Mutations located on both F1 and F2 subunits of the Newcastle disease virus fusion protein confer resistance to neutralization with monoclonal antibodies.

The fusion gene sequence of six Newcastle disease virus escape mutants revealed that residues important for the integrity of antigenic site 1 and antigenic site 2 were located, respectively, on the F2 subunit and within the cysteine-rich domain of the F1 subunit. We further report the antibody-binding capacity of these mutants.     

The nucleotide sequence of the protein subunit of the K88ac fimbriae of porcine enterotoxigenic Escherichia coli

Abstract The nucleotide sequence of the gene encoding the K88ac fimbrial subunit has been determined and the amino acid sequence was derived. In comparison with the two other, previously determined sequences of the K88ab and K88ad sequences, the most striking features of the K88ac protein sequence are the insertion of a lys residue at position 104 and the deletion of three amino residues at positions 165. The differences between the three sequences are discussed with respect to possible structure-functions relationships and antigenic determinants.     

Respiratory syncytial virus fusion glycoprotein: nucleotide sequence of mRNA, identification of cleavage activation site and amino acid sequence of N-terminus of F1 subunit.

The amino acid sequence of respiratory syncytial virus fusion protein (Fo) was deduced from the sequence of a partial cDNA clone of mRNA and from the 5' mRNA sequence obtained by primer extension and dideoxysequencing. The encoded protein of 574 amino acids is extremely hydrophobic and has a molecular weight of 63371 daltons. The site of proteolytic cleavage within this protein was accurately mapped by determining a partial amino acid sequence of the N-terminus of the larger subunit (F1) purified by radioimmunoprecipitation using monoclonal antibodies. Alignment of the N-terminus of the F1 subunit within the deduced amino acid sequence of Fo permitted us to identify a sequence of lys-lys-arg-lys-arg-arg at the C-terminus of the smaller N-terminal F2 subunit that appears to represent the cleavage/activation domain. Five potential sites of glycosylation, four within the F2 subunit, were also identified. Three extremely hydrophobic domains are present in the protein; a) the N-terminal signal sequence, b) the N-terminus of the F1 subunit that is analogous to the N-terminus of the paramyxovirus F1 subunit and the HA2 subunit of influenza virus hemagglutinin, and c) the putative membrane anchorage domain near the C-terminus of F1.     

Kinetic characterization of early immunoreactive intermediates during the refolding of guanidine-unfolded Escherichia coli tryptophan synthase beta 2 subunits.

This paper deals with stopped-flow studies on the kinetics of the regain of immunoreactivity toward five distinct monoclonal antibodies during the folding of the guanidine-unfolded beta 2 subunit of Escherichia coli tryptophan synthase and of two complementary proteolytic fragments of beta, F1 (N-terminal; Mw = 29,000) and F2 (C-terminal; Mw = 12,000). It is shown that, while selected as being "specific" for the native protein, these antibodies are all able to recognize early folding intermediates. The two antigenic determinants carried by the F2 domain and the antigenic site carried by the hinge peptide linking F1 and F2 are present so early during the folding process that their kinetics of appearance could not be followed. On the contrary, the rate constants of appearance of two "native-like" epitopes, carried by F1, could be determined during the folding of beta chains. The rate constant of appearance of the epitope to antibody 19 was found to be k = 0.065 s-1 at 12 degrees C. This value is very similar to that we reported previously for the appearance of an early epitope to the same antibody during the folding of acid-denatured beta chains. Thus, in spite of the important structural differences between guanidine-unfolded and acid-denatured beta chains, the same early folding events seem to be involved in the appearance of this epitope. The rate constant was found to be significantly smaller (k = 0.02 s-1 at 12 degrees C) for the appearance of the epitope to antibody 9. This shows that the regain of immunoreactivity is not concerted within the F1 domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

A conserved region in the F(2) subunit of paramyxovirus fusion proteins is involved in fusion regulation

