Characterization of BoHV-1 gE envelope glycoprotein mimotopes obtained by phage display.

A phage-displayed peptide library was screened using four mAbs directed against bovine herpesvirus 1 (BoHV-1) gE glycoprotein to identify peptides mimicking this glycoprotein. The selected mimotopes allowed us to characterize the epitopes corresponding to the mAbs as continuous and proteinic and to consider using these peptides in further studies. One epitope has been clearly located at the C-terminus of the protein (amino-acids 561-569). The three other mAbs enabled us to stress the immunogenic relevance of the proline-rich motifs of gE. Selected peptides showed no clear sequence identity with gE, but there is a clear link between gE proline-rich regions and the amino-acid composition of the mimotopes. The proline-rich motifs of gE are potentially located in flanking regions involved in the gE/gl glycoprotein complex formation. N-terminal fusion to pill or pVIII filamentous phage protein, C-terminal fusion to the T7 phage capsid protein, biotinylated synthetic peptides and insertion between the non-cleaved CX leader sequence and the C-terminal part of Caulobacter crescentus RsaA protein have been tested in order to increase the valency of a model peptide. We have diverted the C. crescentus expression system and proven its usefulness using the RsaA protein as a scaffold displaying the peptides of interest. Comparison between these different display systems in an indirect ELISA, indicates that the C. crescentus expression and the T7 phage display systems have some major advantages.

Improvement of serological discrimination between herpesvirus-infected animals and animals vaccinated with marker vaccines.

Control/eradication plans of bovine herpesvirus 1 (BHV1) and suid herpesvirus 1 (SHV1) infections involve vaccination with inactivated or attenuated gE-deleted marker vaccines and associated companion serological tests to discriminate naturally infected from vaccinated animals. Blocking or competitive enzyme-linked immunosorbent assays (ELISAs) have been designed for the detection of specific antibodies against BHV1 or SHV1 gE glycoprotein. The antigen source usually consists of a crude viral preparation in which gE is associated with other envelope glycoproteins. Such assays suffer from a lack of specificity which is not due to serological cross-reactions with other pathogens. Interestingly, false-positive results occur with sera collected from multivaccinated cattle or pigs. After multivaccination with a marker vaccine, the binding of the conjugated monoclonal antibody used as a tracer, could be hampered by antibodies directed against the other viral glycoproteins. In order to validate the steric hindrance hypothesis, a simple preadsorption of such samples was carried out with a preparation of antigen devoid of gE, prior to the blocking ELISA itself. The decrease in antibody concentrations against the major glycoproteins, clearly leads to a better discrimination between positive and negative samples; that is between infected and multivaccinated animals, without significant loss of sensitivity. This experiment confirms the steric hindrance hypothesis, therefore serum preadsorption could be an easy way to improve the specificity of currently available diagnostic tests.

Use of a mouse lung challenge model to identify antigens protective against Chlamydia pneumoniae lung infection.

Chlamydia pneumoniae is emerging as a significant human pathogen. Infection causes a range of respiratory tract diseases and is associated with atherosclerosis. A vaccine could provide a considerable public health benefit; however, antigens able to elicit a protective immune response are largely unknown. A panel of open-reading frames (ORFs) from the C. pneumoniae genome sequence was screened for ability to elicit protective responses. Balb/c mice immunized with DNA containing the ORFs were tested for their ability to limit lung infection following an intranasal challenge. Immunization with DNA encoding the major outer membrane protein or an ADP/ATP translocase (Npt1(Cp)) of C. pneumoniae resulted in a reduced bacteria load in the lung after challenge. The identification of these antigens as protective is a significant step toward development of a C. pneumoniae vaccine and demonstrates the feasibility of using a DNA immunization strategy to screen the C. pneumoniae genome for other protective ORFs.

High affinity human antibodies by phage display.

The development of phage display has now made it possible to consider the isolation of human antibodies directly without immunization. Recent advances in the field of human immunogenetics and in phage technology have led to the assembly of 'naive' human repertoires in vitro whose complexity approach that of the natural immune system. Screening of these libraries has allowed the isolation in one step of antibodies with affinities in the nanomolar range.

Full-scale 'naïve' human antibody repertoires assembled from VH and VL variable regions.

