Inserting foreign peptides into the major coat protein of bacteriophage M13.

Foreign DNA fragments were inserted into filamentous phage gene VIII to create hybrid B-proteins with foreign sequences in the amino terminus. The hybrid proteins are incorporated into the virions which retain viability and infectivity. Virions with hybrid B-proteins have the same contour length and the same number of B-protein molecules as virions with natural B-proteins. It was shown that for one of hybrid B-proteins the position of the processing site had changed.

Design of immunogens as components of a new generation of molecular vaccines.

Three new approaches to design effective immunogens are considered. At first, we derived an expression vector from bacteriophage M13 allowing the exposure of short peptides on the virion surface. EIA demonstrates that antibodies against a recombinant phage carrying the antigenic determinant of the HIV-1 gag protein reacted with the 17-kDa core protein of the virus and also with its polyprotein precursor p55 in immunoblotting. In another approach, we chose the hepatitis B core antigen (HBcAg) particle as a vehicle for the presentation of foreign antigenic determinants to the immune system. Chimerical particles of HBcAg containing epitope of the VEE virus were obtained. A vector system for insertion of foreign antigenic determinants and production of both hybrid and wild HBcAg proteins were also obtained. The third approach relies on construction of immunogens from different T- and B-cell epitopes of the HIV-1. We suggested to construct HIV-1 vaccines in a form of the TBI (T- and B-cell epitopes containing Immunogen) with a predetermined tertiary structure, namely, a four-alpha-helix bundle. The gene of the TBI protein consisting of nine HIV-1 epitopes was synthesized and expressed in Escherichia coli cells. Mice immunized with TBI showed humoral and cellular immune responses to HIV-1. Anti-TBI antibodies displayed HIV-1 neutralizing activity. These new approaches offer promise in the development of new effective vaccines.

[Amino acid similarity matrix for detecting the antigenically similar sequences in non-related proteins]. / Matritsa skhodstva aminokislot dlia poiska antigenno blizkikh fragmentov v nerodstvennykh belkakh.

A special matrix of amino acid antigenic similarity for computer detection of the potential antigenic proximity of unrelated proteins is proposed. The matrix was built using the data concerning affinities of amino acid residue interactions between subunits in oligomeric proteins. The diagonal elements of the matrix characterize the recognition of amino acid residues and the non-diagonal ones represent the relative similarity measure of antibody--amino acid residue interactions specificity. The application of the new matrix for comparing proteins allows the hydrophilic potentially immunologically active regions of sequences to be picked out as similar fragments. When the influenza virus hemagglutinin was compared with 116 human proteins, eight fragments were picked out, that could not be determined by means of the routinely used MDM78 matrix. The antigenic similarity matrix for defining the forbidden structures is proposed to be used for preparing the peptidic antiviral vaccines.

[The nature of amino acid substitution in antigenic drift of hemagglutinin N3 and neuraminidase N2 from the influenza virus]. / Izuchenie kharaktera aminokislotnykh zamen v antigennom dreife gemaggliutinina H3 i neiraminidazy N2 virusa grippa.

The nature of amino acid replacements in 16 drift variants of hemagglutinin H3 subtype and 5 drift variants of neuraminidase N2 subtype of the influenza A virus were studied. The dependences of relative replacement frequencies and relative quantities of frequent replacements upon differences of properties of substituted residues are plotted. In contrast to most of the known proteins, amino acid replacements in hemagglutinin and neuraminidase depend weakly on the physico-chemical parameters of amino acids. For the antigenic determinants studied the replacement frequencies were compared to those calculated according to two models: one for conservative replacements and the other for accidental mutation of the genetic code. The differences in the nature of amino acid replacements are found in four antigenic determinants of hemagglutinin. The replacements in experimentally selected proteins are shown to go beyond limitations of natural variants. The explanations of the reasons of low epidemicity of some strains and ineffective attempt to imitate the natural antigenic drift of viruses by using experimental selection are proposed. The causes of time-limited circulation of H3N2 influenza virus subtype are discussed.

[A method of calculatiing immunochemical cross-reactions between homologous proteins]. / Metod rascheta immunokhimicheskogo perekresta mezhdu gomologichnymi belkami.

The effects of amino acid substitutions within the antigenic sites, within the residues close to these sites and within other parts of the molecule on the cross-reaction with the antisera to homologous protein were considered. The method for calculus the values of cross-reactions, based on using primary structures data, X-ray coordinate of one of the homologues and the locations of the antigenic sites is proposed. The values obtained by this method for the cross-reactions of ten myoglobins from various species with antisperm-whale myoglobin sera have a good correlation with the known experimental data. The possibility of using the method to make the location of protein antigenic sites more precise is discussed.

[Design of four-helix protein--a possible vaccine against human immunodeficiency virus (HIV-1)]. / Konstruirovanie chetyrekhspiral'nogo belka--vozmozhnoi vaktsiny protiv virusa immunodefitsita cheloveka (HIV-1).