Paramyxoviruses utilize both an attachment protein and a fusion (F) protein to drive virus-cell and cell-cell fusion. F exists functionally as a trimer of two disulfide-linked subunits: F(1) and F(2). Alignment and analysis of a set of paramyxovirus F protein sequences identified three conserved blocks (CB): one in the fusion peptide/heptad repeat A domain, known to play important roles in fusion promotion, one in the region between the heptad repeats of F(1) (CBF(1)) (A. E. Gardner, K. L. Martin, and R. E. Dutch, Biochemistry 46:5094-5105, 2007), and one in the F(2) subunit (CBF(2)). To analyze the functions of CBF(2), alanine substitutions at conserved positions were created in both the simian virus 5 (SV5) and Hendra virus F proteins. A number of the CBF(2) mutations resulted in folding and expression defects. However, the CBF(2) mutants that were properly expressed and trafficked had altered fusion promotion activity. The Hendra virus CBF(2) Y79A and P89A mutants showed significantly decreased levels of fusion, whereas the SV5 CBF(2) I49A mutant exhibited greatly increased cell-cell fusion relative to that for wild-type F. Additional substitutions at SV5 F I49 suggest that both side chain volume and hydrophobicity at this position are important in the folding of the metastable, prefusion state and the subsequent triggering of membrane fusion. The recently published prefusogenic structure of parainfluenza virus 5/SV5 F (H. S. Yin et al., Nature 439:38-44, 2006) places CBF(2) in direct contact with heptad repeat A. Our data therefore indicate that this conserved region plays a critical role in stabilizing the prefusion state, likely through interactions with heptad repeat A, and in triggering membrane fusion.     

The dimensions of the T lymphocyte glycoprotein leukosialin and identification of linear protein epitopes that can be modified by glycosylation.

Leukosialin (CD43) is a major glycoprotein of T lymphocytes whose extracellular domain of 224 amino acids contains on average one O-linked carbohydrate unit per three amino acids. This suggests an unfolded structure for the extracellular domain which has now been established to extend to a length of 45 nm by transmission electron microscopy following low angle rotary shadowing. The antigenicity of rat leukosialin has been studied using nine monoclonal antibodies (MAbs) whose binding is differentially affected by the cell type on which leukosialin is expressed and by the removal of sialic acid. From these observations it appears that the epitopes are affected by glycosylation, yet seven of the nine MAbs reacted clearly with the extracellular domain of leukosialian expressed in an unglycosylated form in Escherichia coli. The MAbs showing this positive reaction included three of the four antibodies whose epitopes were affected by neuraminidase treatment of leukosialin. It thus appears that linear protein epitopes are recognized and that some of these can be modified in the native structure by glycosylation. The positions of the antigenic determinants have been mapped by expressing fusion proteins of different lengths and the identity of one epitope was proven by the binding of two MAbs to an octapeptide expressed as a fusion protein. For three MAbs, the location of epitopes in the native protein was confirmed by electron microscopy of shadowed leukosialin--Fab complexes. Overall it is concluded that leukosialin is a major component at the periphery of the T lymphocyte and that despite its high level of glycosylation, protein determinants are exposed that could be ligands in cell interactions.     

Expression of foreign epitopes in P-fimbriae of Escherichia coli.

Summary Hypervariable regions (HRs) of the major subunit of F11 fimbriae were exploited for insertion of foreign epitopes. Two insertion vectors were created that contain a unique cloning site in HR1 or HR4 respectively. Several oligonucleotides, coding for antigenic determinants derived from different pathogens, were cloned in both insertion vectors. Hybrid fimbrial subunits were generally shown to be assembled in fimbriae when the length of the inserted peptide did not exceed 14 amino acids. The inserted peptides appeared to be exposed in the fimbrial filament. One hybrid fimbrial protein induced detectable levels of antibodies against the inserted epitope if injected into mice.     

Genetic manipulation of major P-fimbrial subunits and consequences for formation of fimbriae.

The influence of genetic manipulation of the structural genes coding for major P-fimbrial subunits on the formation of fimbriae in Escherichia coli was studied. Deletion of two regions that code for hypervariable parts of the P fimbrillin resulted in strong reduction or total absence of fimbria production. Replacement of deleted amino acids by other amino acid residues restored the formation of fimbriae. The hypervariable regions may be important for biogenesis of fimbriae by imposing correct spacing between conserved regions of the protein. The potential for substituting amino acids in the P-fimbrial subunit opens interesting possibilities for use of fimbriae as carriers of foreign antigenic determinants. An antigenic determinant of foot-and-mouth disease virus (FMDV) was incorporated in the F11 fimbrial subunit. Hybrid fimbriae, recognized by an FMDV-specific neutralizing monoclonal antibody directed against FMDV, were formed.     

Respiratory syncytial virus fusion glycoprotein: further characterization of a major epitope involved in virus neutralization

Competition experiments and biological assays with a panel of 15 monoclonal antibodies confirmed the presence of at least four antigenic sites on the fusion protein of human respiratory syncytial virus, three of which were involved in virus neutralization. One antigenic site, recognized by two strongly neutralizing antibodies, was conserved after reduction and denaturation and shown by immunoblotting to be localized on the F1 fragment of the fusion protein. Cleavage of this protein with staphylococcal protease V8 or papain produced a series of smaller peptides from 11 to 7 kilodaltons that retained this important neutralization determinant. Compared with the other neutralization sites, the epitope defined by monoclonal antibody 7C2 thus appears as the major neutralization epitope. Our peptide mapping results support the hypothesis that this major epitope is composed of a continuous sequence on the viral genome.     

