Detection of a genome-linked protein (VPg) of hepatitis A virus and its comparison with other picornaviral VPgs.

The nucleotide sequence corresponding to the P3 region of the hepatitis A virus (HAV) polyprotein genome was determined from cloned cDNA and translated into an amino acid sequence. Comparison of the amino acid sequences of the genome-linked proteins (VPgs) of other picornaviruses with the predicted amino acid sequence of HAV was used to locate the primary structure of a putative VPg within the genome of HAV. The sequence of HAV VPg, like those of other picornaviral VPg molecules, contains a tyrosine residue as a potential binding site for HAV RNA in position 3 from its N terminus. The potential cleavage sites to generate VPg from a putative HAV polyprotein are between glutamic acid and glycine at the N terminus and glutamic acid and serine or glutamine and serine at the C terminus. A synthetic peptide corresponding to 10 amino acids of the predicted C terminus of HAV VPg induced anti-peptide antibodies in rabbits when it was conjugated to thyroglobulin as a carrier. These antibodies were specific for the peptide and precipitated VPg, linked to HAV RNA, from purified HAV and from lysates of HAV-infected cells. The precipitation reaction was blocked by the synthetic peptide (free in solution or coupled to carrier proteins) and prevented by pretreatment of VPg RNA with protease. Thus, our predicted amino acid sequence is colinear with the nucleotide sequence of the VPg gene in the HAV genome. From our results we concluded that HAV has the typical organization of picornavirus genes in this part of its genome. Similarity among hydrophobicity patterns of amino acid sequences of different picornaviral VPgs was revealed in hydropathy plots. Thus, the VPg of HAV appears to be closely related to VPg1 and VPg2 of foot-and-mouth disease virus. In contrast, HAV VPg has a unique isoelectric point (pI = 7.15) among the picornavirus VPgs.     

Protective immunity to rabbit oral and cutaneous papillomaviruses by immunization with short peptides of L2, the minor capsid protein

The papillomavirus minor capsid protein, L2, has been shown to exhibit immunogenicity, whereby a variety of B-cell epitopes, predominantly in the amino terminus of L2, have been deduced. However, immunity to L2 in vivo has not been examined extensively. Notably, a common neutralization epitope for human papillomavirus (HPV) types 6 and 16 was mapped to amino acids (aa) 108 to 120. The objectives of this study were to derive antisera from rabbits using the corresponding sequences from rabbit viruses and to assess the ability of these peptides to protect against infection. Synthetic peptides consisting of two overlapping sequences each in the region of aa 94 to 122 of the rabbit oral (ROPV) and cottontail rabbit (CRPV) papillomaviruses were used to immunize rabbits. Rabbits were then infected with both ROPV and CRPV and monitored for the development of oral and cutaneous papillomas, respectively. Serum derived from rabbits immunized with either of the two peptides was shown to (i) react to purified L2 from the cognate virus, (ii) specifically recognize L2 within virus-infected cells, and (iii) neutralize virus in vitro. Following viral challenge, cutaneous papilloma growth was completely absent in rabbits immunized with either CRPV peptide. Likewise, ROPV peptide-immunized rabbits were protected from oral papillomatosis. Challenge of CRPV peptide-immune rabbits with the viral genome resulted in efficient papilloma growth, suggesting a neutralizing antibody-mediated mechanism of protection. These results afford in vivo evidence for the immunogenicity provided by a distinct region of L2 and further support previous evidence for the ability of this region to elicit antiviral immunity.     

