Multiple antigenic peptides as vaccine platform for the induction of humoral responses against dengue-2 virus

Dengue is an important agent of human disease for which no licensed vaccine is available to the public. We used multiple antigenic peptides (MAPs) as an antigen carrier for the development of subunit vaccines against dengue-2 virus (DEN-2). Commercially available software (MacVector 7.0) was used to identify potential antigenic B-cell epitopes of E-glycoprotein. A total of 60 BALB/c mice were immunized with 12 recombinant DEN-2-specific MAPs and the humoral immune response was assessed by anti-DEN-2 ELISA and PRNT50 assays. Anti-DEN-2 ELISA showed high levels of anti-DEN-2 antibodies and post-immune sera reduced viral infectivity and prevented infection of monkey kidney cells (LLC-MK2) with live DEN-2 virus. Seven neutralizing DEN-2 epitopes were identified that generated PRNT50 titers of up to 1:160. Our findings show that the MAP platform can be used as an antigen-presenting platform for dengue vaccine development.     

Detection of Infectious bursal disease virus by ELISA using an antipeptide antibody raised against VP3 region

Antigenic determinant analysis was carried out on VP3, one of major immunogenic proteins of Infectious bursal disease virus (IBDV) using computer algorithms. Altogether 17 peptides were synthesized for predicted putative regions and were tested for their reactivity with IBDV-positive polyclonal sera as well as with antisera to other common avian viruses to confirm specificity and to rule out cross reactivity. Of 17 peptides tested, three were selected and synthesized in multiple antigenic peptide (MAP) format. The immunization of rabbits with the three MAPs resulted in high humoral immune response. The purified antipeptide antibodies were screened against native IBDV antigen and the respective titers were determined. Out of the three antisera to MAPs that raised against the MAP3, spanning the amino acids (aa) 974-995 region on the VP3 protein had a very high titer (2048) and reacted specifically with IBDV. Thus, the antiserum to MAP3 detected native virus in enzyme-linked immunosorbent assay (ELISA), revealing the presence of a potential antigenic determinant on the C-terminus of the protein. This study proved that an antipeptide antibody could be used as a safe and specific tool for the diagnosis of IBD in chickens.     

In vitro modification of solid phase multiple antigenic peptides/autoantigens with 4-hydroxy-2-nonenal (HNE) provide ideal substrates for detection of anti-HNE antibodies and peptide antioxidants

The role of free radicals in protein modification and the importance of anti-4-hydroxy-2-nonenal (HNE) antibodies as marker of HNE-mediated cell toxicity has been well documented. Proteins modified by HNE in vitro, prior to immobilization on ELISA plates, have served as substrates for assaying these antibodies. We found preferential binding of HNE-modified versus unmodified proteins to ELISA plates and this prompted us to seek a more reliable assay. We report a method to HNE-modify any cysteine/histidine/lysine-containing protein or multiple antigenic peptide (MAP) following their immobilization on an ELISA plate. To a set of wells, HNE (200 microM) dissolved in PBS is added and incubated for 4 h, followed by regular ELISA. Since HNE was supplied dissolved in ethanol, PBS with appropriate amount of ethanol added was used as control. For inhibition experiments, HNE is incubated with or without inhibitors and then added to the wells. The commercial anti-HNE serum bound only to HNE-modified antigens. Sera from rabbits and mice immunized with HNE-modified 60 kDa Ro autoantigen preferentially bound the modified antigens. Modification of solid phase antigens in this manner makes assaying anti-HNE antibodies unambiguous. Lengthy dialysis procedures or the use of spin columns that lead to antigen loss becomes unnecessary for the separation of free HNE. We were able to HNE-modify various antigens (BSA, the autoantigens Ro, La and Sm/nRNP, 60 kDa Ro and Sm MAPs) using this procedure. Using MAPs, we confirmed the importance of histidine, lysine and cysteine residues in HNE modification. In addition, this method allowed identification of inhibitors of HNE-modification. We obtained 61%, 70% and 74% inhibition of HNE-modification of solid phase Ro MAP 166 substrate using BSA, Ro MAP 482 and Ro MAP 166, respectively. Glycyl-proline dipeptide and a MAP from the Sm autoantigen (PPPGMRPP) showed 0% inhibition of HNE-modification.     