Study of the structural characteristics of the regulatory subunit of cAMP-dependent protein kinase type II using monoclonal antibodies

Monoclonal antibodies to the regulatory subunit of cAMP-dependent protein kinase type II from porcine brain were used to study the antigenic properties of the enzyme regulatory subunit (RII). The monoclonal antibodies were bound to linear antigenic determinants on the protein molecule surface. The cAMP binding to RII interfered with the interaction between monoclonal antibodies and the protein. The use of different proteolytic fragments of RII allowed for the localization of antigenic determinants in the N-terminal moiety of RII.     

Purification and characterization of P fimbriae from an Escherichia coli strain isolated from a septicemic turkey

Abstract A pap + Escherichia coli isolate from a turkey with colisepticemia expressed P fimbriae with a major subunit of an apparent molecular mass of 18 kDa which reacted with anti-F11 serum. This fimbriae was purified and polyclonal antiserum was produced in rabbits. The N-terminal amino acid sequence of the major fimbrial subunit of the avian P fimbriae was identical to that of F11. On immunoblotting, the antiserum against the avian P fimbriae strongly reacted with the major subunit of the homologous fimbriae, with F11, and with F165₁ fimbriae. Some antigenic determinants on the major subunits of F13, F7₁, and F7₂ fimbriae, with a stronger reaction against F13 fimbriae, were also recognized. The F11 antiserum reacted similarly to the antiserum against avian P fimbriae although cross-reactions against F13, F7₁, and F7₂ fimbriae were equivalent. In a competitive enzyme-linked immunosorbent assay, serological differences were observed between the purified avian P fimbriae and F11. Thus, the avian P fimbriae is closely related but not identical to F11 fimbriae which are associated with E. coli isolated from human urinary tract infection.     

Immunochemical characteristics of the subunits of cholera enterotoxin and of thermolabile Escherichia coli enterotoxins of various origins

Antigenic determinants of subunits A and B of cholera enterotoxin (CT), heat-labile enterotoxin from the human E. coli strain (hLT) and heat-labile enterotoxin from the porcine E. coli strain (pLT) were analysed by Ouchterlony double gel-diffusion test against antisera to B subunits of three toxins and antisera to three holotoxins. The results have shown the existence of the following antigenic determinants: in subunits B-1. antigenic determinants, common for B subunits of all three enterotoxins-B(chp); 2. group antigenic determinants, common for B subunits of two toxins in the pair-B(ch), B(hp); 3. antigenic determinants, unique for B subunits of each CT, hLT, pLT-(B(c), B(h), B(p); in subunits A.-1. antigenic determinants, common for A subunits of all three enterotoxins-A.(chp); 2. group antigenic determinants, common for A subunits of two enterotoxins (hLT and pLT/-A(hp); 3. antigenic determinants, unique for A subunit of CT-A(c). On the basis of these results antigenic formulas for subunits of CT, hLT, pLT were proposed.     

Polymorphism of murine major histocompatibility Class I antigen: assignment of putative allodeterminants to distinct positions of the amino acid sequence within the first external domain of the antigen

Forty-five new monoclonal antibodies reacting with the mouse H-2Dd antigen have been established. The specificities of 34 of these antibodies were mapped into the first external domain (N) of the Dd antigen by testing reactivities with the products of mosaic H-2 genes in which the coding sequences of the first and/or the second external domains of the H-2Dd genes were recombined in vitro with the remaining portion of the H-2Ld gene. These antibodies reacted with at least 13 distinct allodeterminants located in the N domain, composed of 91 amino acids, as judged from panel tests carried out on various H-2 haplotypes. To assign possible positions of antigenic determinants of these and other anti-H-2Dd antibodies, we compared primary sequences of seven H-2 antigens and searched for correspondence between the pattern of amino acid substitutions in the N domain, allowing 15 positions to be assigned for the antigenic sites. These putative antigenic determinants were assessed for possible relationships with several parameters of protein secondary structure postulated according to predictive methods. Many of these sites appear to be associated with greatest local hydrophilicity, known to correlate with sites of antibody binding in various proteins. We therefore propose that some of the correspondences found in this work represent structural correlates of allodeterminants.     

