Immunocontraceptive effect of Izumo and enhancement by combination vaccination

Sperm proteins are being investigated for their applications in the development of contraceptive vaccines (CV) in several laboratories. In the present study, various synthetic peptides based upon four sperm proteins, namely Izumo, fertilization antigen-1 (FA-1), YLP(12), and SP56 that are involved in various steps of the fertilization cascade were examined for their immunocontraceptive effect. The synthetic peptides were conjugated to four carrier proteins namely keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), chicken gamma-globulin, and chicken ovalbumin. Female mice were immunized with various peptide vaccines and each booster injection was given with the peptide conjugated to a different carrier protein. Two different fertility trials with different doses of the peptide vaccines were conducted to examine the contraceptive effect. Injection of 150 microg of the peptides (Trial II) caused a significantly higher immune response in serum as well as in the vaginal tract causing enhanced contraceptive effect than those observed after injection with 75 microg of the peptides (Trial I). Immunization with the peptide vaccines based upon Izumo, which is involved in sperm-egg plasma membrane fusion, caused a significant reduction in fertility. The contraceptive effect was enhanced by immunizing with the peptides based upon other antigens (FA-1, YLP(12), and SP56), resulting in an overall 73.33% reduction in fertility. When the antibodies against the peptides disappeared after >9-10 months from circulation and genital tract, all the animals regained fertility. These findings indicate for the first time that the immunization with Izumo and other sperm peptides namely FA-1, YLP(12), and SP56 induces antibodies in serum and genital tract that cause a reversible long-term contraceptive effect in female mice. The data further indicate that the proteins involved in sperm-egg fusion can also be used for contraceptive vaccine development. The contraceptive effects are enhanced by immunizing with the multipeptide vaccines.     

A contraceptive peptide vaccine targeting sulfated glycoprotein ZP2 of the mouse zona pellucida

In this study, we have mapped and characterized a B cell epitope of sulfated glycoprotein ZP2 (ZP2) as a step toward the development of a multi-epitope zona pellucida (ZP) vaccine. Recombinant polypeptides expressed by random deoxyribonuclease-digested fragments of ZP2 cDNA were screened for binding to IE-3, a monoclonal antibody to murine ZP2. Positive clones contained cDNA inserts encoding polypeptide corresponding to ZP2(103-134). When normal or ovariectomized female mice were immunized with three overlapping peptides that span this region of ZP2 (101-120, 111-130, 121-140), only ZP2(121-140) elicited IgG antibodies that reacted with mouse ovarian ZP, indicative of the presence of native B epitope and helper T cell epitope in ZP2(121-140). To more finely map the ZP2 B cell epitope, a random peptide display library was screened with the IE-3 antibody, and a consensus tetramer sequence VxYK that matched the ZP2(123-126) sequence VRYK was located. Competitive immunofluorescence analysis with single alanine-substituted VxYK peptides ranked the relative contribution of the three critical B cell epitope residues as Y > V > K. A chimeric peptide was constructed that contained the YRYK motif of ZP2 and a bovine RNase T cell epitope. Although (C57BL/6xA/J) F1 (B6AF1) female mice immunized with the chimeric peptide developed ZP antibody response, this peptide elicited antibody only in mice of the histocompatibility complex (MHC) H-2(k or b) haplotype. In contrast, ZP2(121-140) peptide elicited antibody in inbred mice with three additional mouse MHC haplotypes. Moreover, although ZP2(121-140) contained a T cell epitope, no oophoritis was observed after immunization of B6AF1 mice with ZP2(121-140) in complete Freund's adjuvant (CFA). In a preliminary trial, female B6AF1 mice immunized with ZP2(121-140) in CFA had reduced litter sizes as compared with mice injected with CFA alone.     