Very large 'naïve' human antibody repertoires have been obtained from RT-PCR cloned VH and VL variable regions. They are used as starting material for the assembly of medium sized combinatorial libraries or so called multicombinatorial libraries. In nonimmunized individuals immunoglobulin messenger RNAs are poorly expressed, which can be a serious limitation for cloning efficiency. To overcome this problem two complementary strategies have been used: a nonspecific polyclonal activation of B cells, and a secondary PCR amplification technique to ensure recovery of Ig messengers in large amount and without introducing any bias.

A new phage display system to construct multicombinatorial libraries of very large antibody repertoires.

We present an easy and efficient technique for the construction of large phage-displayed antibody (Ab) repertoires through the recombination of two separate heavy (VH) and light (VL) chain gene libraries. Here, the system has been applied to the display of a chimpanzee anti-HIV gp160 Ab. The process, which makes use of lambda phage att recombination sites, leads to the irreversible physical association between plasmid and phagemid vectors carrying, respectively, VL and VH sequences. The heat-inducible expression of the Int recombinase allows perfect control of recombination. Selection of the recombinant phagemid is made possible by the assembly, in vivo, of a genetic marker (chloramphenicol resistance) created only after the correct recombination event. Theoretically, all possible associations between the VL and VH sequences should be obtained, and it should be possible to generate multicombinatorial libraries of close to 10(12) clones.

Sequence of the Kluyveromyces lactis beta-galactosidase: comparison with prokaryotic enzymes and secondary structure analysis.

The LAC4 gene encoding the beta-galactosidase (beta Gal) of the yeast, Kluyveromyces lactis, was cloned on a 7.2-kb fragment by complementation of a lacZ-deficient Escherichia coli strain. The nucleotide sequence of the structural gene, with 42 bp and 583 bp of the 5'- and 3'-flanking sequences, respectively, was determined. The deduced amino acid (aa) sequence of the K. lactis beta Gal predicts a 1025-aa polypeptide with a calculated M(r) of 117618 and reveals extended sequence homologies with all the published prokaryotic beta Gal sequences. This suggests that the eukaryotic beta Gal is closely related, evolutionarily and structurally, to the prokaryotic beta Gal's. In addition, sequence similarities were observed between the highly conserved N-terminal two-thirds of the beta Gal and the entire length of the beta-glucuronidase (beta Glu) polypeptides, which suggests that beta Glu is clearly related, structurally and evolutionarily, to the N-terminal two-thirds of the beta Gal. The structural analysis of the beta Gal alignment, performed by mean secondary structure prediction, revealed that most of the invariant residues are located in turn or loop structures. The location of the invariant residues is discussed with respect to their accessibility and their possible involvement in the catalytic process.

Epitope mapping of HLA-B27 and HLA-B7 antigens by using intradomain recombinants.

To study the HLA-B7 and HLA-B27 antigenic determinants, hybrid genes between these two alleles were constructed by in vivo recombination in Escherichia coli. After transfection of these genes into P815 (high transfection efficiency recipient) murine cells, the bindings of Bw6, HLA-B7, and HLA-B27 allele-specific mAb were studied, as well as that of human anti-HLA-B7 and anti-HLA-B27 monospecific alloantisera. Most of the HLA-B7 antigenic determinants were assigned to the first external domain of the molecule. Four different epitopic areas could be defined: the Bw6 epitope was associated with residues 82 and 83; the BB7.1 epitope to amino acids 63, 67, and 70; the MB40.2 and MB40.3 epitope to amino acid sequence 177-180, and human alloantisera identified as an epitope associated with residue 9. HLA-B27 antigenicity studied by TM-1 mAb was found to involve residues 77 and 80 in the alpha-1 domain. Results obtained with human monospecific alloantisera allowed the definition of an additional allospecific site associated with the NH2 terminal part on the alpha-1 domain of HLA-B27. Epitope mapping fits with data obtained by sequence comparisons and is discussed with reference to the crystallographic three-dimensional structure of the HLA-A2 molecule.

Hybrid genes between HLA-A2 and HLA-A3 constructed by in vivo recombination allow mapping of HLA-A2 and HLA-A3 polymorphic antigenic determinants.