The use of monoclonal antibody probes for the detection of infectious bursal disease virus antigens

Two monoclonal antibodies (MAb), 2E6 and 2G10, were used against infectious bursal disease virus (IBVD) P3009 in an immuno-dot assay to detect IBDV antigens from cell culture, and from bursa and spleen tissue samples of chickens. The limit of viral antigens detected by using both MAb probes was 48 ng. The probes were used to detect five serotype 1 IBDV isolates and one serotype 2 IBDV strain. The result indicated that these probes had broad specificity. The probes, however, did not cross-react with viral antigens prepared from seven unrelated avian viruses. The probes detected IBDV antigens in bursa and spleen tissues collected from chicks as early as 2 days after inoculation. The IBDV antigens in bursa tissue samples from six poultry farms were detected by both probes. However, tissue samples from two of six farms did not react with probe 2E6. The data suggest that it is possible to use two MAb probes for diagnosis of IBDV infection in poultry farms.     

Application and limitations of the multiple antigen peptide (MAP) system in the production and evaluation of anti-peptide and anti-protein antibodies.

The multiple antigen peptide (MAP) system has been proposed as a novel and valuable approach for eliciting antibodies to peptides and developing synthetic vaccines. The MAP system consists of a small immunogenically inert core matrix of lysine residues with alpha- and epsilon-amino groups for anchoring multiple copies of the same or different synthetic peptides. Several MAP systems, each containing eight copies of 6-15 residue-long peptides derived from the terminal and central regions of various proteins were analyzed in this study. The immunogenicity of MAPs was compared to that of the same peptides linked to carrier protein by means of conventional conjugation procedures. The various peptide antisera were tested in ELISA with homologous peptides conjugated to a carrier protein via their C terminal (as in the MAP system) or their N terminal end, or with their parent proteins. The antigenic properties of MAPs were studied with anti-peptide sera obtained by classical methods and with anti-protein sera. The results showed that the MAP system was an efficient antigen in ELISA except when the peptide corresponded to a C terminal epitope. However, the value of MAPs for raising anti-peptide antibodies cross-reactive with the cognate protein appeared much more limited. In the case of one N terminal peptide, the MAP construction was not immunogenic while the conventionally conjugated peptide induced antibodies that reacted strongly with the corresponding protein. In the case of the two C terminal peptides tested, the antibodies raised against MAP constructs reacted well with homologous MAPs but did not cross-react with the whole protein. Only in the case of a peptide from an internal domain of histone H2A did immunization with a MAP generate antibodies that cross-reacted with the protein.     

Characterization of the antigenic,immunogenic, and pathogenic variation of infectious bursal disease virus due to propagation in different host systems (bursa,embryo, and cell culture). II. Antigenicity at the epitope level

Antigenic variation of infectious bursal disease virus (IBDV) due to propagation in different host systems (bursa of Fabricius, embryos, or cell cultures) was determined by enzyme-linked immunosorbent assay (indirect and antigen capture) and western blot analysis. To conduct this study, we used 27 non-neutralizing anti-VP 2 monoclonal antibodies, a reference panel of nine neutralizing monoclonal antibodies, and 13 neutralizing anti-IBDV chicken polyclonal antibodies. Changes occurred in neutralizing, cross-reactive, conformation-dependent epitopes on the VP 2 protein of IBDV. Interestingly, non-neutralizing, cross-reactive, conformation-dependent and confirmation-independent epitopes also changed on VP 2 . These epitope changes were directly associated with the method used to propagate IBDV. These results demonstrate that different host systems may play an important role in the antigenicity of IBDV.     

