Selection of glutamate-rich protein long synthetic peptides for vaccine development: antigenicity and relationship with clinical protection and immunogenicity

Antibodies against three long synthetic peptides (LSPs) derived from the glutamate-rich protein (GLURP) of Plasmodium falciparum were analyzed in three cohorts from Liberia, Ghana, and Senegal. Two overlapping LSPs, LR67 and LR68, are derived from the relatively conserved N-terminal nonrepeat region (R0), and the third, LR70, is derived from the R2 repeat region. A high prevalence of antibody responses to each LSP was observed in all three areas of endemic infection. Levels of cytophilic immunoglobulin G (IgG) antibodies against both GLURP regions were significantly correlated with protection from clinical P. falciparum malaria. Protected children from the Ghana cohort possessed predominantly IgG1 antibodies against the nonrepeat epitope and IgG3 antibodies against the repeat epitope. T-cell proliferation responses, studied in the cohort from Senegal, revealed that T-helper-cell epitopes were confined to the nonrepeat region. When used as immunogens, the LR67 and LR68 peptides elicited strong IgG responses in outbred mice and LR67 also induced antibodies in mice of different H-2 haplotypes, confirming the presence of T-helper-cell epitopes in these constructs. Mouse antipeptide antisera recognized parasite proteins as determined by immunofluorescence and immunoblotting. This indicates that synthetic peptides derived from relatively conserved epitopes of GLURP might serve as useful immunogens for vaccination against P. falciparum malaria.     

Plaque reduction test: an alternative method to assess specific antibody response to pIII-displayed peptide of filamentous phage M13

Phage-displayed peptide systems have been used to identify the immunogenic epitopes and to develop the design of peptide-based or peptide-displaying phages themselves as vaccine candidates. To estimate the humoral immunity of phage-based vaccine, it is necessary to evaluate the antibody response specifically directed at the displayed peptide. Enzyme-linked immunosorbent assays (ELISAs) and Western blot analysis are commonly used for this purpose. However, using these methods, it is not easy to distinguish the antibody response against phage coat protein or the antibody response specific to the displayed peptide. The purified anti-Mycoplasma hyopneumoniae IgG was used to screen heptapeptides displaying on the pIII coat protein of M13 phage. Four selected phage clones were chosen to immunize mice. In order to evaluate the specific antibody response that is directed against heptapeptides, advantage was taken of the natural property of M13 phage to infect Escherichia coli, which is mediated by the pIII coat protein binding with the F pili of E. coli, and plaque reduction tests were performed to assess the specificity of antibody response. By comparing the number of plaques produced by the different phages (which are the same except for the displayed peptides) neutralized by the antiserum, we could demonstrate that the specificity of antibody response is directed against the peptide displayed on pIII coat protein. The results described here indicate that plaque reduction test is a convenient and more precise method to detect the antibody against the phage-displayed peptide.     

Characterization of murine coronavirus neutralization epitopes with phage-displayed peptides

Phage-displayed peptide libraries were used to map immunologically relevant epitopes on the surface (S) glycoprotein of a neurotropic murine coronavirus (MHV-A59). Three in vitro virus-neutralizing and in vivo protective mAbs against either continuous or discontinuous epitopes on the S glycoprotein were used to screen 12 different peptide libraries expressed on the pVIII major coat protein of the fd filamentous bacteriophage. Consensus sequences that matched short sequences within the S glycoprotein were identified. The sequence of a tight-binding, mAb-selected peptide suggested the location of a discontinuous epitope within the N-terminal S1 subunit. Several tightly binding phage were amplified and used directly as immunogens in BALB/c and C57BL/6 mice. Partial protection of C57BL/6 mice against a lethal acute virus infection was achieved with a phage preparation that displayed a linear epitope. Protection correlated with the presence of sufficient levels of specific antiviral antibodies recognizing the same immunodominant domain and 13-mer peptide, located within the C-terminal S2 subunit, as the selecting mAb. Thus, the direct use of phage-displayed peptides to evaluate protective antiviral immune responses complements their use to characterize antibody-binding epitopes. This is the first evaluation of protective immunization induced by mAb-selected phage-displayed peptides.     