Successful approach to the development of safe and effective synthetic vaccines requires that different B- and T-cell epitopes of the infectious agent be included into the vaccine construction. It is suggested that vaccines should be constructed as proteins with both optimal epitope composition and predetermined tertiary structure. Based on analysis of B-cell and T-cell epitope properties, a possibility to use one well-known protein spatial motif--four-alpha-helix bundle--for vaccine construction is substantiated. Antigenic determinants of cellular immunity (amphipathic alpha-helices) and humoral immunity (flexible hydrophilic loop regions) can be used as blocks for vaccine design. Nonloop B-epitopes and nonhelical T-epitopes may be introduced in the protein N- and C-terminal regions. General principles of PTS-vaccine construction have been applied to anti-HIV-1 vaccine design. Experimentally studied T- and neutralizing B-cell epitopes from HIV-1 proteins were analyzed. The sequence of one possible four-alpha-helix protein vaccine has been constructed. Predicted secondary structure and T- and B-cell epitopes of this protein coincided with the planned ones. The amino acid composition of the protein was found to be consistent with the composition of globular water-soluble proteins. The gene of the protein with codon composition optimal for expression in E. coli has been synthesized. The advantages and limitations of this approach to vaccine design are discussed.

[Development of rules for vaccine engineering based on the variability of peptide fragments in closely related proteins]. / Razrabotka pravil dlia vaktsinnoi inzhenerii na osnove analiza variabel'nosti peptidnykh fragmentov v blizkorodstvennykh belkakh.

Based on the protein sequence data bank (PIR), the "variable fragment" bank, comprising pairs of closely related proteins, containing one or more strongly differing sites of primary structures was formed. The bank includes 465 "variable fragments" of 383 protein pairs. Amino acid residues composition of "variable fragments" was examined and indexes of potential amino acid residues variability was formed. An analysis of amino acid fragments replaceability was carried out by substituting the N-, C-terminal, or middle part of a chain), the fragments length differences and physico-chemical properties of residues, such as volume, hydrophobicity, polarity, isoelectric point, etc. Some general empirical rules of peptide insertions in carrier-proteins were created based on these analyses. The rules are directed for performing modifications maintaining the common structure and function of the carrier-protein molecule. The selection scheme for determining the regions suitable for modification and the criteria for defining the width of acceptable modifications in this regions were suggested. The use of potential variability profile for detecting regions suitable for peptide insertion was considered on the model of hepatitis B surface protein.

[Directed reconstruction of the influenza virus hemagglutinin gene]. / Napravlennaia rekonstruktsiia gena gemaggliutinina virusa grippa.

A new approach to create chimeric genes by directed exchange of oligonucleotide fragments was developed. By oligonucleotide-directed mutagenesis a few deletion mutants of the influenza virus hemagglutinin (HA) gene were obtained. These variants of HA gene contain unique restriction sites in DNA regions coding for the A and B epitopes of the HA molecule. The obtained special vectors may be used for cloning DNA fragments coding for new amino acid sequences in internal sites of the HA gene.

[Preparation of a mutant human tumor necrosis factor with increased resistance to proteases]. / Poluchenie mutantnogo faktora nekroza opukholei cheloveka s povyshennoi ustoichivost'iu k proteazam.

The potential proteolysis sites of human TNF are considered. By site-directed mutagenesis the Arg-31 residue of mature TNF was substituted by Gln. The analysis of cytotoxicity of initial and mutant (R31Q) proteins on mouse L929 fibroblasts did not reveal any differences in biological activity. For the mutant protein a change in proteolysis dynamics was shown in contrast to the natural variant: mutant TNF displayed increased stability when treated with trypsin.

[The use of filamentous phage M13 in protein engineering]. / Ispol'zovanie nitchatogo faga M13 dlia belkovoi inzhenerii.

M13B1 vector based on the filamentous phage M13 has been constructed. M13B1 phage carries the gene of resistance to ampicillin and contains the unique site of recognition for BamHI restriction endonuclease in gene VIII coding for the major coat protein. BamHI restriction site has been inserted into the gene of the major coat protein by means of oligonucleotide directed mutagenesis. The synthetic DNA fragment coding for the model peptides has been inserted through BamHI site into the M13B1 DNA. The possibility of inserting foreign peptides into the N-terminus at maintaining the viability of hybrid phages has been shown. The differences in specificity of the recombinant phage maturation have been determined by analysing the amino acid sequence of B-protein.

[Construction and expression in Escherichia coli of a gene of hybrid hemagglutinin H1-H3 of influenza virus]. / Konstruirovanie i ékspressiia v Escherichia coli gena gibridnogo gemaggliutinina podtipa H1-H3 virusa grippa.

The hybrid gene of influenza virus hemagglutinin (HA) of the H1-subtype, carrying the sequence coding for the fragment of H3-subtype antigenic site B, was constructed. The product of expression of this gene in E. coli was obtained as a fusion protein with beta-galactosidase. The chimeric protein was shown to retain the antigenic properties of HA of H1-subtype and to interact specifically with antibodies against the synthetic peptide corresponding to the B site fragment of HA of the H3-subtype.

[Primary structure of the mutant genes of human leukocyte interferon alpha2]. / Opredelenie pervichnoi struktury mutantnykh genov leikotsitarnogo interferona alpha2 cheloveka.