Use of linear and multiple antigenic peptides in the immunodiagnosis of acute hepatitis A virus infection

The reactivities of two panels of anti-HAV human sera from geographically distinct areas (Chile and Spain) to synthetic peptides from the VP1, VP2 and VP3 hepatitis A virus capsid proteins were examined by an enzyme-linked immunosorbent assay (ELISA) procedure. Two and four branched multiple antigenic peptides (MAPs) and palmitoylated peptides were compared with free synthetic sequences for the detection of IgM anti-HAV antibodies in the two panels of human sera. Our results showed that acute hepatitis A patient sera recognized preferentially homogeneous two branched MAPs and palmitic acid conjugated peptides. The palmitoyl-derived VP3(110-121) peptide and the corresponding dimeric MAP were the most sensitive and appropriate for serological studies of HAV-infected patients by ELISA, sensitivity and specificity being higher than 90% and 95%, respectively. These peptide-based tests open up new avenues in the development of peptide-based immunosorbent assays for the detection of acute HAV disease.     

A monoclonal antibody-based agar gel precipitin test for antigenic assessment of infectious bursal disease viruses.

Five monoclonal antibodies (MAbs) directed at different neutralization epitopes of infectious bursal disease virus (IBDV) precipitated IBDV antigens in double immunodiffusion. Based on the positive or negative reaction of some of the MAbs in agar gel precipitin tests (AGPT), four different antigenic species of IBDV could be discerned. Although the antigen-capture enzyme-linked immunosorbent assays (AC-ELISA) were more sensitive than AGPTs for differentiating IBDV types, use of the MAbs in AGPT did offer a useful and accessible technique for differentiating wild type IBDV directly from infected tissues taken in the field.     

Limited antigenic variation among recent infectious bursal disease virus isolates from France

Summary.  Seven neutralizing monoclonal antibodies (Mabs) to infectious bursal disease virus (IBDV) were used in an antigen-capture ELISA for the antigenic characterization of 58 IBDV isolates obtained in France since 1989. Fifty-six isolates exhibited an antigenic profile which was different from reference strain Faragher 52/70, and similar to French very virulent IBDV strain 89 163 (no binding of two Mabs). Two strains (3.4%), isolated in 1991 and 1994, showed additional antigenic modifications resulting in suppressed or reduced binding activity of three other Mabs. The two atypical viruses were not re-identified in field samples subsequently collected, hence showing that antigenic variants of IBDV do not tend to replace the antigenically dominant 89 163-like strains that have prevailed in France since 1989. Received March 14, 1997 Accepted June 2, 1997     

Precipitation of bovine rotavirus by polyethylene [corrected] glycol (PEG) and its application to produce polyclonal and monoclonal antibodies

Many protocols are available to produce monoclonal antibodies (MAbs) against bovine rotavirus (BRV) using purified virus particles but those methods are time-consuming and produce substantial loss of virus structure or infectivity. Polyethylene gycol (PEG) viral precipitation was investigated as a possible alternative method to obtain purified virus and viral antigen for monoclonal and polyclonal antibodies production. The antigenic mass inoculated into rabbits was sufficient to obtain a polyclonal hyperimmune serum with neutralizing activity and a wide range of humoral responses to different viral proteins with minimum cellular contamination. This antigenic mass inoculated into mice was capable of producing seven MAbs with different biochemical characteristics (Western-blot; indirect immunofluorescence and serum neutralization assays). When evaluated as diagnostic tools to detect BRV antigen in feces the MAbs were effective for detecting rotavirus in naturally infected calves. This work demonstrate that PEG precipitation could be an useful procedure for obtaining viral antigen to produce polyclonal and MAbs against BRV. The antigenic mass obtained, the viral infectivity, and the conserved protein pattern suggest that this methodology could be applied to this and to other viruses, reducing time or possible loss of antigens involved in viral purification.     