Seroreactive recombinant herpes simplex virus type 2-specific glycoprotein G.

The herpes simplex virus type 2 (HSV-2) genome codes for an envelope protein, glycoprotein G (gG), which contains predominantly type 2-specific epitopes. A portion of this gG gene has been expressed as a fusion protein in Escherichia coli. Expression was regulated by a lambda phage pL promoter. The 60,000-molecular-weight recombinant protein was purified by ion-exchange chromatography. Amino acid sequence analysis confirmed the N terminus of the purified protein. Mice immunized with recombinant gG developed antibodies reactive with native HSV-2 protein, but not with HSV-1 protein, in an indirect immunofluorescence assay. The serological activity of this purified recombinant gG protein was evaluated by immunoblot assay. This protein was reactive with an HSV-2 gG monoclonal antibody. It was also reactive with HSV-2 rabbit antiserum but not with HSV-1 rabbit antiserum. Of 15 patient serum samples known to have antibody to HSV-2, 14 were reactive with this recombinant type 2-specific gG protein, and none of 15 HSV antibody-negative patient serum samples showed reactivity. In agreement with the expected prevalence of HSV-2 infection, 27.6% of 134 serum samples from random normal individuals had antibodies reactive with recombinant gG. This recombinant gG protein may be of value in detecting HSV-2-specific antibody responses in patients infected with HSV-2.     

Baculovirus-expressed glycoprotein G of herpes simplex virus type 1 partially protects vaccinated mice against lethal HSV-1 challenge.

The DNA sequence encoding the complete HSV-1 glycoprotein G (gG) was inserted into a baculovirus transfer vector and recombinant viruses expressing gG were isolated. Three gG-related recombinant baculovirus expressed peptides of 37, 42, and 44 kDa were detected by Western blotting using monoclonal antibody to gG. The 42- and 44-kDa species were susceptible to tunicamycin, Endoglycosidase H (Endo-H), and N-glycosidase F (PNGase F) treatments, suggesting that they were glycosylated. Although only very low levels (approximately 1:10) of HSV-1-neutralizing antibody were produced in mice vaccinated with the baculovirus gG, these mice were partially protected from lethal challenge with HSV-1 (75-78% survival) and this level of protection was highly significant (P = 0.002). This is the first report to show that vaccination with HSV-1 gG can provide mice with any level of protection against lethal HSV-1 challenge.     

Utilisation of bacteriophage display libraries to identify peptide sequences recognised by equine herpesvirus type 1 specific equine sera

Three filamentous phage random peptide display libraries were used in biopanning experiments with purified IgG from the serum of a gnotobiotic foal infected with equine herpesvirus-1 (EHV-1) to enrich for epitopes binding to anti-EHV-1 antibodies. The sequences of the amino acids displayed were aligned with protein sequences of EHV-1, thereby identifying a number of potential antibody binding regions. Presumptive epitopes were identified within the proteins encoded by genes 7 (DNA helicase/primase complex protein), 11 (tegument protein), 16 (glycoprotein C), 41 (integral membrane protein), 70 (glycoprotein G), 71 (envelope glycoprotein gp300), and 74 (glycoprotein E). Two groups of sequences, which aligned with either glycoprotein C (gC) or glycoprotein E (gE), identified type-specific epitopes which could be used to distinguish between sera from horses infected with either EHV-1 or EHV-4 in an ELISA using either the phage displaying the peptide or synthetic peptides as antigen. The gC epitope had been previously identified as an immunogenic region by conventional monoclonal antibody screening whereas the gE antibody binding region had not been previously identified. This demonstrates that screening of phage display peptide libraries with post-infection polyclonal sera is a suitable method for identifying diagnostic antigens for viral infections such as EHV-1.     

