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.     

Purified protein derivative (PPD) as an immunogen carrier elicits high antigen specificity to haptens.

The effectiveness of the carrier protein in eliciting antigen-specific antibodies was investigated. The effect of the carrier protein was independent of the conjugation chemistry involved. Keyhole limpet hemocyanin (KLH), purified protein derivative (PPD), and ovalbumin (OVA) were used as carrier proteins in the immunization of mice. Three antigens were studied: LY170881 (a small drug molecule), 4-[1'-cyanobenz(f)isoindolyl]butyric acid (CBI-butyric acid), and a seven residue peptide GPGRGPG (KLE1). The serum antibody response to the antigen or antigen:BSA conjugate was superior in the case where the PPD:antigen conjugates were used as the immunogen when compared to KLH and OVA. The specificity of the antibodies to the respective antigens vs cross-reactivity with the carrier protein was investigated. PPD-coupled antigen immunized mice generated a higher percentage of antigen-specific hybridomas compared to the other carrier proteins. These findings confirmed PPD as the best carrier molecule for the production of both polyclonal and monoclonal antibodies.     

DNA topological change in the hyperthermophilic archaeon Pyrococcus abyssi exposed to low temperature

Abstract Mice were immunized with resin-bound peptides whose sequences have been proposed to be part of exposed loops in Salmonella typhi outer membrane protein OmpC. To screen hybridomas for monoclonal antibodies against those epitopes, we designed fusion proteins where the candidate peptide sequence was attached to the amino end of cholera toxin B-subunit (CTB). The constructed fusion proteins allowed the efficient selection of positive clones by GM1-ELISA. Selected antibodies recognized purified OmpC and whole Salmonella bacteria. This suggests a native structure of our genetically attached peptides in agreement with immunological properties reported for previous CTB recombinant fusion proteins. In a more general context, CTB hybrids could be used to screen for antibodies towards immunogenic epitopes in other systems. This might turn out to be particularly useful when producing antibodies against peptide sequences in microorganisms whose handling is difficult or that pose inherent health risks.     

A Japanese encephalitis virus peptide present on Johnson grass mosaic virus-like particles induces virus-neutralizing antibodies and protects mice against lethal challenge

Protection against Japanese encephalitis virus (JEV) is antibody dependent, and neutralizing antibodies alone are sufficient to impart protection. Thus, we are aiming to develop a peptide-based vaccine against JEV by identifying JEV peptide sequences that could induce virus-neutralizing antibodies. Previously, we have synthesized large amounts of Johnson grass mosaic virus (JGMV) coat protein (CP) in Escherichia coli and have shown that it autoassembled to form virus-like particles (VLPs). The envelope (E) protein of JEV contains the virus-neutralization epitopes. Four peptides from different locations within JEV E protein were chosen, and these were fused to JGMV CP by recombinant DNA methods. The fusion protein autoassembled to form VLPs that could be purified by sucrose gradient centrifugation. Immunization of mice with the recombinant VLPs containing JEV peptide sequences induced anti-peptide and anti-JEV antibodies. A 27-amino-acid peptide containing amino acids 373 to 399 from JEV E protein, present on JGMV VLPs, induced virus-neutralizing antibodies. Importantly, these antibodies were obtained without the use of an adjuvant. The immunized mice showed significant protection against a lethal JEV challenge.     

Expression,purification, and characterization of minimized chicken riboflavin carrier protein from a synthetic gene in Escherichia coli

Minimized proteins have long been used to elicit immune response to particular regions of a protein antigen. Most efforts to derive minimized proteins have employed synthetic peptide fragments. Here we describe molecular cloning and production of a minimized chicken riboflavin carrier protein (mini-RCP) sequence that harbours all the four neutralizing epitopes but lacks the sequences that otherwise elicit undesirable antibodies. The gene encoding mini-RCP is engineered by contiguous alignment of nucleotide sequences coding for selected epitopes of chicken RCP separated by leucyl alanine residues. The gene has been constructed from eight oligonucleotides by employing overlapping PCR strategy and expressed in Escherichia coli, using the T7 promoter system. The recombinant protein could be purified to homogeneity by a single step Ni2+ affinity chromatography. Western blot experiments using epitope specific antisera confirm that the corresponding linear amino acid sequences are available for immunorecognition in the engineered protein. This methodology enables continuous production and purification in bulk amounts of the minimized RCP as a source of candidate immunocontraceptive vaccine in mammals.     

