA14-14-2, a strain licensed as a live, attenuated vaccine in China. Full-length RNA transcripts of the YF/JE chimaera were used to transfect Vero cells. The progeny virus (named 'ChimeriVax-JE'), was used to define safety after intracerebral (i.c.) inoculation of rhesus monkeys. Monkeys (N = 3) inoculated with a high dose (6.6 log10 pfu) developed a brief viremia, showed no signs of illness, developed high titers of anti-JE neutralizing antibody, and had minimal brain and spinal cord lesion scores according to criteria specified in the WHO monkey neurovirulence test. A control group of 3 monkeys that received a lower dose (4.2 log10 pfu) of commercial YF 17D vaccine had slightly higher lesion scores. To develop a lethal monkey model of JE for vaccine protection tests, we inoculated groups of monkeys i.c. or intranasally (i.n.) with a JE virus strain found to be highly neurovirulent and neuroinvasive for mice. Monkeys inoculated i.c., but not i.n., developed severe encephalitis after an incubation period of 8-13 days. The ChimeriVax-JE virus was passed in a cell line acceptable for human use (diploid fetal rhesus lung) and 4.3 or 5.3 log10 pfu were inoculated into groups of 3 monkeys by the subcutaneous route. All 6 animals developed brief viremias (peak titer < 2.0 log10 pfu/ml) and subsequently had anti-JE but no yellow fever neutralizing antibodies. On day 64, the monkeys were challenged i.c. with 5.5 log10 pfu of virulent JE virus. The immunized animals had no detectable viremia post-challenge, whereas 4 unimmunized controls became viremic. Only 1 of 6 (17%) vaccinated monkeys but 4 of 4 (100%) unvaccinated controls developed encephalitis. Histopathological examination 30 days after challenge confirmed that the protected, immunized animals had no or minimal evidence of encephalitis. These data demonstrated the ability of the ChimeriVax-JE to induce a rapid humoral immune response and to protect against a very severe, direct intracerebral virus challenge. Target areas of neuronal damage and inflammation in monkeys infected IC with wild-type JE, the chimaeric virus and YF 17D were similar, indicating that the histopathological scoring system used for the WHO yellow fever monkey neurovirulence test will be applicable to control testing of chimaeric seed viruses and vaccines.

Hydrogen bonding and circular dichroism of bacteriochlorophylls in the Rhodobacter capsulatus light-harvesting 2 complex altered by combinatorial mutagenesis.

We have investigated the spectroscopic properties of two classes of light-harvesting 2 (LH2, B800-850) mutants of Rhodobacter capsulatus obtained by combinatorial mutagenesis to the C-terminal half of the beta-apoprotein: a pseudoLH2 (pLH2) class, in which the 800-nm absorption was normal but the 850-nm peak was blue-shifted by up to 14 nm, and the other a pseudoLH1 (pLH1) class, which lacked the 800-nm absorption band and showed 850-nm absorption red-shifts of up to 30 nm. In several of the pLH1 antennae, carotenoid depletion contributed to the phenotype, while in the pLH2 complexes there was some carotenoid enrichment. A number of mutants from each class have also been characterized by low-temperature absorption and fluorescence spectroscopy, resonance Raman spectroscopy, and circular dichroism. In all of the mutants investigated, the B850 bacteriochlorophyll a binding site remained intact, conserving both the hydrogen bonding environment of the chromophores and their conformation and liganding. In contrast, the intensity of the CD spectra of pLH1 complexes was considerably reduced, relative to that of wild-type or pLH2 complexes, consistent with alterations in the interactions between pigments and in their relative orientation. Elevated fluorescence polarization over the red wing of the B850 band in the pLH2 complexes indicated a reduction of exciton mobility within the ring of BChl molecules. Possible structural alterations governing the spectral properties of the different mutants are discussed.

Context dependence of phenotype prediction and diversity in combinatorial mutagenesis.