Selection of an immunogenic and protective epitope of the PsaA protein of Streptococcus pneumoniae using a phage display library

Streptococcus pneumoniae is an important pathogen that causes disease in young and elderly individuals. The currently available polysaccharide vaccines have limited efficacy in those age groups most susceptible to pneumococcal infections. This study focuses on mapping the epitopes of a surface protein of S. pneumoniae by biopanning a 15 mer phage display library using 5 different monoclonal antibodies (MAbs) against the Pneumoccal surface adhesin A (PsaA). PsaA is a component of the bacterial cell wall that is highly species specific and is involved in bacterial adherence and virulence. Biopanning of the phage display library reveals three distinct epitopes on the PsaA protein. The sequence homology of these epitopes ranges from two to six amino acids when compared to the native PsaA protein type 2. Two of these epitopes have been evaluated for their immunogeneicity in mice. The peptide selected by the MAbs 8G12, 6F6, and 1B7 is referred to as the consensus peptide and is immunogenic in mice. Optimal anti-PsaA response is observed in mice immunized with 50microg of the consensus peptide complexed to proteosomes in 1:1 ratio. The anti-PsaA response is significantly lower than the response to the PsaA native protein. The peptide selected by monoclonal antibody 4E9 in its lipidated form is significantly protective in mice challenged with S. pneumoniae serotype 2 when compared to mice immunized with the native protein. These results show that the selected epitopes of PsaA protein are immunogenic and protective in mice. These epitopes need to be evaluated further as alternatives to currently available vaccines.     

A branched, synthetic oligopeptide corresponding to a region of glycoprotein G of HSV-1 reacts sensitively and specifically with HSV-1 antibodies in an ELISA

Herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2), which are common worldwide, are so similar that antibodies directed against one serotype may crossreact with antigens from the other one. Methods for specific detection of antibodies against HSV-1 or HSV-2 are based upon the antigenicities of glycoproteins G. However, due to the cost, the available commercial methods may not readily be used in developing countries. A different enzyme-linked immunosorbent assay (ELISA) method, based upon a synthetic oligopeptide corresponding to an immunogenic region in glycoprotein G of HSV-2, has been used recently and successfully for detection of HSV-2 antibodies. In the present study, the sequences of a newly identified immunogenic and type-specific region in glycoprotein G of HSV-1 was used to synthesize three different, branched oligopeptides. The performances of these peptides in an ELISA were investigated by testing Scandinavian and African sera which were characterized by commercial ELISA and Western blotting methods and divided into four groups either lacking HSV antibodies, containing antibodies against one or the other virus, or against both types. The peptide which corresponded in sequence to the immunodominant region was as specific and sensitive by an ELISA as were the commercial methods. The method is inexpensive and reliable.     

Virus-derived tubular structure displaying foreign sequences on the surface elicit CD4+ Th cell and protective humoral responses

Particulate vector systems for the presentation of immunogenic epitopes provide an alternate and powerful approach for the delivery of immunogens of interest. In this article, we have exploited a viral protein of unknown function, bluetongue virus (BTV) nonstructural protein NS1, which forms distinct tubular aggregates in infected cells, as an immunogen delivery system. Tubules are helical assemblies of NS1 protein that present the C-terminus of the protein to the outer edge effectively displaying appended residues in a regular and repeating array akin to the coat of a filamentous phage. To assess the breadth of response induced following tubule-based immunization, two different immunodominant foreign peptides were inserted at the C-terminus of NS1 and chimeric tubules generated following expression in the baculovirus expression system. Both constructs, one carrying a peptide of foot and mouth disease virus (FMDV) (aa 135-144 of VP1) and the other, a peptide of influenza A virus (aa 186-205 of HA), effectively assembled into tubules and were easily purified. Subsequently, using in vitro assay systems, we demonstrated that each purified chimeric particle was capable of eliciting strong immune responses. Further, NS1-FMDV chimeric tubules could induce a potent immune response that could protect against disease.     