Mapping of dominant B-cell epitopes of a human zona pellucida protein (ZP1)

Zona pellucida (ZP) glycoproteins contain numerous antigenic determinants including carbohydrate, protein, and conformational epitopes; and the immunogenicity of these complex glycoproteins varies in different mammalian hosts. Studies have now shown that antibodies from primates immunized with a cDNA-expressed recombinant rabbit ZP protein (the homologue of the human ZP1 [hZP1]) inhibit sperm binding to the ZP without altering ovarian function, unlike immunization with ZP3 and ZP2 protein families. The ZP1 protein or peptides derived from it (recombinant or synthetic) are therefore primary candidates for use in designing safe and reversible human and animal contraceptive vaccines. In order to define peptide epitope(s) that may be critical for eliciting an immune response sufficient to effect immunological contraception without causing any adverse effects on ovarian physiology, studies have been carried out to identify immunodominant B-cell epitopes of the ZP1 protein. The amino acid sequence of the hZP1 was used to design a set of 94 (15-mer) biotinylated peptides having an overlap of 9 amino acids. Using these peptides in a modified enzyme-linked immunoassay, antibodies in sera from rabbits or baboons immunized with native porcine ZP protein were screened for ZP1 peptide recognition. These studies demonstrate that there are a limited number of peptides recognized by primate antibodies but that the overlapping peptides sharing the sequence GPLTLELQI are recognized by both rabbit and baboon antibodies regardless of the adjuvant system used to induce the immune response. This peptide is 100% conserved in amino acid sequence between the human and pig, although the rabbit protein has two conserved amino acid substitutions (100% similar, 77% identical). Because this peptide is immunogenic as well as antigenic in primates, it could play a major role in the development of human contraceptive vaccines.     

Major histocompatibility complex-linked control of the murine immune response to myelin basic protein

Recent experiments have shown that different regions of myelin basic protein (MBP) are encephalitogenic for different inbred strains of mice. It was therefore of interest to determine whether the immune response to MBP was MHC associated, and if so, what subregion controlled this response. Because PL/J and A/J mice were good responders to mouse MBP and C57Bl/10SN were not, B10.PL(73NS) and B10.A mice were immunized with mouse MBP under conditions designed to induce EAE. These strains were found to be highly susceptible. Intra-H-2 recombinant mice were then assessed for susceptibility. B10.A(4R) and B10.MBR were susceptible, whereas B10.A(5R) were resistant. Thus, EAE induced by purified MBP is under the control of the MHC, and the response maps to the I-A subregion. Production of IL 2 in vitro by T cells from MBP-primed mice in the presence of antigen and adherent cells was blocked by monoclonal antibody to the I-A, but not the I-E, subregion. When the specificity of the encephalitogenic response was tested, peptide 1-37 was active in B10.PL(73NS) and B10.A mice, whereas peptide 89-169 was active in A.SW, SWR, and B10.T(6R) strains. Serum from mice immunized with MBP peptides was assayed for antibody content. PL, B10.PL, and B10.A mice made a good antibody response to peptides 1-37 and 43-88 but were nonresponsive to peptide 89-169. SJL, A.SW, SWR, and B10.T(6R) mice responded well to peptide 89-169 but were poorly responsive to peptides 1-37 and 43-88.     

Response of cynomolgus macaques to immunization against a synthetic peptide from the human zona pellucida

Abstract: This study tested immunogenicity of a synthetic peptide hZP3_327–341 from a human zona pellucida (ZP) glycoprotein. After antibody response to various peptide-carrier conjugates was assessed in mice, two female cynomolgus macaques were immunized with the peptide conjugated to keyhole limpet hemocyanin (KLH). A control macaque was immunized with KLH. The peptide was immunogenic in both species, and included both B and T cell epitopes since low to moderate titers of peptide-specific antibodies and a T cell proliferative response were measured. Profiles of ovarian steroid metabolites indicated unchanged ovarian function in the macaques, but only the control conceived when bred. Ovarian histology was normal except that immunoglobulin was bound to ZP in follicles of the peptide-immune macaques. ZP from these females bound sperm and induced acrosome reactions at rates equal to those of an untreated control. The results support the feasibility of an immunocontraceptive vaccine based on autologous ZP peptides.     