N-glycans of F protein differentially affect fusion activity of human respiratory syncytial virus

The human respiratory syncytial virus (Long strain) fusion protein contains six potential N-glycosylation sites: N27, N70, N116, N120, N126, and N500. Site-directed mutagenesis of these positions revealed that the mature fusion protein contains three N-linked oligosaccharides, attached to N27, N70, and N500. By introducing these mutations into the F gene in different combinations, four more mutants were generated. All mutants, including a triple mutant devoid of any N-linked oligosaccharide, were efficiently transported to the plasma membrane, as determined by flow cytometry and cell surface biotinylation. None of the glycosylation mutations interfered with proteolytic activation of the fusion protein. Despite similar levels of cell surface expression, the glycosylation mutants affected fusion activity in different ways. While the N27Q mutation did not have an effect on syncytium formation, loss of the N70-glycan caused a fusion activity increase of 40%. Elimination of both N-glycans (N27/70Q mutant) reduced the fusion activity by about 50%. A more pronounced reduction of the fusion activity of about 90% was observed with the mutants N500Q, N27/500Q, and N70/500Q. Almost no fusion activity was detected with the triple mutant N27/70/500Q. These data indicate that N-glycosylation of the F2 subunit at N27 and N70 is of minor importance for the fusion activity of the F protein. The single N-glycan of the F1 subunit attached to N500, however, is required for efficient syncytium formation.     

N-linked glycans with similar location in the fusion protein head modulate paramyxovirus fusion

N-linked glycans not only orchestrate the folding and intracellular transport of viral glycoproteins but also modulate their function. We have characterized the three glycans attached to fusion (F) proteins of the morbilliviruses canine distemper virus and measles virus. The individual Morbillivirus glycans have similar functional properties: the glycan at position 68 is essential for protein transport, and those at positions 36 and 75 modulate fusion (numbering according to the Newcastle disease virus [NDV] F protein sequence). Based on the crystal structure of the NDV F protein, we then predicted the locations of the Morbillivirus glycans: the glycan at position 36 is located in the F protein head, and those at positions 68 and 75 are located near the neck-stalk interface. NDV position 36 is not occupied by a glycan; the only glycan in that F protein head also has a fusion control function and grows from residue 366, located only 6 A from residue 36. We then exchanged the glycan at position 36 with the glycan at position 366 and showed functional complementation. Thus, structural information about the F proteins of Paramyxoviridae coupled with functional analysis disclosed a location in the protein head into which fusion-modulating glycans independently evolved.     

The relative abundance of two measles virus fusion protein peptide-DR1 complexes expressed by B cells is independent of the form of the antigen

The relative processing and presentation efficiency of two DR1-restricted determinants from the fusion protein (F protein) of measles virus (MV) was determined using three forms of antigen (Ag): MV, an F protein recombinant vaccinia virus, and a chimerical polypeptide between the glutathione S-transferase and the F protein (GST-F protein). First, it was shown that these different preparations of F protein have distinct processing requirements. In MV-infected B cells, the F254 determinant (contained within the F protein sequence 254-268) relies on protein synthesis for its presentation, while the F314 determinant (contained within the F protein sequence 314-328) is also presented in the absence of protein synthesis. By contrast, in GST-F protein-pulsed B cells, presentation of both determinants is dependent on protein synthesis. Then, it was established that, independently of the form of the Ag, the F314 determinant was considerably more (18- to 36-fold) efficiently processed and presented than the F254 determinant. These results indicate that determinants from the same protein are displayed by antigen-presenting cells at widely different levels and they may also suggest that this is an intrinsic characteristic of the determinants, rather than a feature controlled by the processing pathways followed by the Ag.     

Evaluation of Naturally Acquired IgG Antibodies to a Chimeric and Non-Chimeric Recombinant Species of Plasmodium vivax Reticulocyte Binding Protein-1: Lack of Association with HLA-DRB1*/DQB1* in Malaria Exposed Individuals from the Brazilian Amazon