Identification of an immunodominant sequential epitope in glycoprotein G of herpes simplex virus type 2 that is useful for serotype-specific diagnosis

A series of 67 oligopeptides that spanned the open reading frame of herpes simplex virus type 2 (HSV-2) glycoprotein G (gG2) were synthesized and tested for reactivity with 173 serum specimens collected from 117 individuals. The oligopeptides were made as multiple antigenic peptides consisting of four copies of a unique sequence attached to a branched lysine core and separated from the core by four glycine residues. The sera included HSV antibody-negative samples as well as sera from individuals from whom HSV had been isolated. Isolated viruses were typed by indirect fluorescence using a panel of type-specific monoclonal antibodies. One peptide, corresponding to residues 561 to 578 of gG2, did not react with any sera lacking HSV-specific antibodies or with sera from HSV-1-infected individuals, but did react with sera from HSV-2-infected individuals. For sera taken seven or more days after initialclinical lesions, the detection rate of the peptide was 92% (47/51), comparable with the 98% (50/51) of truncated glycoprotein D, a sensitive type-common reagent. We conclude that this peptide, of structure (PEEFEGAGDGEPPEDDDSG₄)K₃A, is an immunodominant type-specific epitope for human antibodies and should be useful for type-specific serodiagnosis of HSV-2. Surprisingly, the epitope lies within one of the most conserved regions of gG1 and gG2. The test can distinguish an initial HSV-2 infection in the presence of a preexisting HSV-1 infection. J. Med. Virol. 56:79–84, 1998. © 1998 Wiley-Liss, Inc.     

The use of peptides from glycoproteins G-2 and D-1 for detecting herpes simplex virus type 2 and type-common antibodies.

BACKGROUND: identification and discrimination of latent herpes simplex virus (HSV) infection relies on antibody identification. The inclusion of synthetic peptides with HSV glycoproteins provides means for stable and discriminatory assays for population studies. OBJECTIVE: to determine whether virus-specific synthetic peptides might identify HSV type 2 (HSV-2) antibodies in the presence of the cross-reactive and more common HSV type 1 (HSV-1) antibodies. STUDY DESIGN: the capacity of synthetic peptides as HSV antigens was analyzed in enzyme immunoassay (EIA) using well characterized human serum cohorts. The HSV peptide assays were evaluated in comparison with two commercial HSV-2 assays. RESULTS: a combination of two C-terminal HSV-1 glycoprotein D (gD-1) peptides detected type-common HSV immunoglobulin G (IgG) with high sensitivity (95%) and specificity (93%). Peptides derived from the C-terminus of HSV-2 glycoprotein G (gG-2) had a high HSV-2 type-specificity. Inclusion of both gD-1 and gG-2 peptides gave a sensitivity for human anti-HSV-2 IgG that was similar to that of assays including different amounts of native gG-2. With western blotting as a standard, the sensitivity of the peptide assay ranged between 86% for HSV-2 seropositive persons and 61% for HSV-2 seroconverters. Addition of a small amount of native gG-2 to the peptide assay tended to increase the specificity. CONCLUSION: HSV gG and gD peptides show promise as type-specific and type-common HSV antigens. These peptides are more stable and reproducibly prepared than native or recombinant glycoproteins and may be considered for inclusion in future HSV serodiagnostic assays.     

Peptide based enzyme-linked immunoassays for detection of anti-HSV-2 IgG in human sera

Glycoprotein G of HSV-2 (gG2) and a peptide, corresponding to a previously recognised immunodominant epitope spanning residues 561-578 of the protein, were compared directly for type-specific serodiagnosis of HSV-2. The protein was affinity purified and obtained in a commercially available EIA kit while the peptide, previously designated as peptide 55, was made as a multiple antigenic peptide. A panel of 100 characterised serum samples (60 HSV-2 positive, 20 HSV-1 positive and 20 HSV negative) was screened using the two antigens. The intact protein and peptide 55 showed the same sensitivity for antibodies in the serum of HSV-2 infected individuals, reacting with 96.7% (58/60) of the samples. The peptide did not react with any of the HSV-1 positive or HSV negative sera. In contrast, gG2 gave a number of false positive results, reacting with 20% (4/20) of the HSV-1 positive sera and 10% (2/20) of the HSV negative sera. The superior performance of peptide 55, together with the very much lower costs of its production, compared with gG2 suggest that the peptide will become the antigen of choice in enzyme immunoassays for type-specific serodiagnosis of HSV-2.     