Two different combinatorial mutagenesis experiments on the light-harvesting II (LH2) protein of Rhodobacter capsulatus indicate that heuristic rules relating sequence directly to phenotype are dependent on which sets or groups of residues are mutated simultaneously. Previously reported combinatorial mutagenesis of this chromogenic protein (based on both phylogenetic and structural models) showed that substituting amino acids with large molar volumes at Gly beta 31 caused the mutated protein to have a spectrum characteristic of light-harvesting I (LH1). The six residues that underwent combinatorial mutagenesis were modeled to lie on one side of a transmembrane alpha-helix that binds bacteriochlorophyll. In a second experiment described here, we have not used structural models or phylogeny in choosing mutagenesis sites. Instead, a set of six contiguous residues was selected for combinatorial mutagenesis. In this latter experiment, the residue substituted at Gly beta 31 was not a determining factor in whether LH2 or LH1 spectra were obtained; therefore, we conclude that the heuristic rules for phenotype prediction are context dependent. While phenotype prediction is context dependent, the ability to identify elements of primary structure causing phenotype diversity appears not to be. This strengthens the argument for performing combinatorial mutagenesis with an arbitrary grouping of residues if structural models are unavailable.

Red-shifted excitation mutants of the green fluorescent protein.

Using optimized combinatorial mutagenesis techniques and Digital Imaging Spectroscopy (DIS), we have isolated mutants of the cloned Aequorea victoria green fluorescent protein (GFP) that show red-shifted excitation spectra similar to that of Renilla reniformis GFP. Selective excitation of wild-type versus Red-Shifted GFP (RSGFP) enables spectral separation of these proteins. Six contiguous codons spanning the tyrosine chromophore region were randomized and sequence analysis of the mutants revealed a tyrosineglycine consensus. These mutants will enable the simultaneous analysis of two promoters or proteins per cell or organism. In consideration of the multitude of applications which are developing for GFP alone, we envisage that spectrally shifted fluorescent proteins will be of value to a diversity of research programs, including developmental and cell biology, drug-screening, and diagnostic assays.

Searching sequence space to engineer proteins: exponential ensemble mutagenesis.

We describe an efficient method for generating combinatorial libraries with a high percentage of unique and functional mutants. Combinatorial libraries have been successfully used in the past to express ensembles of mutant proteins in which all possible amino acids are encoded at a few positions in the sequence. However, as more positions are mutagenized the proportion of functional mutants is expected to decrease exponentially. Small groups of residues were randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins. By using optimized nucleotide mixtures deduced from the sequences selected from the random libraries, we have simultaneously altered 16 sites in a model pigment binding protein: approximately one percent of the observed mutants were functional. Mathematical formalization and extrapolation of our experimental data suggests that a 10(7)-fold increase in the throughput of functional mutants has been obtained relative to the expected frequency from a random combinatorial library. Exponential ensemble mutagenesis should be advantageous in cases where many residues must be changed simultaneously to achieve a specific engineering goal, as in the combinatorial mutagenesis of phage displayed antibodies. With the enhanced functional mutant frequencies obtained by this method, entire proteins could be mutagenized combinatorially.

Recursive ensemble mutagenesis.

We have developed a generally applicable experimental procedure to find functional proteins that are many mutational steps from wild type. Optimization algorithms, which are typically used to search for solutions to certain combinatorial problems, have been adapted to the problem of searching the 'sequence space' of proteins. Many of the steps normally performed by a digital computer are embodied in this new molecular genetics technique, termed recursive ensemble mutagenesis (REM). REM uses information gained from previous iterations of combinatorial cassette mutagenesis (CCM) to search sequence space more efficiently. We have used REM to simultaneously mutate six amino acid residues in a model protein. As compared to conventional CCM, one iteration of REM yielded a 30-fold increase in the frequency of 'positive' mutants. Since a multiplicative factor of similar magnitude is expected for the mutagenesis of additional sets of six residues, performing REM on 18 sites is expected to yield an exponential (30,000-fold) increase in the throughput of positive mutants as compared to random [NN(G,C)]18 mutagenesis.