Engineering an Antibody V Gene-Selective Vaccine.

The ligand-binding surface of the B cell receptor (BCR) is formed by encoded and non-encoded antigen complementarity determining regions (CDRs). Genetically reproducible or 'public' antibodies can arise when the encoded CDRs play deterministic roles in antigen recognition, notably within human broadly neutralizing antibodies against HIV and influenza virus. We sought to exploit this by engineering virus-like-particle (VLP) vaccines that harbor multivalent affinity against gene-encoded moieties of the BCR antigen binding site. As proof of concept, we deployed a library of RNA bacteriophage VLPs displaying random peptides to identify a multivalent antigen that selectively triggered germline BCRs using the human VH gene IGVH1-2*02. This VLP selectively primed IGHV1-2*02 BCRs that were present within a highly diversified germline antibody repertoire within humanized mice. Our approach thus provides methodology to generate antigens that engage specific BCR configurations of interest, in the absence of structure-based information.

[Genetically engineered mutants of the envelope protein of the RNA-containing bacteriophage]. / Gennoinzhenernye mutanty belka obolochki RNK-soderzhashchego bakteriofaga.

Expression of the coat protein gene of RNA bacteriophage fr in Escherichia coli cells leads to the formation of capsid-like structures of ca. 25 nm in diameter, which are immunologically indistinguishable from the native phage fr capsids. The modification strategy of the coat protein gene by gene engineering technique was developed in order to localize coat protein regions, which are exposed on the capsid surface and are capable to include foreign amino acid inserts without an appreciable effect on the capsid self-assembly. The oligonucleotide linkers, coding short amino acid sequences and bearing also convenient restriction sites, were synthesized and inserted into different regions of the coat protein gene. The mutant proteins, containing insertions of 2-12 amino acids in potentially exposed regions, were obtained. It was shown that N- and C-terminal insertions, as well as the insertion into codon 51 in the RNA-binding region, do not prevent the self-assembly. The regions (codons 96 and 112) were also revealed, insertions in them decreased drastically the protein yield as a consequence of a block in the self-assembly.

"Pseudolysogenization" by RNA phage Q beta.

We isolated fairly stable lysogenic-like bacteria from a lysogenic state established between an amber mutant for the maturation protein gene of RNA phage Q beta (Q beta am 205) and its nonpermissive host BE110. These bacteria contained few mature phages intracellularly (less than 10(-3) plaque forming unit per cell), continued to grow with a potentiality to produce Q beta am 205 spontaneously, and showed an immunity-like response against homologous phage infection. These characteristics were maintained by growth in liquid medium containing anti-Q beta serum. We designated these cells as pseudolysogenic bacteria. The relative amounts of RNA genomes in these pseudolysogenic cells (about 10(2) infectious RNA strands per cell) indicated that the RNA genomes could replicate in nonpermissive cells and be distributed in daughter cells synchronizing well with cell division.

Generation of antibody diversity subsequent to antigenic stimulation.

Experiments are presented in which spleen cells from donor mice, exhibiting a clonally very restricted early antibody response to a small RNA bacteriophage, are transferred at limiting cell dose into irradiated syngeneic recipients. Antibodies produced in such recipients were analyzed with regard to isoelectric points (pI's). Most antibodies in the recipients were found to differ from the donor, but the pI range into which they fall is frequently fairly narrow and related to the pI of the donor. It is suggested that most recipient clones may be derived from a common ancestral precursor cell by somatic mutation in immunoglobulin genes, conceivably subsequent to antigenic stimulation. If such a process should indeed account for the observed diversity, it would imply an unusually high mutation rate.

Short synthetic CDR-peptides forming the antibody combining site of the monoclonal antibody against RNA bacteriophage fr neutralize the phage activity.