A peptide mimotope of type 8 pneumococcal capsular polysaccharide induces a protective immune response in mice

Increasing antibiotic resistance and a rising patient population at risk for infection due to impaired immunity underscore the importance of vaccination against pneumococci. However, available capsular polysaccharide vaccines are often poorly immunogenic in patients at risk for pneumococcal disease. The goal of this study was to explore the potential of peptide mimotopes to function as alternative vaccine antigens to elicit a type-specific antibody response to pneumococci. We used a human monoclonal immunoglobulin A (IgA) antibody (NAD) to type 8 Streptococcus pneumoniae capsular polysaccharide (type 8 PS) to screen a phage display library, and the phage PUB1 displaying the peptide FHLPYNHNWFAL was selected after three rounds of biopanning. Inhibition studies with phage-displayed peptide or the peptide PUB1 and type 8 PS showed that PUB1 is a mimetic of type 8 PS. PUB1 conjugated to tetanus toxoid (PUB1-TT) induced a type 8 PS-specific antibody response in BALB/c mice, further defining it as a mimotope of type 8 PS. The administration of immune sera obtained from PUB1-TT-immunized mice earlier (days 14 and 21) and later (days 87 and 100) after primary and reimmunization resulted in a highly significant prolongation of the survival of naive mice after pneumococcal challenge compared to controls. The survival of PUB1-TT-immunized mice was also prolonged after pneumococcal challenge nearly 4 months after primary immunization. The efficacy of PUB1-TT-induced immune sera provides proof of principle that a mimotope-induced antibody response can protect against pneumococci and suggests that peptide mimotopes selected by type-specific human antibodies could hold promise as immunogens for pneumococci.     

Selection of phage-displayed peptides recognised by monoclonal antibodies directed against the lipopolysaccharide of Brucella

Panning and screening of various phage display libraries with monoclonal antibodies (mAbs) directed against the O-chain of the lipopolysaccharide (LPS) of Brucella sp. allowed the identification of peptidic mimotopes of some O-chain epitopes. Four mAbs were tested. The A76-12G12 mAb, which is specific for LPS of all strains of Brucella, either A- or M-dominant, did not yield any peptidic mimotope, despite a specific yield enrichment during the rounds of panning. The B66-4F9 mAb, that recognises an epitope common to both Brucella sp. and Yersinia enterocilitca O:9 strains, allowed the selection of only one phage clone that was shown to be an antigenic but not immunogenic mimotope. The B66-2C8 and A15-6B3 mAbs, respectively, specific for the LPS of A-dominant and M-dominant Brucella sp., yielded several sequences, which allowed the determination of consensus sequences. These consensus will be of high interest for the construction of second generation libraries. For the best binding peptides, competition with LPS for the binding to the mAb is detected, which suggests that the peptides bind to the paratope of the mAb. The phages selected from the libraries were used to immunise mice, and a weak antibody response directed against LPS has been observed for some peptides. These data suggest that a subset of the selected peptides are immunogenic mimotopes of the LPS epitopes.     

Induction of anti-carbohydrate antibodies by phage library-selected peptide mimics

One of the prerequisites for the development of polysaccharide subunit vaccines is the induction of an efficient immune response to carbohydrate antigens like lipopolysaccharide (LPS) or capsular polysaccharide antigens of pathogens. In an attempt to overcome the problems that arise from the T-independent immune response induced by such antigens, selecting peptide sequences that mimic protective carbohydrate epitopes has been proposed. In this study, we investigate a new selection strategy for immunogenic peptide mimics using the phage displayed peptide library technology. Two monoclonal antibodies (mAb) of the A isotype (mIgA), mIgA C5 and mIgA I3, specific for the O-antigen (O-Ag) part of the human pathogen Shigella flexneri serotype 5a LPS and protective against homologous infection were used to screen two phage-displayed nona-peptide libraries in pVIII. Using mIgA C5, 13 different specific clones were selected, and 6 using mIgA I3; 5 of the latter also interacted in enzyme-linked immunosorbent assay with the first mAb. All of the 19 clones selected were separately used to immunize mice, but only 2 of them, p100c (mIgA I3-specific) and p115 (interacting with both mIgA) were able to induce anti-O-Ag antibodies. The immune response was specific for the O-Ag of the S. flexneri serotype 5a, and also selectively recognized the corresponding bacterial strain. The amino acid sequences of p100c and p115 immunogenic peptide mimics were YKPL-GALTH (flanked by two Cys residues) and KVPPWARTA, respectively. These results are the first example of immunogenic mimicry of carbohydrates by phage-displayed peptides, and indicate a new strategy of selection of immunogens for the development of anti-polysaccharide vaccines.     