Tat-neutralizing antibodies in vaccinated macaques

The human immunodeficiency virus Tat protein is essential for virus replication and is a candidate vaccine antigen. Macaques immunized with Tat or chemically modified Tat toxoid having the same clade B sequence developed strong antibody responses. We compared these antisera for their abilities to recognize diverse Tat sequences. An overlapping peptide array covering three clade B and two clade C Tat sequences was constructed to help identify reactive linear epitopes. Sera from Tat-immunized macaques were broadly cross-reactive with clade B and clade C sequences but recognized a clade B-specific epitope in the basic domain. Sera from Tat toxoid-immunized macaques had a more restricted pattern of recognition, reacting mainly with clade B and with only one clade B basic domain sequence, which included the rare amino acids RPPQ at positions 57 to 60. Monoclonal antibodies against the amino terminus or the domain RPPQ sequence blocked Tat uptake into T cells and neutralized Tat in a cell-based transactivation assay. Macaques immunized with Tat or Tat toxoid proteins varied in their responses to minor epitopes, but all developed a strong response to the amino terminus, and antisera were capable of neutralizing Tat in a transactivation assay.     

Analysis of antibody response to synthetic peptides derived from the fusion protein of measles virus

A computer program combining of hydrophilicity, flexibility, surface probability, secondary structure and antigenic index parameters of the amino acid sequence of measles virus (MV) fusion protein was used to select four possible epitopes. Rabbits were immunized with the synthesized peptides conjugated to purified protein derivative using the homobifunctional cross-linker bis-sulfosuccinimidyl suberate. Immune stimulating complexes were prepared with the peptides conjugated to the purified protein derivative carrier using a dialysis method. All antisera raised in rabbits against the peptide conjugates had a high titer to the homologous peptides and reacted well with denatured MV as tested by plate ELISA. None of the sera had neutralizing antibody. Human sera positive for MV antibody reacted strongly with the synthesized peptides indicating that the selected locations function as partial antigenic sites. Antisera against peptide conjugates reacted weakly in immunofluorescence and none of these antisera reacted with purified MV proteins in Western blot. The results obtained in this study indicated that although the computer program could not predict epitopes important for the neutralization of the MV, the predicted epitopes are useful for detecting antibodies against MV.     

HTLV-II-specific antisera raised in rabbits immunized with a synthetic peptide of HTLV-II envelope protein.

In order to discriminate HTLV-II from HTLV-I, HTLV-II-specific polyclonal antibodies against a synthetic peptide of HTLV-II envelope sequence were raised in rabbits. We immunized two adult rabbits with a KLH-conjugated synthetic peptide corresponding to the amino acid sequence 171-196 of the HTLV-II envelope sequence, which is a specific region for HTLV-II as evaluated with an ELISA method. The resulting rabbit antisera to the synthetic peptide reacted with gp46 of HTLV-II lysates in Western blot analysis but not with that of HTLV-I. Flow cytometric analysis and immunohistochemical study revealed that these affinity purified antisera recognized some HTLV-II-producing cell lines examined, but not HTLV-I-producing cell lines or other cell lines uninfected by HTLV. These findings indicate that these antisera specifically recognized the envelope glycoprotein (gp46) of HTLV-II and suggest the specificity of this region in the immune response to HTLV-II. Such antisera are useful in distinguishing between HTLV-I and HTLV-II infection and in determining the presence of individual HTLV-II-infected cells both in vivo and in vitro, including non-lymphoid cells. They may also assist in the elucidation of the pathogenesis of HTLV-II.     

Peptide vaccine against canine parvovirus: identification of two neutralization subsites in the N terminus of VP2 and optimization of the amino acid sequence.

The N-terminal domain of the major capsid protein VP2 of canine parvovirus was shown to be an excellent target for development of a synthetic peptide vaccine, but detailed information about number of epitopes, optimal length, sequence choice, and site of coupling to the carrier protein was lacking. Therefore, several overlapping peptides based on this N terminus were synthesized to establish conditions for optimal and reproducible induction of neutralizing antibodies in rabbits. The specificity and neutralizing ability of the antibody response for these peptides were determined. Within the N-terminal 23 residues of VP2, two subsites able to induce neutralizing antibodies and which overlapped by only two glycine residues at positions 10 and 11 could be discriminated. The shortest sequence sufficient for neutralization induction was nine residues. Peptides longer than 13 residues consistently induced neutralization, provided that their N termini were located between positions 1 and 11 of VP2. The orientation of the peptides at the carrier protein was also of importance, being more effective when coupled through the N terminus than through the C terminus to keyhole limpet hemocyanin. The results suggest that the presence of amino acid residues 2 to 21 (and probably 3 to 17) of VP2 in a single peptide is preferable for a synthetic peptide vaccine.     