Possible relationship between antigenic properties and isolation history of HSV-1 strains

29 monoclonal anti-herpes simplex virus (HSV)-1 antibodies were produced and characterized with regard to virus neutralizing activity, intracellular or cell-surface location of viral antigens and, where possible, molecular weight of the viral protein recognized. 13 antibodies recognized viral antigens expressed on the surface of infected cells and 16 were directed to intracellular viral components. Only two antibodies exhibited virus neutralizing activity. Application of these antibodies to an antigenic comparison of standard laboratory HSV-1 strains F, HFEM, mP, Glasgow-17 and MAC revealed unique antigenic differences among these strains. The antibodies were further used in an antigenic comparison of 45 human HSV-1 isolates with defined isolation history. Except for two paired isolates from left and right trigeminal ganglia of two human cadavers, the antibody panel revealed antigenic differences among all isolates, including paired isolates from three additional cadavers. Overall, isolates from different human donors showed greater antigenic dissimilarity from each other in cell-surface associated than in intracellular antigens. The data suggest the possibility of a correlation between antigenic and biologic properties of HSV-1.     

New multiple antigenic peptide-based enzyme immunoassay for detection of simian immunodeficiency virus infection in nonhuman primates and humans

Infections with human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2, respectively) are zoonotic infections. In Africa, the potential exists for additional cross-species transmissions from at least 33 different species of simian immunodeficiency virus (SIV)-infected nonhuman primates (NHPs) through hunting and butchering of these animals for food. Here we describe a highly sensitive and specific enzyme immunoassay (EIA) with chemically modified, multiple antigenic peptides (MAPs) developed for the detection and discrimination of antibodies to SIV genetic lineages. The SIV EIA was developed by using a comprehensive array of MAPs covering two envelope gene regions from all of the SIV lineages for which env sequences were available. Assay sensitivity was evaluated by using 63 plasma or serum samples obtained from primates naturally or experimentally infected with SIVs from 10 genetic lineages. Assay specificity was determined by using 97 known SIV-negative plasma specimens from these same species. Also used in the evaluations were 369 human samples: 198 HIV seronegative, 170 HIV-1 and/or HIV-2 seropositive, and 1 from a human SIVsm infection. Overall assay sensitivity and specificity were 100% with both immunodominant region (IDR) and V3 region MAPs. Although SIV env sequences from talapoin monkeys were not available for specific MAP inclusion, 5 (100%) of 5 SIVtal-infected samples were detected through cross-reactivity with other SIV IDR MAPs used in the assay. The one human SIVsm infection was identified. In conclusion, our SIV MAP EIA proved to be highly sensitive and specific for detecting SIV infections in NHPs and humans. As shown with SIV-infected talapoin monkeys, this assay has the potential to detect previously unidentified SIV strains and should be suitable for sentinel surveillance for potential new cross-species transmissions of SIVs to humans.     

Serologic analysis of dogs, horses, and cottontail rabbits for antibodies to an antigenic flagellar epitope of Borrelia burgdorferi.

Enzyme-linked immunosorbent assays (ELISA) and immunoblots using either whole-cell lysates of Borrelia burgdorferi or an antigenic region of flagellin (41-G) as the antigen were performed, and the abilities of the two assays to detect antibodies to this spirochete in dog, cottontail rabbit, and horse sera were compared. Assays using whole-cell B. burgdorferi lysates as the antigen were more sensitive for detecting antibodies. ELISA with 41-G as the antigen were specific for Borrelia antibodies but were not as sensitive as the assays with whole-cell lysates coated to the solid phase. Use of recombinant full-length flagellin, rather than 41-G, as the antigen in immunoblots increased the sensitivity of each assay. However, antibodies to other bacterial antigens cross-react with whole flagellin and may account for false-positive results. Antibodies to B. burgdorferi outer surface protein A or B were usually undetected when the sera were tested by immunoblotting methods. Borrelia lysates or the 41-G antigen may be used in ELISA or immunoblots to document host exposure to this spirochete. The use of 41-G as the antigen may increase the specificity of an assay or help confirm the serologic diagnosis of Lyme borreliosis in dogs, horses, and cottontail rabbits.     

Identification of phase-specific antigenic fractions of Coxiella burnetti by enzyme-linked immunosorbent assay.