Expression of two S-ribonucleases of Petunia inflata using baculovirus expression system.

We have previously shown that three Petunia inflata S-proteins, products of the multiallelic S-gene of the self-incompatibility system, are ribonucleases. Here we report the expression of cDNAs for two of these S-proteins using the baculovirus expression system. S2- and S3-proteins were found in both supernatants and lysates of Spodoptera frugiperda cells infected with recombinant baculoviruses. Both recombinant S-proteins contained glycosylated (25 kD) and nonglycosylated (23 kD) forms. Recombinant S2- and S3-proteins were purified from insect cell cultures, and the amino-terminal sequences determined from glycosylated S2- and S3-proteins indicated that the leader peptide encoded by each cDNA was correctly removed. Both glycosylated and nonglycosylated forms of S2- and S3-proteins exhibited ribonuclease activity.     

Study of Peptide Mimetics of Hepatitis A Virus Conjugated to Keyhole Limpet Hemocyanin and as Multiple Antigen Peptide System

Mimotopes mimic binding properties of natural antigen epitopes. They could be used for vaccine design, drugs development, and diagnostic assays. We have previously identified four bacteriophages displaying hepatitis A virus (HAV) mimotopes from a phage-display peptide library by affinity selection on serum antibodies from hepatitis A patients. Three of these HAV mimotopes showed similarity in their amino acid sequences with at least one of the VP3 and VP1 antigenic proteins of HAV and the four induced specific anti-HAV antibodies. In the present work, four conjugations were done. In each of them, a linear peptide (46, 53, 54 or 56) containing the amino-acid sequence of the corresponding mimotope was conjugated to keyhole limpet hemocyanin (KLH). Conjugation products were named: 46KLH, 53KLH, 54KLH and 56KLH. A two-arm multiple antigen peptide (MAP) system containing peptide sequence 46, and a second MAP containing two copies of peptide sequence 56 were synthesized and dimerized, to obtain the heterodimeric four-arms MAP (named MAP46-56) containing two copies of peptides 46 and 56. Mice were immunized with peptides conjugated to KLH and MAP46-56 to evaluate the ability of these two forms of mimotope presentation, to elicit antibodies that bind to the original antigen. KLH conjugated peptides rendered the highest levels of anti-peptide antibodies and were the only ones that induced specific anti-HAV antibodies. The results of immunizations showed that for the mimotopes chosen here, conjugation to a carrier protein was the most effective option to induce antibodies that cross-reacted with the natural antigen.     

Antibody to synthetic somatostatin-28(1-12): immunoreactivity with somatostatin in brain is dependent on orientation of immunizing peptide

Rabbits were immunized with synthetic peptides representing the neurotransmitter dodecapeptide somatostatin-28(1-12) (SANSNPAMAPRE) coupled to the carrier protein keyhole limpet hemocyanin (KLH) at either the amino or the carboxyl terminus. Although all rabbits produced high-titer antisera to immunizing peptide, as assayed by ELISA, only rabbits immunized with peptide coupled to carrier at the amino terminus yielded antibodies that bound to native somatostatin in mouse brain slices. This effect of peptide coupling orientation on epitope specificity of peptide antisera is likely to be significant to other investigators who use predetermined peptide sequences to generate immunohistochemical reagents.     

Identification, isolation, and N-terminal sequencing of style glycoproteins associated with self-incompatibility in Nicotiana alata.

S-Gene-associated glycoproteins (S-glycoproteins) from styles of Nicotiana alata, identified by non-equilibrium two-dimensional electrophoresis, were purified by cation exchange fast protein liquid chromatography with yields of 0.5 to 8 micrograms of protein per style, depending on the S-genotype of the plant. The method relies on the highly basic nature of the S-glycoproteins. The elution profiles of the different S-glycoproteins from the fast protein liquid chromatography column were characteristic of each S-glycoprotein, and could be used to establish the S-genotype of plants in outbreeding populations. In all cases, the S-genotype predicted from the style protein profile corresponded to that predicted from DNA gel blot analysis using S-allele-specific DNA probes and to that established by conventional breeding tests. Amino-terminal sequences of five purified S-glycoproteins showed a high degree of homology with the previously published sequences of N. alata and Lycopersicon esculentum S-glycoproteins.     