Immune response genes modulate serologic responses to Vibrio cholerae TcpA pilin peptides.

Cholera is an enteric disease caused by Vibrio cholerae. Toxin-coregulated pilus (TCP), a type 4 pilus expressed by V. cholerae, is a cholera virulence factor that is required for host colonization. The TCP polymer is composed of subunits of TcpA pilin. Antibodies directed against TcpA are protective in animal models of cholera. While natural or recombinant forms of TcpA are difficult to purify to homogeneity, it is anticipated that synthesized TcpA peptides might serve as immunogens in a subunit vaccine. We wanted to assess the potential for effects of the immune response (Ir) gene that could complicate a peptide-based vaccine. Using a panel of mice congenic at the H-2 locus we tested the immunogenicity of TcpA peptide sequences (peptides 4 to 6) found in the carboxyl termini of both the classical (Cl) and El Tor (ET) biotypes of TCP. Cl peptides have been shown to be immunogenic in CD-1 mice. Our data clearly establish that there are effects of the Ir gene associated with both biotypes of TcpA. These effects are dynamic and dependent on the biotype of TcpA and the haplotypes of the host. In addition to the effects of the classic class II Ir gene, class I (D, L) or nonclassical class I (Qa-2) may also affect immune responses to TcpA peptides. To overcome the effects of the class II Ir gene, multiple TcpA peptides similar to peptides 4, 5, and 6 could be used in a subunit vaccine formulation. Identification of the most protective B-cell epitopes of TcpA within a particular peptide and conjugation to a universal carrier may be the most effective method to eliminate the effects of the class II and class I Ir genes.     

Detection of human serum antibodies against type-specifically reactive peptides from the N-terminus of glycoprotein B of herpes simplex virus type 1 and type 2 by surface plasmon resonance

A single-step surface plasmon resonance protocol for the detection of antibodies against herpes simplex virus type 1 and type 2 (HSV-1, HSV-2) in human sera was established using the BIAcore system. Two peptides from corresponding segments of the N-terminus of HSV-1 and HSV-2 glycoprotein B (gB), i.e. peptide gB-1 (60-73) (GAAPTGDPKPKKNK) and peptide gB-2 (55-68) (SPATTKARKRKTKK), were identified as immunogenic. Employing both peptides as diagnostic antigens in the surface plasmon resonance assay, a sensitivity for the detection of HSV-1 and HSV-2 type-specific antibodies of 83 and 86%, respectively, was achieved as compared with immunoblotting as a reference method. Peptide gB-1 (60-73) allowed the discrimination between HSV-1 and HSV-2 type-specific antibodies with a specificity of 67%, whereas peptide gB-2 (55-68) reacted in a strictly HSV-2 type-specific manner. It is concluded that peptides from the N-terminus of gB-1 and gB-2 are recognized predominantly by human sera in an HSV-specific manner. Peptide gB-2 (55-68) can be employed successfully for the determination of type-specific antibodies against HSV-2.     