Antigenic fractions of Coxiella burnetii phase variants were identified with an enzyme-linked immunosorbent assay (ELISA). Immune sera from guinea pigs immunized with Formalin-inactivated phase I or phase II whole cells were used to measure the antigenic activity of whole cells and various soluble and particulate preparations. Phase-specific antigens of C. burnetii whole cells and fractions were compared by dose-response curves at different (antigen and antibody) dilutions. Water-soluble extracts prepared by meta-periodate, ether, and phenol extraction of phase I whole cells yielded antigenic fractions which reacted with anti-phase I antibodies. The extraction of phase I whole cells with dimethyl sulfoxide, trichloracetic acid, and Formalin yielded antigenic fractions which detected antibodies in both anti-phase I and -phase II sera. Interestingly, the trichloracetic acid extract of phase I whole cells also contained a component which bound nonimmune immunoglobulin. The sera of animals immunized with whole cells of the phase II Australian QD strain reacted with lipopolysaccharides of the phase I and phase II Nine Mile strains. Therefore, variations in lipopolysaccharide structure among phase variants of C. burnetii were detected as cross-reactions with immune sera from an interspecific strain. Comparisons of immunofluorescence, microagglutination, and the complement fixation assays with the ELISA indicated greater sensitivity and specificity of the ELISA for the measurement of phase-specific antigens and antibodies.     

Enzyme-linked immunosorbent assays for sperm antibody detection and antigenic analysis

Enzyme-linked immunosorbent assays for estimation of antibodies against human sperm and for determination of antigenic reactivity of spermatozoal proteins were established. Sperm immobilized on PHA-coated microtiter plates or solubilized spermatozoal antigens adsorbed on poly(L)-lysine coated microtiter plates were used as the solid phase. Assay of sperm antibodies was performed by incubation of the test samples with the solid phase followed by incubation with anti-Ig conjugated to peroxidase. Sigmoidal antibody dilution curves were obtained with rabbit and mouse anti-sperm sera. The ELISA was effectively used to screen production of anti-sperm antibodies by mouse myeloma x splenocyte hybridomas. The sensitivity of this ELISA for sperm antibodies was more than 1000-fold greater than the classical tray sperm immobilization test, and was comparable in sensitivity to a radioimmunoassay using 125I-labeled protein A as the tracer. Sperm immobilized on PHA-coated plates exhibited significantly greater antigenic reactivity in both the ELISA and RIA compared with methanol fixed sperm. In a competitive inhibition ELISA, linear Logit-log dose-response curves were obtained with detergent solubilized spermatozoal antigens. The assay was used to monitor the purification of the solubilized spermatozoal antigens by chromatofocussing; a more than 60-fold increase of antigenic potency of purified sperm antigen compared with unfractionated sperm extract was evident in the competitive ELISA.     

Comparing tandem repeats and multiple antigenic peptides as the antigens to detect antibodies by enzyme immunoassay.

Short synthetic peptides are useful alternatives to whole lysate or recombinant proteins as the antigens used for serodiagnosis of bacterial or viral infections. However, certain known antigenic peptides displayed low seroreactivities in direct enzyme immunoassay. This was believed to be due to the low coating efficiency, a constrained orientation, or loss of flexibility required for optimal antibody binding. Using a model peptide system derived from the V3-loop of HIV-1 gp120, we demonstrated that low antigenicity could be overcome by using either tandem repeats (TR) or multiple antigenic peptides (MAPs) which contained the same amino acid sequence as the monomeric peptide. In our model system, a four-branch MAP was a better choice compared to the tandem repeats because of the MAP's slightly higher sensitivity and lower cost of production.     

Chicken recombinant antibodies against infectious bursal disease virus are able to form antibody-virus immune complex.