HLA-A2 and -A3 genes have been modified in their third exon (second domain) by using in vivo recombination. In this method Escherichia coli are transfected with a plasmid which contains two highly homologous sequences (e.g., the third exons of HLA-A2 and -A3) and has been linearized by cleavage between these two sequences. Circularization takes place in the bacteria by homologous recombination leading to hybrid A2-A3 sequences. The analysis by DNA sequencing of a number of such recombinants shows that they indeed occur by homologous recombination (no insertions or deletions) and that the probability of crossing over decreases as the distance from the free end of DNA in the homologous region increases. No double recombinants were observed. These hybrid exons were reinserted into either HLA-A2 or HLA-A3 genes, thus generating a panel of functional hybrid genes containing one or several HLA-A2 specific substitutions in an HLA-A3 background or vice versa. These genes were expressed by transfection into murine P815-high transfection efficiency recipient cells. Serologic analysis leads to the conclusion that expression of polymorphic antigenic determinants specific for HLA-A2 (detected with M58, A2A28M1, and CR11.351 mAb) is linked to the presence of threonine residue (amino acid (AA) 142) and/or histidine residue (AA 145) and valine residue (AA 152). The expression of specific HLA-A3 polymorphic determinants (recognized by GAP-A3 mAb) is correlated with the existence of a asparagine residue (AA 127) and a aspartic residue (AA 161). But aspartic residue 161 contributes with glutamic acid residue 152 in the formation of the A3 epitope recognized by the anti-A3 mAb X1.23.2.

Mapping of HLA epitopes recognized by H-2-restricted cytotoxic T lymphocytes specific for HLA using recombinant genes and synthetic peptides.

Immunization of DBA/2 (H-2d) mice with syngeneic P815 tumor cell transfectants that express HLA class I genes elicits CTL that recognize HLA in the context of H-2Kd molecules. Anti-HLA-CW3 CTL cross-react to a variable extent on the related alleles A3 and A24. Using a panel of target cells expressing native or recombinant HLA genes, we could map the epitope recognized by a CTL clone specific for CW3 to the second external (alpha 2) domain of CW3. Moreover, the epitope recognized by this clone could be mimicked by incubating P815 (HLA negative) target cells with a synthetic peptide corresponding to the C-terminal 12 amino acids of the CW3 alpha 2 domain (residues 171 to 182). Other independent anti-CW3 CTL clones with different fine specificities recognized the same CW3 peptide. In contrast, CTL clones specific for HLA-A24 or HLA-A3 that did not lyse P815-CW3 transfectants did not recognize this peptide. The CW3 peptide could be recognized on other tumor cell targets that were also of H-2d origin, but not on those of H-2b or H-2k origin. The requirement for the expression of H-2Kd by the target cells was directly demonstrated using L cell Kd transfectants. Our results suggest that the CTL response of DBA/2 mice immunized with P815-CW3 transfectants is predominantly Kd restricted and focused on epitopes contained within the 12 C-terminal amino acids of the alpha 2 domain.

Transfection of murine LMTK- cells with purified HLA class I genes. VII. Association of allele- and locus-specific serological reactivities with respectively the first and second domains of the HLA-B7 molecule.

The individual contributions of the first two external domains of the HLA-B7 heavy chain to the expression of allele-specific (B7) and locus-specific (B and C) antigenic determinants were investigated using hybrid class I genes. Hybrid genes were constructed in vitro by exon shuffling between the parent genes HLA-B7, HLA-Cw3, HLA-A3, and H-2Kd, and their expression was monitored following transfection into mouse L cells. The results show that most allele-specific antigenic determinants are associated with the first external domain of the B7 heavy chain, whereas all the locus-specific antigenic determinants tested map to the second external domain.

An HLA-C-specific DNA probe.

Analysis of available nucleotide sequence data for class I HLA genes has established that the seventh intron is one of the gene regions which expresses the highest degree of locus specificity (the percentage sequence divergence between nonallelic genes minus the percentage sequence divergence between allelic genes). We have subcloned short DNA sequences including this region from the HLA-Cw3 gene. Two clones, pC250 and pC800, were tested by hybridizing them at high stringency to a panel of clones containing class I HLA genes. Under conditions permitting a strong hybridization signal with a C-locus gene, pC800 also expressed a weak but significant hybridization to other class I genes, while pC250 appeared to hybridize exclusively to the C-locus gene. Hybridization of the pC250 probe at high stringency to Hind III-digested genomic DNA from a panel of unrelated individuals and homozygous typing cell lines revealed a single band in all cases. However, equivalent hybridization against Eco RI-digested DNA revealed two hybridization bands, one at 7.9 kb which correlated with the serologically defined Cw5 and Cw8 alleles, and one at 7.6 kb which correlated with the Cw1, Cw2, Cw3, Cw4, Cw6, and Cw7 alleles.