Strong immunogenicity of a multicomponent peptide vaccine developed with the branched lysine oligopeptide method for human immunodeficiency virus infection

We synthesized one V3 peptide each from HTLV-III_B, Thai A and Thai B, conjugating then to the T cell epitope of the env region, and we also synthesized a p17 protein peptide of the gag region (HGP-30). These peptide were then coupled to 8-lysine copolymers using N-succinimidyl maleimido carboxylate (M_r = ca 60 000). We designated this the branched lysine oligopeptide method. The large peptide complexes constructed from these four macromolecular peptide were used with aluminium hydroxide or complete Freund's adjuvant to immunize mice and rabbits four times. ELISA assay with aluminium hydroxide or complete Freund's adjuvant to immunize mice and rabbits four times. ELISA assay showed high titres of anti-peptide antibodies to each V3loop peptide and the HGP-30 peptide. Strong inhibition of CD4⁺ dependent cell fusion was obtained with these antisera when III_b, Thai A and Thai B strains of human immunodeficiency virus (HIV) were used. Strong anti-fusion inhibition was also observed with two other HIV strains. In addition, an increase of the anti-HIV effect was observed when we used sera obtained by multicomponent vaccine immunization. The same kind of inhibition was also observed in p24 assay systems using these immunized antisera. Activation of IL-2 production in lymphocytes was observed in p24 assay systems using these immunized antisera. Activation of IL-2 production in lymphocytes was observed in mice immunized with this vaccine. These results suggest that immunization with macromolecular peptide complexes can result in strong immunogenicity towards HIV-1.     

Radioimmunoassay of human prolactin based on a 13 amino acid synthetic analog of the amino terminus

A 13 amino acid analog of the human prolactin amino terminus was synthesized, substituting tyrosine for valine at residue 13. The peptide was coupled to crystalline bovine serum albumin for antisera production. The peptide was used for iodination with 125I, and displacement curves were found to be parallel when human prolactin and the synthetic peptide were compared as standards. The radioimmunoassay using the synthetic peptide has the advantages of purity in its roles as hapten in the antigen and as labelled peptide, of ease of iodination of the peptide, of its stability after iodination, and of obviating the need for native human prolactin. The radioimmunoassay is suitable for the measurement of human prolactin concentration in plasma.     

Antibodies against the chemically synthesized genome-linked protein of poliovirus react with native virus-specific proteins

The genome-linked protein (VPg) of poliovirus has been chemically synthesized, coupled to bovine serum albumin carrier and injected into rabbits. An antibody response was elicited not only by the full-length synthetic VPg peptide, but also by a synthetic 14-amino acid carboxy-terminal peptide. All antisera reacted with virus-specific proteins from HeLa cells infected with poliovirus. Three of these proteins have previously been implicated by others as precursors of VPg. No free cytoplasmic VPg could be detected, and the antibodies did not react with radiolabeled proteins from uninfected cells.     

Mapping of homologous, amino-terminal neutralizing regions of human T-cell lymphotropic virus type I and II gp46 envelope glycoproteins.