Selection of Phage-displayed Peptides Recognised by Monoclonal Antibodies Directed against the Lipopolysaccharide of Brucella

Panning and screening of various phage display libraries with monoclonal antibodies (mAbs) directed against the O-chain of the lipopolysaccharide (LPS) of Brucella sp. allowed the identification of peptidic mimotopes of some O-chain epitopes. Four mAbs were tested. The A76–12G12 mAb, which is specific for LPS of all strains of Brucella, either A-or M-dominant, did not yield any peptidic mimotope, despite a specific yield enrichment during the rounds of panning. The B66–4F9 mAb, that recognises an epitope common to both Brucella sp. and Yersinia enterocilitca O:9 strains, allowed the selection of only one phage clone that was shown to be an antigenic but not immunogenic mimotope. The B66–2C8 and A15–6B3 mAbs, respectively, specific for the LPS of A-dominant and M-dominant Brucella sp., yielded several sequences, which allowed the determination of consensus sequences. These consensus will be of high interest for the construction of second generation libraries. For the best binding peptides, competition with LPS for the binding to the mAb is detected, which suggests that the peptides bind to the paratope of the mAb. The phages selected from the libraries were used to immunise mice, and a weak antibody response directed against LPS has been observed for some peptides. These data suggest that a subset of the selected peptides are immunogenic mimotopes of the LPS epitopes.     

Comparative Performance of a Novel Herpes Simplex Virus Type 2-Specific Enzyme-Linked Immunosorbent Assay Using a Targeted Chain Oligopeptide,Peptide 55

Herpes simplex virus (HSV) glycoprotein G (gG2) has been used as the basis of many serological assays for the detection of HSV type 2 (HSV-2)-specific antibodies. In the present study, an enzyme-linked immunosorbent assay (ELISA), the Pathozyme Viro HSV-2 immunoglobulin G (IgG) ELISA (Omega Diagnostics, Alva, United Kingdom), based on an immunodominant epitope of gG2 presented in a branched-chain format (peptide 55), was compared with two commercially available gG2-specific assays, the Bioelisa HSV-2 IgG assay (Biokit, S.A., Barcelona, Spain) and the HerpesSelect HSV-2 IgG assay (Focus Diagnostics, Cypress, CA). A panel of 218 well-characterized serum samples was tested. Thirty-one samples were determined to be HSV-2 IgG antibody positive and 164 samples were determined to be negative with all three kits. The levels of concordance between the tests were 95.9% between the Omega and HerpeSelect assays, 90.8% between the Omega and Bioelisa assays, and 94.5% between the HerpeSelect and Bioelisa assays. Twenty-three samples gave discordant results. Western blot results showed that of these, the results for 77% were correctly identified by the Omega assay, the results for 68% were correctly identified by the HerpeSelect assay, and the results for 13.6% were correctly identified by the Bioelisa assay. Although there was a high level of agreement between the results obtained by the three assays and no false-positive results were detected by any of the three kits, confirmation of the results for samples with discordant results by Western blotting suggested that the peptide 55-based Omega assay is the most sensitive and specific assay among the assays evaluated.Genital herpes is one of the most common sexually transmitted infections worldwide. Historically, herpes simplex virus type 2 (HSV-2) has predominantly been associated with genital infections; however, recent reports suggest that a considerable and increasing number of genital isolates are of HSV-1 (10, 24). The clinical course of primary genital herpes infections among patients infected with HSV-1 and HSV-2 are similar; however, there are differences in epidemiology and natural history of the diseases caused by the two viral subtypes (9). HSV-2 primary infection during pregnancy has been related to spontaneous abortion, prematurity, congenital infection, and neonatal herpes. Several recent studies have reported that genital herpes may increase the susceptibility of acquiring human immunodeficiency virus by persons with high-risk behavior, because HSV-2 can cause breaks in the genital mucosal barrier (16).HSV-1 and HSV-2 have approximately 83% nucleotide sequence similarity, and for some proteins they share more than 85% identity (4). Both serotypes therefore show extensive serological cross-reactivity. This has impeded seroepidemiologic studies of the two viruses. The major antigenic determinants are glycoprotein antigens exposed on the virion surface (22), and although HSV-1 and HSV-2 are known to express more than 11 glycoproteins, 10 of these glycoproteins express multiple epitopes common to each type. A variety of studies have concluded that only glycoprotein G (gG2) expresses epitopes specific for HSV-2 (14, 21). Consequently, this glycoprotein has been used as the basis of type-specific serological assays for the diagnosis of HSV-2 infection (1, 2, 7, 19, 20). However, epitope mapping of gG2 by Levi et al. (11) suggested that some epitopes may cross-react with HSV-1-specific antibody.In an effort to develop even greater selectivity for HSV-2, several research groups have studied HSV-2 type-specific epitopes within gG2. Regions comprising amino acids 350 to 427 and 525 to 587 and also regions comprising amino acids 625 to 641 and 676 to 699 were identified by two independent groups (6, 11), and a secreted protein of gG2 comprising amino acids 23 to 340 was described by Liljeqvist et al. (13) and Gorander et al. (5). Marsden et al. (15) developed multiple antigenic peptides corresponding to residues 561 to 578 of gG2, designated peptide 55. This peptide comprises four peptide copies attached to a branched lysine core and separated from the lysine core by four glycine residues. The sensitivity of the peptide for the detection of HSV-2 antibody was evaluated by screening a panel of HSV-2-positive human serum samples previously characterized by virus isolation and typed by indirect immunofluorescence with a type-specific monoclonal antibody. Peptide 55 was found to be sensitive and specific, and no false-positive results were detected with HSV-1 antibody-positive or HSV-1 or HSV-2 antibody-negative samples.Oladepo et al. (18) compared the performance of an assay with peptide 55 with that of a commercially available HSV-2-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) kit based on affinity-purified gG2 (Gull Laboratories) and found that peptide 55 had the same sensitivity; however, it was more specific than the complete protein. Nilsen et al. (17) tested the performance of an ELISA based on peptide 55 and compared it with the performance of two different assays, an ELISA developed by Ho et al. (8) based upon native gG2 selected by affinity to Helix pomatia lectin, and an ELISA based on affinity-purified gG2 (Gull Laboratories), and found that the performance of the assay with peptide 55 was better than that of the other ELISA methods.In the present study, an ELISA (Pathozyme Viro HSV-2 ELISA IgG; Omega Diagnostics Ltd., Scotland) based on peptide 55 was compared with two other commercially available HSV-2-specific IgG ELISAs, the HerpeSelect HSV-2 ELISA (Focus Diagnostics, Cypress, CA), which uses purified recombinant type-specific gG2 antigen, and the Bioelisa HSV-2 IgG-specific assay (Biokit, S.A., Barcelona, Spain), which uses affinity-purified gG2 protein, for the abilities to detect and serologically differentiate HSV-2 antibody. The three assays are referred to in this study as the HerpeSelect, Bioelisa, and Omega assays, respectively.     