The construction of a mouse hybridoma FR52 secreting neutralizing monoclonal antibody specific for RNA bacteriophages fr, MS2 and GA is reported. The genes encoding the variable domains of the monoclonal antibody FR52 heavy and light chains were cloned and sequenced and the corresponding complementarity determining region (CDR) peptides were chemically synthesized. The CDR-peptides were tested for their ability to neutralize the activity of RNA phage fr and related RNA phages MS2 and GA. The CDR-derived peptides H2, L2 and L3 interacted with the fr phage particles and neutralized fr phage activity. Two of these peptides--H2 and L3 also had the ability to neutralize partly the activity of related bacteriophage MS2, but L1 and especially L3 neutralize the activity of the RNA phage GA. These results provide an excellent system for further antibody-antigen interaction studies and raise the possibility that simple CDR-peptides may serve as a new class of anti-viral molecules.

Characterization of an intergroup serological mutant from group II RNA phage GA.

Starting from the group II RNA phage GA which has an amber mutation in the maturation protein cistron, a spontaneous mutant of group II phage GA, whose serological and electrophoretic properties became similar to those of group I phage MS2, was isolated and analyzed. The mutant has now become sensitive to anti-MS2 serum and resistant to anti-GA serum. Analysis of the nucleotide sequence of the coat protein gene revealed that G----A transition was the main change. The deduced amino acid sequence showed that five amino acids were substituted in the mutant, and three of the five became identical to MS2, resulting in increased molecular weight of the coat protein. However, it did not complement MS2. These results suggested that the serological change from group II phage GA type to group I phage MS2 type is induced spontaneously at high frequency by minor nucleotide changes in coat protein gene, and confirmed the previous results at the RNA level that MS2 and GA were related although the closeness between them seems somewhat remoter than that of groups III and IV (18, Inokuchi et al, unpublished data for the nucleotide sequence of group IV phage SP).

Protein P4 of double-stranded RNA bacteriophage phi 6 is accessible on the nucleocapsid surface: epitope mapping and orientation of the protein.

Protein P4, an early protein of double-stranded RNA bacteriophage phi 6, is a component of the virion-associated RNA polymerase complex and possesses a nucleoside triphosphate (NTP) phosphohydrolase activity. We have produced and characterized a panel of 20 P4-specific monoclonal antibodies. Epitope mapping using truncated molecules of recombinant P4 revealed seven linear epitopes. The accessibility of the epitopes on the phi 6 nucleocapsid (NC) surface showed that at least the C terminus and an internal domain, containing the consensus sequence for NTP binding, protrude the NC shell. Four of the NC-binding antibodies distorted the integrity of the NC by releasing protein P4 and the major NC surface protein P8. This finding suggests a close contact between these two proteins. The dissociation of the NC led to the activation of the virion-associated RNA polymerase. The multimeric status of the recombinant P4 was similar to that of the virion-associated P4, indicating that no accessory virus proteins are needed for its multimerization.

Analysis of RNA phage fr coat protein assembly by insertion, deletion and substitution mutagenesis.

A structure-function analysis of the icosahedral RNA bacteriophage fr coat protein (CP) assembly was undertaken using linker-insertion, deletion and substitution mutagenesis. Mutations were specifically introduced into either pre-existing or artificially created restriction enzyme sites within fr CP gene expressed in Escherichia coli from a recombinant plasmid. This directs synthesis of wild type protein that undergoes self-assembly and forms capsid-like particles indistinguishable morphologically and immunologically from native phage particles. A series of fr CP variants containing sequence alterations in the regions which are (i) exposed on the external surface of capsid or (ii) located on the contacting areas between CP subunits were obtained and their assembly properties investigated. The majority of mutants demonstrated reduction of assembly ability and formed either CP dimers (mutations at residues 2, 10, 63 or 129) or both dimer and capsid structures (residue 2 or 69). The exceptions were variants demonstrating normal assembly and containing insertions at residues 2, 50 or 129 of the fr CP. A third type of assembled structure was formed by a variant with a single amino acid substitution I104T. The alpha A-helix region (residues 97-111) is particularly sensitive to mutation and any alteration in this region decreases accumulation of mutant protein in E. coli. The relative contributions of particular fr CP domains in maintenance of capsid structural integrity as well as the possible capsid assembly mechanism are discussed.