Twelve synthetic peptides containing hydrophilic amino acid sequences of human T-cell lymphotropic virus type I (HTLV-I) envelope glycoprotein were coupled to tetanus toxoid and used to raise epitope-specific antisera in goats and rabbits. Low neutralizing antibody titers (1:10 to 1:20) raised in rabbits to peptides SP-2 (envelope amino acids [aa] 86 to 107), SP-3 (aa 176 to 189), and SP-4A (aa 190 to 209) as well as to combined peptide SP-3/4A (aa 176 to 209) were detected in the vesicular stomatitis virus-HTLV-I pseudotype assay. Higher-titered neutralizing antibody responses to HTLV-I (1:10 to 1:640) were detected with pseudotype and syncytium inhibition assays in four goats immunized with a combined inoculum containing peptides SP-2, SP-3, and SP-4A linked to tetanus toxoid. These neutralizing anti-HTLV-I antibodies were type specific in that they did not inhibit HTLV-II syncytium formation. Neutralizing antibodies in sera from three goats could be absorbed with peptide SP-2 (aa 86 to 107) as well as truncated peptides containing envelope aa 90 to 98, but not with equimolar amounts of peptides lacking envelope aa 90 to 98. To map critical amino acids that contributed to HTLV-I neutralization within aa 88 to 98, peptides in which each amino acid was sequentially replaced by alanine were synthesized. The resulting 11 synthetic peptides with single alanine substitutions were then used to absorb three neutralizing goat antipeptide antisera. Both asparagines at positions 93 and 95 were required for adsorption of neutralizing anti-HTLV-I antibodies from all three sera. Peptide DP-90, containing the homologous region of HTLV-II envelope glycoprotein (aa 82 to 97), elicited antipeptide neutralizing antibodies to HTLV-II in goats that were type specific. In further adsorption experiments, it was determined that amino acid differences between homologous HTLV-I and HTLV-II envelope sequences at HTLV-I aa 95 (N to Q) and 97 (G to L) determined the type specificity of these neutralizing sites. Thus, the amino-terminal regions of HTLV-I and -II gp46 contain homologous, linear, neutralizing determinants that are type specific.     

Preparation of rabbit antibodies to 4,4'-dimethoxytriphenylmethyl, the protective group in oligonucleotide synthesis.

Antibodies were raised in 2 rabbits by immunization with carrier proteins covalently bound to deoxyguanosine bearing a 4,4'-dimethoxytriphenylmethyl group protecting the 5'-hydroxy terminus of deoxyribose. After several injections with such complexes, immune sera were tested with an immuno-enzymatic method using as antigens several compounds containing the hapten, as well as synthetic oligonucleotides bearing, or not, this protective group at the 5' terminus. One of the two antisera appeared to recognize the dimethoxytrityl group bound to carrier molecules, and thus might find a useful application for the detection, quantitation, and control of oligonucleotides obtained by automatic synthesis.     

Antibodies against a synthetic peptide of the poliovirus replicase protein: reaction with native, virus-encoded proteins and inhibition of virus-specific polymerase activities in vitro.

A carboxy-terminal peptide of the poliovirus replicase protein (p63) was chemically synthesized, coupled to bovine serum albumin carrier, and injected into rabbits. The resulting antisera reacted with six virus-specific proteins from HeLa cells infected with poliovirus: NCVP 0b, NCVP 1b, NCVP 2, a protein of about 60,000 daltons, p63, and NCVP 6b. The identity of the 60,000-dalton protein is not known, but the other results were consistent with previous experimental approaches which demonstrated that p63 and the other four polypeptides have common coding sequences. An amino-terminal peptide of p63 failed to elicit an immune response in rabbits. Antibodies raised against the p63 carboxy-terminal peptide inhibited poliovirus replicase and polyuridylic acid polymerase activities in vitro, providing strong support for earlier suggestions that these activities are a property of a single virus-specific polypeptide.     

Aldehyde modification of peptide immunogen enhances protein-reactive antibody response to toxic shock syndrome toxin-1.