The development of modular constructs that include antigenic regions targeted by protective immune responses is an attractive approach for subunit vaccine development. However, a main concern of using these vaccine platforms is how to preserve the antigenic identity of conformational B cell epitopes. In the present study we evaluated naturally acquired antibody responses to a chimeric protein engineered to contain a previously defined immunodominant domain of the Plasmodium vivax reticulocyte binding protein-1 located between amino acid positions K₄₃₅-I₇₇₇. The construct also includes three regions of the cognate protein (F₅₇₁-D₅₈₇, I₁₇₄₅-S₁₇₈₆ and L₂₂₃₅-E₂₂₆₃) predicted to contain MHC class II promiscuous T cell epitopes. Plasma samples from 253 naturally exposed individuals were tested against this chimeric protein named PvRMC-RBP1 and a control protein that includes the native sequence PvRBP1_23-751 in comparative experiments to study the frequency of total IgG and IgG subclass reactivity. HLA-DRB1 and HLA-DQB1 allelic groups were typed by PCR-SSO to evaluate the association between major HLA class II alleles and antibody responses. We found IgG antibodies that recognized the chimeric PvRMC-RBP1 and the PvRBP1_23-751 in 47.1% and 60% of the studied population, respectively. Moreover, the reactivity index against both proteins were comparable and associated with time of exposure (p<0.0001) and number of previous malaria episodes (p<0.005). IgG subclass profile showed a predominance of cytophilic IgG1 over other subclasses against both proteins tested. Collectively these studies suggest that the chimeric PvRMC-RBP1 protein retained antigenic determinants in the PvRBP1_435–777 native sequence. Although 52.9% of the population did not present detectable titers of antibodies to PvRMC-RBP1, genetic restriction to this chimeric protein does not seem to occur, since no association was observed between the HLA-DRB1* or HLA-DQB1* alleles and the antibody responses. This experimental evidence strongly suggests that the identity of the conformational B cell epitopes is preserved in the chimeric protein.     

Prospects and achievements of genetic engineering in development of antiviral vaccines

Proceeding from the known data various theoretical and experimental approaches to the construction of gene-engineering vaccines are considered. Gene-engineering subunit vaccines of the first generation are based on isolation of the genes coding for the synthesis of full length capsid proteins with the main antigenic determinants and their subsequent expression in suitable recipient cells. Initial idea of the microbiological synthesis as the main way for production of any antiviral vaccines was not confirmed by the later development. Now for this type of vaccines eucaryotic systems are widely employed using the animal virus vectors and the animal cell cultures. Gene-engineering subunit vaccine of the second generation appears to be a chimeric protein with built-in antigenic determinants of different viruses and maximal immunogenicity in monomeric form. The last point reopens the perspective to use a microbiological synthesis for the production of antiviral vaccines. Besides that the chemically synthesized polypeptide antiviral vaccine will be used widely. In gene-engineering subunit vaccines of the third generation it is possible to use not the natural antigenic determinants which often are characterized by high level of the primary structure changes but artificial (non-natural) antigens, that are the capsid protein conservative regions which under natural conditions of infection or immunization do not induce the protective antiviral antibodies. The recombinant DNA technology in addition to subunit type vaccine allows to construct living vaccines which represent a DNA-containing attenuated virus with build-in natural or synthetic gene of the capsid or chimeric protein with antigenic determinants of another viral species.     

Influence of the alpha-, beta- and gamma-subunits of the energy-transducing adenosine triphosphates from Micrococcus lysodeikticus in the immunochemical properties of the protein and in their reconstitution studied by a radioimmunoassay method.

We developed a sensitive and specific radioimmunoassay of the energy-transducing adenosine triphosphatase (F1-ATPase, EC 3.6.1.3) of Micrococcus lysodeikticus and extended the assay to the alpha-, beta- and gamma-subunits of the enzyme. We isolated these subunits and studied cross-reactions. We found the immunochemical properties of alpha- and beta-subunits to differ, and gamma-subunits showed an intermediate behaviour between that of alpha- and beta-subunits. Our findings indicate that each subunit of M. lysodeikticus F1-ATPase has its own identity and that conformational antigenic determinants and/or co-operative antigenic sites-arise from subunit assembly. Equimolecular amounts of alpha- and beta-subunits (up to three copies of each) reconstituted partially the immunochemical properties of the ATPase molecule, and addition of 2 mol of gamma-subunit per mol of alpha 3 beta 3 complex improved reconstitution. Our findings describe the first reconstitution of biological activity of this ATPase by assembly of the isolated subunits, and provide support for earlier proposals on the stoicheiometry of the alpha 3 beta 3 gamma 2 type for M. lysodeikticus F1-ATPase. The radioimmunoassay method affords opportunities to study the homologies between different energy-transducing ATPases and their constituent polypeptides before the primary structure of these complex proteins has been determined.     

Role of N-linked glycosylation of the Hendra virus fusion protein

The Hendra virus fusion (F) protein contains five potential sites for N-linked glycosylation in the ectodomain. Examination of F protein mutants with single asparagine-to-alanine mutations indicated that two sites in the F(2) subunit (N67 and N99) and two sites in the F(1) subunit (N414 and N464) normally undergo N-linked glycosylation. While N-linked modification at N414 is critical for protein folding and transport, F proteins lacking carbohydrates at N67, N99, or N464 remained fusogenically active. As N464 lies within heptad repeat B, these results contrast with those seen for several paramyxovirus F proteins.