Virus particles exposed to specific anti-virus antibodies result in the formation of immune complexes (Icx). Recent vaccination strategies have employed this feature, and an infectious bursal disease virus (IBDV) vaccine based on Icx has been released and is expected to replace conventional IBDV vaccines. We evaluated whether chicken recombinant antibodies (rAb) specific for IBDV, rather than conventional chicken anti-IBDV sera, could be used to generate Icx. Out of 14 rAb expressed as soluble single-chain variable fragments (scFv), nine were able to completely neutralize Bursavac, a live IBDV vaccine, when tested in ovo. When these rAb were mixed with IBDV and inoculated into either 18-day-old embryos, or 1-day-old or 2-week-old specific pathogen free chicks, a rAb.IBDV complex was formed. These Icx were similar to those produced by polyclonal chick anti-IBDV sera and IBDV. Following inoculation of the rAb.IBDV complex, the virus was rendered non-infectious for 5 to 7 days. After this time virus was released from the Icx, resulting in infection of the inoculated chicks and subsequent induction of an immune response and protection against virulent IBDV challenge. The results indicated that genetically derived antibodies can replace polyclonal sera in the formulation of Icx vaccines.     

Multiple antigenic peptides facilitate generation of anti-prion antibodies

Recent reports have demonstrated the ability of anti-prion antibodies to inhibit PrPSc propagation. Due to the relatively poor immunogenic properties of both PrPC and PrPSc, the generation of anti-prion antibodies still causes a significant problem in the development of immunotherapeutic strategies. This study examines the potential of multiple antigenic peptides (MAPs) to raise an antibody response to prion derived sequences in mice. The MAP was constructed of a four spiked ring. Two spikes containing human or mouse derived prion amino acid sequences and two spikes containing the universally promiscuous tetanus toxoid sequence (aa 830-844) which was used to assist T-cell-dependent B-cell antibody production. Following vaccinations with the MAP or MAP plus adjuvant, sera were taken and antibody titres assessed. The MAP containing only the mouse sequence failed to elicit a significant antibody response. MAPs containing human prion sequences elicited antibody production to the corresponding prion sequence. Further analysis also demonstrated that these peptides were able to generate antibody responses that recognize conserved human and mouse sequences. These homologous sequences contain the heralded PrPSc specific sequence 'Tyr-Tyr-Arg' and therefore these MAPs may have some therapeutic potential.     

多种单抗联合检测HIV抗原

目的 建立多种单抗联合早期检测HIV抗原的夹心ELISA方法.方法 以SAS盐析沉淀法和亲和层析法纯化抗HIV-1 p24、gp41、gp120及抗HIV-2 gp36的腹水型单克隆抗体(McAb),用高碘酸钠法将纯化的McAb以HRP进行标记.建立针对单个抗原的双抗体夹心ELISA法,对其灵敏度及特异性进行检测.将筛选得到的4株捕获McAb按比例混合作为捕获抗体,4株酶标McAb按比例混合作为检测抗体,建立多种单抗联合检测HIV抗原的夹心ELISA方法,检测混合HIV抗原.结果 按确定的最优反应条件建立的多种McAb联合夹心ELISA方法,检测到的最高稀释度的HIV混合抗原中各抗原的终浓度分别为:重组HIV-1 p24:0.625 pg/ml,gp41:6.25 ng/ml,gp120:6.25 ng/ml;HIV-2 gp36:9.25 ng/ml.结论 建立了具有高度敏感性的鸡尾酒式多种单抗联合检测HIV抗原的夹心ELISA法,为早期榆测HIV抗原提供了新的思路,为后续的研究奠定了一定基础.     

Rapid diagnosis of influenza by detecting viral M protein in an immunoenzyme test

Previously developed ELISA test system with sensitivity about 0.5 ng/ml of virus protein M was used for influenza virus detection in nasopharyngeal washings of patients with acute respiratory virus diseases (ARVD). Altogether 184 specimens from patients with ARVD and 31 from patients with other infections were examined in the pre-epidemic and epidemic periods of 1983. In parallel, virus antigens were detected by direct immunofluorescence test (IF). The total frequency of viral antigen detection by ELISA and IF coincided (63% of the specimens examined). With the specimens collected early in the disease (2-3 days) the rate of virus findings by ELISA rose to 82%. Direct detection of viral antigen in nasopharyngeal washings by means of an objective immunochemical method seems to be promising for a large-scale rapid diagnosis of influenza.