Efficacy of antibodies against a chimeric synthetic peptide encompassing epitopes of bonnet monkey (Macaca radiata) zona pellucida-1 and zona pellucida-3 glycoproteins to inhibit in vitro human sperm-egg binding

Immunocontraception achieved by immunization with zona pellucida (ZP) glycoproteins is invariably associated with ovarian dysfunction. Use of ZP glycoprotein-based synthetic peptides as immunogens has been proposed to overcome adverse side effects on ovaries. In the present study, a chimeric peptide encompassing the epitopes of bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1; amino acid residues 251-273) and ZP glycoprotein-3 (bmZP3; amino acid residues 324-347), separated by a tri-glycine spacer, was synthesized and conjugated to diphtheria toxoid (DT). Immunization of female BALB/cJ mice and bonnet monkeys with the chimeric peptide led to generation of antibodies that reacted with the chimeric peptide, individual bmZP1 & bmZP3 peptides, and also recombinant bmZP1 and bmZP3 proteins expressed by E. coli in an ELISA. Indirect immunofluorescence studies revealed that the immune serum also recognized human as well as bonnet monkey ZP. A significant inhibition of human sperm binding to ZP was observed with antibodies generated against the chimeric peptide in mice (P = 0.0001) as well as monkeys (P = 0.0002) in a hemizona assay (HZA). The inhibition efficacy was significantly higher than that observed by using antibodies against the individual bmZP1 and bmZP3 peptides. Interestingly, no ovarian pathology was observed in female bonnet monkeys immunized with the chimeric peptide. These studies have demonstrated that the chimeric peptide encompassing peptides of multiple ZP glycoproteins may be a promising candidate antigen for designing immunocontraceptive vaccines.     

Unusual pattern of surface marker expression on peripheral lymphocytes from aged humans suggestive of a population of less differentiated cells

SJL/J, PL/J, and (SJL/J x PL/J)F1 mice were immunized with bovine, guinea pig, mouse, or rat myelin basic proteins (MBP) in adjuvant containing Mycobacterium tuberculosis H37Ra. Twenty-four and 72 hr later, Bordetella pertussis vaccine was given i.v. All MBP tested induced experimental allergic encephalomyelitis (EAE) in SJL/J and F1 mice; however, bovine MBP was inactive in PL/J mice. Each strain was immunized in a similar manner with peptic peptides, residues 1-37, 43-88, and 89-169 of guinea pig MBP. In contrast to the SJL/J strain, which has been shown to recognize a major encephalitogenic determinant in peptide 89-169, PL/J and F1 mice responded primarily to an encephalitogenic determinant within peptide 1-37. Analysis of antibody levels showed that SJL/J mice made no antibody to peptide 1-37, although anti-peptide 89-169 antibodies were consistently found. Conversely, PL/J mice responded well to peptide 1-37, but only an occasional animal made a significant response to peptide 89-169. (SJL/J x PL/J)F1 mice were more susceptible to EAE than either parental strain, as shown by the percentage of animals showing neurologic signs and by clinical severity.     

Cell-mediated immunoreactivity of tumor-bearing mice with myelin basic protein and related peptides

Spleen cells (SC) from tumor-bearing mice and mice immunized with porcine myelin basic protein (MBP) reacted in vitro in E-rosette augmentation assays with MBP and certain of its constituent peptides. Peptides 1-115, 43-169, 64-83, 113-121, and 153-161 reacted significantly with both types of SC, while peptide 1-19 reacted only with SC from MBP-immunized mice. The phenomenon of specific inhibition of peptide reactivity by a moderate excess of a related protein was used to identify peptides as accessible epitopes of that protein. Peptide 113-121 was specifically inhibited by excess MBP when reacted with both types of SC, whereas peptide 64-83 was inhibited by excess MBP only when reacted with SC from MBP-immunized mice. These reactions suggest that the immunizing antigen in tumor-bearing mice is related to MBP but differs in epitopes associated with peptides 1-19 and 64-83.     