Restriction of anti-peptide antibody specificity by enzyme-modified Sepharose-peptide immunoadsorbents

Sepharose-peptide immunoadsorbents, employed for the isolation of specific antibodies from the sera of rabbits immunized with carrier protein-peptide conjugates, were digested with suitable proteolytic enzymes, in order to obtain the splitting of a part of the peptide bound to the gel. This new modified immunoadsorbent can be advantageously used for the isolation of antibody subsets, that do not cross-react with related peptides exhibiting high sequence homology with the immunogens.     

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.     

A proteomics approach for the identification and cloning of monoclonal antibodies from serum

We describe a proteomics approach that identifies antigen-specific antibody sequences directly from circulating polyclonal antibodies in the serum of an immunized animal. The approach involves affinity purification of antibodies with high specific activity and then analyzing digested antibody fractions by nano-flow liquid chromatography coupled to tandem mass spectrometry. High-confidence peptide spectral matches of antibody variable regions are obtained by searching a reference database created by next-generation DNA sequencing of the B-cell immunoglobulin repertoire of the immunized animal. Finally, heavy and light chain sequences are paired and expressed as recombinant monoclonal antibodies. Using this technology, we isolated monoclonal antibodies for five antigens from the sera of immunized rabbits and mice. The antigen-specific activities of the monoclonal antibodies recapitulate or surpass those of the original affinity-purified polyclonal antibodies. This technology may aid the discovery and development of vaccines and antibody therapeutics, and help us gain a deeper understanding of the humoral response.     

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.     

Ribosome display enhanced by next generation sequencing: A tool to identify antibody-specific peptide ligands

Detection of antibodies in serum has many important applications. Our goal was to develop a facile general experimental approach for identifying antibody-specific peptide ligands that could be used as the reagents for antibody detection. Our emphasis was on an approach that would allow identification of peptide ligands for antibodies in serum without the need to isolate the target antibody or to know the identity of its antigen. We combined ribosome display (RD) with the analysis of peptide libraries by next generation sequencing (NGS) of their coding RNA to facilitate identification of antibody-specific peptide ligands from random sequence peptide library. We first demonstrated, using purified antibodies, that with our approach-specific peptide ligands for antibodies with simple linear epitopes, as well as peptide mimotopes for antibodies recognizing complex epitopes, were readily identified. Inclusion of NGS analysis reduced the number of RD selection rounds that were required to identify specific ligands and facilitated discrimination between specific and spurious nonspecific sequences. We then used a model of human serum spiked with a known target antibody to develop NGS-based analysis that allowed identification of specific ligands for a target antibody in the context of an overwhelming amount of unrelated immunoglobins present in serum.     

Localization and synthesis of an antigenic determinant of herpes simplex virus glycoprotein D that stimulates the production of neutralizing antibody.

An antigenic determinant capable of inducing type-common herpes simplex virus (HSV)-neutralizing antibodies has been located on glycoprotein D (gD) of HSV type 1 (HSV-1). A peptide of 16 amino acids corresponding to residues 8 to 23 of the mature glycoprotein (residues 33 to 48 of the predicted gD-1 sequence) was synthesized. This peptide reacted with an anti-gD monoclonal antibody (group VII) previously shown to neutralize the infectivity of HSV-1 and HSV-2. The peptide was also recognized by polyclonal antibodies prepared against purified gD-1 but was less reactive with anti-gD-2 sera. Sera from animals immunized with the synthetic peptide reacted with native gD and neutralized both HSV-1 and HSV-2.     

GABARAPL1 antibodies: target one protein, get one free!

Atg8 is a yeast protein involved in the autophagic process and in particular in the elongation of autophagosomes. In mammals, several orthologs have been identified and are classed into two subfamilies: the LC3 subfamily and the GABARAP subfamily, referred to simply as the LC3 or GABARAP families. GABARAPL1 (GABARAP-like protein 1), one of the proteins belonging to the GABARAP (GABA(A) receptor-associated protein) family, is highly expressed in the central nervous system and implicated in processes such as receptor and vesicle transport as well as autophagy. The proteins that make up the GABARAP family demonstrate conservation of their amino acid sequences and protein structures. In humans, GABARAPL1 shares 86% identity with GABARAP and 61% with GABARAPL2 (GATE-16). The identification of the individual proteins is thus very limited when working in vivo due to a lack of unique peptide sequences from which specific antibodies can be developed. Actually, and to our knowledge, there are no available antibodies on the market that are entirely specific to GABARAPL1 and the same may be true of the anti-GABARAP antibodies. In this study, we sought to examine the specificity of three antibodies targeted against different peptide sequences within GABARAPL1: CHEM-CENT (an antibody raised against a short peptide sequence within the center of the protein), PTG-NTER (an antibody raised against the N-terminus of the protein) and PTG-FL (an antibody raised against the full-length protein). The results described in this article demonstrate the importance of testing antibody specificity under the conditions for which it will be used experimentally, a caution that should be taken when studying the expression of the GABARAP family proteins.     