Introduction of aldehyde groups into protein conjugates enhanced the immune response to a coupled peptide without the use of strong adjuvants. Synthetic peptides representing the N-terminal (residues 1-16) and internal (residues 53-65) epitopes of toxic shock syndrome toxin-1 (TSST-1) were coupled to carrier protein, and carbonyl tags were introduced by Amadori reaction with glycolaldehyde. Modified and unmodified antigens in alum were used to immunize rabbits and the reactivities of antisera were compared. Aldehyde modification augmented the response detected by ELISA, which included enhanced binding to peptides and to native TSST-1. In western blot, TSST-1 was detected by antiserum elicited to the N-terminal peptide, but not that generated to the peptide representing the internal sequence. The same antiserum also neutralized TSST-1 activity in a lymphocyte proliferation assay. The circular dichroism spectrum of the N-terminal peptide indicated a propensity for helical conformation, similar to the structure at the corresponding sequence of the native protein. These data suggest that aldehyde modification can boost immunogenicity of peptide-based vaccines, generating epitope-specific immune responses against the cognate protein antigens without using potent adjuvants.     

Antigenic and immunogenic epitopes shared by human papillomavirus type 16 and bovine, canine, and avian papillomaviruses.

All types of papillomaviruses (PV) share common, so-called group-specific epitopes. To identify the major group-specific epitopes, we immunized 26 guinea pigs or rabbits with purified bovine PV type 1 (BPV), canine PV, or avian PV from the common chaffinch. The resulting hyperimmune sera, as well as a commercially available rabbit antiserum to BPV and seven monoclonal antibodies to BPV, were tested in an enzyme-linked immunosorbent assay with a set of 66 overlapping 20-amino-acid peptides representing the complete sequence of the major capsid proteins (L1 and L2) of human PV type 16 (HPV 16). Sera from the same animals before immunization were used as controls. The minimal reactive epitopes within each peptide were further characterized by testing of truncated peptides. The cross-reactive epitopes were clustered in two regions of L1, an internal region (at positions 171 to 235), which contained three epitopes, and the more reactive region at the carboxy terminus (at positions 411 to 475), which contained six epitopes. The most reactive of the HPV 16 broadly cross-reactive epitopes was a carboxy-terminal epitope which had the sequence DTYRF and which reacted with nine of the antisera to BPV, canine PV, or avian PV, with the commercially available rabbit antiserum to BPV, and also with a mouse monoclonal antibody to BPV. Antipeptide antisera to all of the HPV 16 L1 peptides and to the most antigenically reactive of their truncated analogs were made in guinea pigs. Antipeptide antisera reactive with BPV were obtained for three of the cross-reactive epitopes, and one of these antisera allowed highly sensitive detection of group-specific PV antigen by immunoperoxidase staining.     

Identification and distribution of immunoreactive peptide YY in the human, canine, and murine gastrointestinal tracts: species-related antibody recognition differences

A radioimmunoassay using two antisera (antibody 80 and antibody 213) from rabbits immunized with porcine peptide YY has been characterized for both sensitivity and specificity. To determine the distribution of peptide YY in the gut, fresh tissue specimens from the human and canine gut were separated into mucosal-submucosal and muscularis externa layers by microdissection. These tissues and transmural specimens from murine gut were acid-extracted and neutralized, followed by radioimmunoassay using each antiserum. Immunoreactive peptide YY in canine and murine gut was present in similar concentration and distribution using each antiserum, with highest concentrations in the mucosal-submucosal layer of the descending colon. Using antibody 213, immunoreactive peptide YY throughout the human gut was measured only at the lower detection limit of the radioimmunoassay. By contrast, using antibody 80, peptide YY in human gut was present in a distribution similar to canine and murine gut. Using antibody 80, one major immunoreactive species was identified with C18 reverse-phase high-performance liquid chromatography in extracts of human, canine, and murine colon. These results suggest species-related antibody recognition differences. The similar concentrations of peptide YY in canine and murine gut determined with the two antisera are consistent with the hypothesis that the amino acid sequences of canine and murine peptide YY are similar to porcine peptide YY. Using antibody 213, the low concentrations of immunoreactive peptide YY found in human gut are consistent with the hypothesis that human and porcine peptide YY have different amino acid sequences. Antisera prepared by immunization with porcine PYY must therefore be carefully characterized prior to studies using human sera or human tissue extracts.     