Comparison of phage pIII, pVIII and GST as carrier proteins for peptide immunisation in Balb/c mice

Carrier proteins are important for improving the efficiency of synthetic peptide vaccines. Recently, genetically-based systems such as filamentous phage display or glutathione S-transferase (GST)-fusion proteins have been employed for immunisation. Whilst these carrier systems can facilitate the evaluation of a potential vaccine by reducing the time and cost of production, their relative efficacy and the kinetics of the immune response to each carrier has not been directly compared. In this study, we have displayed the epitopes of the anti-ErbB-2 Mabs N12 (C531-A586, EP531) and N28 (T216-C235, EP216) on phage minor coat protein pIII, major coat protein pVIII and GST. Balb/c H-2(d) mice were immunised with the constructs and the sera were tested after the initial, the 3rd, 5th and 6th immunisations for an anti-peptide, an anti-ErbB-2 and an anti-carrier response. The specificity of the antibody response was also mapped using synthetic peptides. It was found that GST was the best of the three carriers, both in terms of the magnitude and the kinetics of the induced anti-peptide and anti-ErbB-2 response. Multiple (five) administrations of the immunogens were necessary to obtain a high titre of antibodies specific for ErbB-2. It was further noted that whilst an anti-EP531 response was induced using all three carriers, EP216 was not immunogenic irrespective of the carrier used. The lack of immunogenicity of EP216 implies it does not contain a H-2(d) T cell recognition site. All three carriers provide a useful system for vaccination and consequently facilitate the identification of T-cell epitopes in Balb/c inbred mice.