Development of a enterovirus diagnostic assay system for diagnosis of viral myocarditis in humans

The coxsackieviruses type B3 (CVB3) are members of the genus Enterovirus of the family Picornaviridae. They are the commonest cause of chronic myocarditis and dilated cardiomyopathy. However, there is still no effective method for diagnosing CVB3 infection in humans. Here, a fast and accurate system that uses a capsid‐protein‐specific peptide sequence to detect CVB3 in the sera of patients with viral myocarditis was established. The peptide sequence was selected from the whole CVB3 capsid protein sequence by computationally predicting fragments with high antigenicity and low hydrophobicity. Two of eight possible peptide sequences were selected and commercially synthesized. The synthesized peptides encoded either the VP2 or VP1 capsid protein and induced immunoglobulin G antibody expression in immunized rabbits. Anti‐VP2 and anti‐VP1 sera detected the viral proteins extracted from CVB3‐infected HeLa cells. The newly synthesized peptides successfully induced antibody production. These peptides, applied in an ELISA system, detected anti‐CVB3 antibodies in virus‐infected mouse serum. Moreover, an ELISA system based on the VP2 peptide detected CVB3 infection in patients with positively identified CVB3‐induced fulminant myocarditis. These results indicate that these new peptides specifically interact with anti‐CVB3 IgG antibodies in mouse and human sera. This ELISA system should be useful for the clinical diagnosis of enterovirus‐induced myocarditis.     

HIV p24-specific helper T cell clones from immunized primates recognize highly conserved regions of HIV-1.

We have investigated Th cell recognition of the HIV core protein p24 by using CD4+ T cell clones derived from cynomolgus macaques immunized with hybrid HIV p24: Ty virus-like particles (VLP). T cell lines from two immunized animals responded to p24: Ty-VLP, control Ty-VLP, purified p24, and whole inactivated HIV, indicating the presence of T cells specific for p24 as well as the Ty carrier protein. The HIV determinants recognized by the T cell lines were identified by using a series of overlapping peptides synthesized according to the sequence of p24. Both T cell lines recognized peptide 11 (amino acids 235-249) and peptide 14 (amino acids 265-279). In addition, one T cell line also responded to peptide 9 (amino acids 215-229). Definitive identification of two T cell epitopes on p24 was confirmed at the clonal level: from a total of four T cell clones generated from one of the T cell lines, two respond specifically to peptide 11 and two to peptide 14. The T cell clones were CD4+ and MHC class II-restricted and secreted IL-2 in response to stimulation with purified p24, inactivated HIV or a single synthetic peptide. The specificity of the Th clones for variant peptides demonstrated cross-reactivity with two simian immunodeficiency virus isolates, but only limited responses to HIV-2 sequences. However, the Th cell epitopes identified on p24 are highly conserved between 12 HIV-1 isolates and were recognized by both of the immunized primates. These sequences may therefore be useful for priming a broadly reactive immune response to HIV-1.     

Conservation among vertebrate immunoglobulin chains detected by antibodies to a synthetic joining segment peptide

We used affinity purified antibodies produced against a synthetic peptide sequence corresponding to the entire J beta of a human T cell receptor gene to screen sera of man, mouse and other vertebrates to determine the presence of cross-reactive molecules. Little evidence for free alpha/beta heterodimers was found, but the antibody reacted with light chains of many vertebrate species, including characterized myeloma proteins of man and mouse. Some vertebrate orders, notably Aves, lacked polypeptide chains cross-reactive with J beta, but detectable determinants occurred in primitive vertebrates such as the galapagos shark (Carcharhinus galapagensis). In addition to the strong cross-reaction with purified light chains, human heavy chains reacted weakly with the antibody. The cross-reaction correlated with the sequence of the denatured immunoglobulins and was inhibitable with free peptide. These results establish the similarity of T cell receptor beta chains to immunoglobulin chains and support the conclusion that J region sequences were conserved, not only within mammalian immunoglobulins and T cell receptors, but in vertebrate evolution.