The HLA-AW24 gene: sequence, surroundings and comparison with the HLA-A2 and HLA-A3 genes.

A cosmid clone containing two class I sequences was found to cause expression of the HLA-AW24 protein after transfection into mouse L cells. The restriction map of this cosmid shows extensive homology over 26 kb with the map of the HLA-A3 region obtained from cosmids of the same library, constructed with DNA from an HLA-A3/HLA-AW24 heterozygote, but diverges over the remaining 14 kb. The HLA-AW24 gene was subcloned from this cosmid and its nucleotide sequence was determined. Amino acid and, more strikingly, nucleotide sequence comparisons with other HLA alleles indicate that the A locus alleles are more closely related to each other than to alleles from other HLA loci. A very skewed distribution of silent substitutions is apparent, and the occurrence of clustered multiple substitutions hints at gene-conversion-like events.

Allelism in the HLA class I multigene family.

Recent data on the structure of functional HLA class I genes shows that, at least at the HLA-A locus, the allelic genes are more related to each other than to HLA genes from other loci. This "A-ness" is discernible at the protein sequence level but much more evident when nucleotide sequences are compared; the homology is particularly striking in the 3' non-coding region. Genes coding for the same HLA specificity in different genetic backgrounds show no obvious difference, although in one case the 3' flanking regions are clearly different; the restriction maps around the HLA-A3 and HLA-AW24 genes are also compared to see whether the chromosomal environments of these two genes are recognizably similar.

Complete nucleotide sequence of a gene encoding a functional human class I histocompatibility antigen (HLA-CW3).

The HLA-CW3 gene contained in a cosmid clone identified by transfection expression experiments has been completely sequenced. This provides, for the first time, data on the structure of HLA-C locus products and constitutes, together with that of the gene coding for HLA-A3, the first complete nucleotide sequences of genes coding for serologically defined class I HLA molecules. In contrast to the organisation of the two class I HLA pseudogenes whose sequences have previously been determined, the sequence of the HLA-CW3 gene reveals an additional cytoplasmic encoding domain, making the organisation of this gene very similar to that of known H-2 class I genes and also the HLA-A3 gene. The deduced amino acid sequences of HLA-CW3 and HLA-A3 now allow a systematic comparison of such sequences of HLA class I molecules from the three classical transplantation antigen loci A, B, C. The compared sequences include the previously determined partial amino acid sequences of HLA-B7, HLA-B40, HLA-A2 and HLA-A28. The comparisons confirm the extreme polymorphism of HLA classical class I molecules, and permit a study of the level of diversity and the location of sequence differences. The distribution of differences is not uniform, most of them being located in the first and second extracellular domains, the third extracellular domain is extremely conserved, and the cytoplasmic domain is also a variable region. Although it is difficult to determine locus-specific regions, we have identified several candidate positions which may be C locus-specific.

Complete nucleotide sequence of a functional class I HLA gene, HLA-A3: implications for the evolution of HLA genes.

The complete nucleotide sequence of an active class I HLA gene, HLA-A3, has been determined. This sequence, together with that obtained for the HLA-CW3 gene, represents the first complete nucleotide sequence to be determined for functional class I HLA genes. The gene organisation of HLA-A3 closely resembles that of class I H-2 genes in mouse: it shows a signal exon, three exons encoding the three extracellular domains, one exon encoding the transmembrane region and three exons encoding the cytoplasmic domain. The complete nucleotide sequences of the active HLA genes, HLA-A3 and HLA-CW3, now permit a meaningful comparison of the nucleotide sequences of class I HLA genes by alignment with the sequence established for a HLA-B7-specific cDNA clone and the sequences of two HLA class I pseudogenes HLA 12.4 and LN- 11A . The comparisons show that there is a non-random pattern of nucleotide differences in both exonic and intronic regions featuring segmental homologies over short regions, which is indicative of a gene conversion mechanism. In addition, analysis of the frequency of nucleotide substitution at the three base positions within the codons of the functional genes HLA-A3, HLA-B7 and HLA-CW3 shows that the pattern of nucleotide substitution in the exon coding for the 3rd extracellular domain is consistent with strong selection pressure to conserve the sequence. The distribution of nucleotide variation in the other exons specifying the mature protein is nearly random with respect to the frequencies of substitution at the three nucleotide positions of their codons. The evolutionary implications of these findings are discussed.