Nucleotide sequences of 10 mutant genes of human leukocyte interferon alpha 2 (IFN) with the use of 4 oligonucleotide primers containing ethyl substituents at phosphate groups were determined. To design primer sequences, an approach based on the local similarity profile of the IFN gene and M13mp7 vector DNA is described.

[Construction of original artificial immunogens--candidate vaccines against HIV-1]. / Konstruirovanie original'nykh iskusstvennykh immunogenov--kandidatov v vaktsiny protiv HIV-1.

An original method for making effective artificial vaccines has been developed. Immunogens are virus-like particles containing a DNA molecule covered with recombinant proteins carrying the pathogen epitopes. The recombinant proteins are exposed on the surface of the particle and are attached to DNA via spermidine-polygluquine-glutathione or galactopyranoside conjugates (depending on the hybrid protein composition). Two variants of artificial immunogens-candidates for antiHIV vaccine have been prepared.

[Cloning proviral DNA sequences of the human T-cell leukemia virus (HTLV-1) from the genome of cultured MT-4 cells]. / Klornirovanie provirusnykh DNK-posledovatel'nostei virusa T-kletochnogo leikoza cheloveka tipa 1 (HTLV-1) iz genoma kul'tury kletok MT-4.

MT-4 cell line is a continuous strain of human T lymphocytes expressing defective noninfective subviral HTLV-1 particles. A fragment of sequence encoding the p24 protein and gene for envelope protein (env) have been obtained from genomic DNA of this culture by polymerase chain reaction. Both HTLV-1 fragments were cloned in bacterial vectors, and the nucleotide sequence of these regions was determined. The cloned DNA fragment encoding the p24 has only four point nucleotide exchanges. Analysis of the env gene structure revealed that the sequence had several amino acid exchanges and two deletions (13 bp and 70 bp).

[Use of a phage peptide library in mapping the group-specific hemagglutinating domain of alpha-virus glycoprotein E2]. / Ispol'zovanie fagovoi biblioteki peptidov v kartirovanii gruppospetsificheskogo gemaggliutiniruiushchego domena glikoproteina E2 al'fa-virusa.

Phage display peptide library f88-4/15 (G. P. Smith, USA) was used for mapping the hemagglutination activity domain of glycoprotein E2 of alphaviruses. Using affinity selection and ELISA, we selected the clones binding monoclonal antibody 4H5 to Venezuelan equine encephalomyelitis virus and inhibiting alphavirus hemagglutinating activity. Analysis of the similarity between the peptides amino acid sequences with the alphavirus glycoprotein E2 sequences revealed a structural motive of 4 amino acid residues (HTSR) which was identified in the 85-88 region. Bacteriophages F36 and F19 contained motives corresponding to 102-SXXM-105 and 109-AXXP-112 regions in alphavirus proteins E2. These data permit us to propose that the detected regions are fragments of a group-specific alphavirus hemagglutination domain.

[Structural and physico-chemical properties of amino acids of functionally important regions of proteins]. / Strukturnye i fiziko-khimicheskie svoistva aminokislot funktional'no vazhnykh uchastkov belkov.

A correlation of the localization of functionally important regions with places having low and high values on twelve profiles built on a basis of amino acid sequences was analysed using a broad set of proteins. The profiles of hydrophilicity, resemblance to the sequences of human proteins, flexibility, mutability and others were considered. The resemblance profile was plotted by the program fixing short similar fragments in the testing protein and 92 human ones. The active centres were shown to be located in the primary structure regions having relatively low values on the resemblance profiles. Similar effect was observed in the mutability and alpha-helicity profiles. The potential functionally important sites of the human leukocyte interferon and interleukin-2 isolated on the basis of the analysis of this profiles were in accord with the available literary data.

Mutations in fd phage major coat protein modulate affinity of the displayed peptide.

Multibillion-clone libraries of phages displaying guest peptides fused to the major coat protein pVIII (landscape libraries) are a rich source of probes for proteinaceous and non-proteinaceous targets. As opposed to the pIII-type fusion phages, which display peptides as independent structural domains, the guest peptides in the pVIII-fusion phages can be structurally and functionally influenced by contiguous subunits. To decipher the impact of the locale of a guest peptide on its affinity characteristics, we constructed a library of phages carrying beta-galactosidase-binding peptide ADTFAKSMQ at the N-terminus of the pVIII protein surrounded by random amino acids. It was found that mutagenesis of amino acids 12-19 (domain C) has polar effects on target binding affinity of the displayed peptide. The phages with highest affinity are characterized by: (i) a net electrostatic charge around -1 of domain C of the mutated phages at pH 7.0; (ii) a lower radius of cylinder coaxial to alpha-helix formed by domain C; (iii) a lower higher occupied molecular orbital (HOMO) of domain C leading to a decreased formation of hydrogen bonds and (iv) positively charged surface and torsion energy of domain C, which may require a conformational transition of N-terminal peptide ADTFAKSMQ for its binding with beta-galactosidase. Influence of the guest peptide on the diversity of mutations in the neighboring landscape area was also observed.