Production of anti-IFN-beta sera with chemically synthetic IFN-beta peptide fragment (1 - 13)

Region specific antisera for human fibroblast interferon (IFN-β) was obtained from New Zealand white rabbits immunized with the immunogen which was the synthetic tridecapeptide corresponding to the N-terminal sequence of native human IFN-β coupled to bovine serum albumin. A sensitive and specific radioimmunoassay for IFN-β was developed and these antisera was successfully applied in the purification of human IFN-β by affinity chromatography, resulting in about 100-fold purification in one step.     

Production and characteristics of human tissue plasminogen-activator antibodies with region-oriented synthetic peptides.

We selected six peptide sequences as belonging to potential epitopes of tissue plasminogen activator (tPA) using, as the main criterion for their choice, the location of the peptide sequences on the surface of the protein molecule. The six peptides (corresponding to amino acids 4-8, 11-16, 96-101, 272-277, 371-376 and 514-519) were synthesized, coupled to carrier proteins and injected into rabbits. All of these peptides elicited antibodies and 15-75% binding of the corresponding iodinated peptide was obtained with a 1:100 dilution of antiserum. Only two anti-(peptide) sera [anti-(tPA96-101) and anti-(tPA272-277)] reacted with intact tPA and its heavy chain in Western immunoblotting analysis. These two peptides sequences and fragment tPA11-16 appear to be involved in the structure of native antigenic epitopes of tPA, since they were recognized and antibodies present in antisera raised against native tPA. There was no interaction between anti-(tPA4-8) and anti-(tPA371-376) sera with intact one-chain or two-chain tPA. In the case of anti-(tPA4-8) cleavage of one-chain tPA to two-chain tPA and reduction of disulfide bonds exposed this epitope.     

The cysteine bonding in TSH receptor and it function in autoantibodies recognition

The majority of epitopes for TSH receptor (TSHR) stimulating autoantibodies are clustered around the Nterminal region of the TSH receptor. The characteristic feature of this region is the presence of four cysteine residues. It was proposed that cysteines in positions 29 and 41 in the receptor are connected by disulfide bonds and they are the target for receptor stimulating antibodies. The present study was aimed to check this possibility. The synthetic peptides: peptide corresponding to the part of TSHR containing the above 29-41 cysteine bond, the peptide similar to this peptide but without disulfide bond and the control peptide, containing sequence absent in the receptor were used for rabbit immunization. The thyroid status of all immunized rabbits was the same. Rabbits immunized with peptides related to TSHR generated antisera reactive with TSHR in immunoenzymatic assay. To check specificity of this reaction the influence of the peptides and the antisera on TSH binding to the receptor in competitive assay (TRAK) and their influence on adenylate cyclase activity were studied. It was found that neither synthetic peptides nor antiserum from any rabbit influenced TSH binding to the receptor in TRAK. In contrast low, but significant adenylate cyclase stimulating activity was noticed for antisera from two of six rabbit immunized by peptide containing the disulfide bond. We concluded that such a bond between cysteine residues 29 and 41 are present in TSHR in the site of stimulating antibodies epitope.     

Immunoreactive helodermin-like peptides in rat: a new class of mammalian neuropeptides related to secretin and VIP

Helodermin is a peptide from the venom of the lizard Heloderma suspectum (Gila Monster) showing a high degree of sequence similarity with VIP, PHI and secretin in its N-terminal moiety. The present data support the presence of peptide(s) closely related to helodermin in the brain, gut and salivary glands of rat. In our radioimmunoassays, we routinely used one of the three specific antisera obtained from rabbits that were immunized against lizard helodermin coupled to bovine serum albumin with carbodiimide. Heat- and acid-stable immunoreactive helodermin-like material was more abundant in striatum, hippocampus and anterior pituitary than in cerebral cortex and hypothalamus. High levels of helodermin-like material were also present in salivary glands, duodenum and jejunum. When submitted to gel permeation chromatography on a TSK-G 2000 SW column, the apparent molecular radius of most of the immunoreactive material ranged from 6 to 12 KDa.