Serum Antibody Repertoire Profiling Using In Silico Antigen Screen

Serum antibodies are valuable source of information on the health state of an organism. The profiles of serum antibody reactivity can be generated by using a high throughput sequencing of peptide-coding DNA from combinatorial random peptide phage display libraries selected for binding to serum antibodies. Here we demonstrate that the targets of immune response, which are recognized by serum antibodies directed against sequential epitopes, can be identified using the serum antibody repertoire profiles generated by high throughput sequencing. We developed an algorithm to filter the results of the protein database BLAST search for selected peptides to distinguish real antigens recognized by serum antibodies from irrelevant proteins retrieved randomly. When we used this algorithm to analyze serum antibodies from mice immunized with human protein, we were able to identify the protein used for immunizations among the top candidate antigens. When we analyzed human serum sample from the metastatic melanoma patient, the recombinant protein, corresponding to the top candidate from the list generated using the algorithm, was recognized by antibodies from metastatic melanoma serum on the western blot, thus confirming that the method can identify autoantigens recognized by serum antibodies. We demonstrated also that our unbiased method of looking at the repertoire of serum antibodies reveals quantitative information on the epitope composition of the targets of immune response. A method for deciphering information contained in the serum antibody repertoire profiles may help to identify autoantibodies that can be used for diagnosing and monitoring autoimmune diseases or malignancies.     

Molecular analysis of the antigenicity and immunogenicity of recombinant zona pellucida antigens in a primate model.

The studies reported here are the first to demonstrate that recombinant zona pellucida (ZP) proteins will elicit a humoral immune response that recognizes native ZP proteins. Three cDNAs encoding rabbit ZP protein antigens expressed in bacteria were used to immunize cynomolgus monkeys. Four groups of six monkeys each were immunized with bacterially expressed cro-beta-galactosidase recombinant proteins encoded by a full-length cDNA (rc55) encoding the 55-kDa rabbit ZP recombinant protein (rec55), two partial cDNAs (rc75a and rc75b) encoding two recombinant peptides (rec75a and rec75b) of the 75-kDa rabbit ZP protein, and the plasmid-encoded cro-beta-galactosidase control protein. Initial immunizations with these fusion proteins using the muramyl dipeptide adjuvant did not elicit significant levels of antibodies to native or recombinant ZP proteins. Further immunizations were therefore carried out using recombinant ZP proteins conjugated to either protein A or keyhole limpet hemocyanin. Antibodies were detected in the groups immunized with the rec55 and rec75a; however, no antibodies were generated against the rec75b protein. These antibodies have been characterized by two-dimensional PAGE immunoblotting and shown to recognize antigenic domains associated with two of the native rabbit ZP proteins. Reprobes of these immunoblots with sheep anti-total native rabbit ZP proteins, affinity-purified on pig ZP, further demonstrate that a fourth distinct rabbit ZP antigen may be present. The characterization of species-conserved antigenic domains of mammalian ZP proteins is important for studies of the functional regions of ZP proteins and is critical for the design of safe and effective contraceptive vaccines.     

Antigens of Ambrosia elatior (short ragweed) pollen. III. Crossed radioimmunoelectrophoresis of ragweed-allergic patients' sera with special attention to quantification of IgE responses

Short ragweed allergenic extract has been studied by means of crossed radioimmunoelectrophoresis (CRIE) with the use of sera from 37 allergic patients and the relevant control sera. In this study 22 of 52 antigens, detectable in crossed immunoelectrophoresis (CIE) against polyspecific rabbit anti-ragweed IgG, were able to bind specific human IgE to their corresponding immunoprecipitates. This binding was semiquantified by comparison with the binding of a standard serum pool. Nine antigens were identified as important allergens, including the previously isolated components, AgE, AgK, and Ra6. Certain allergens (e.g., AgE, AgK, and Ag 31) bound IgE in almost all patients' sera, whereas others showed a bimodal distribution for sera of responder and nonresponder patients. The total CRIE score was found to correlate significantly both with ragweed-specific serum IgE antibody determined by RAST (rs = 0.88; p less than 0.001) and with total IgE level (rs = 0.55; p less than 0.01). Patient's CRIE scores to AgE also correlated significantly with their specific IgE antibody to AgE measured by RIA (r = 0.47; p less than 0.01) and with skin-test sensitivity to AgE (r = 0.44; p less than 0.05). It was concluded that CRIE is well suited for identification of important ragweed allergens without the previous need for laborious isolation procedures.     

Monoclonal antibodies to rabbit and pig zonae pellucidae distinguish species-specific and shared antigenic determinants

Monoclonal antibodies against rabbit or porcine zonae pellucidae (ZP) demonstrate species-specific and shared antigenic determinants. In addition, these antibodies are used to characterize the biochemical nature of these determinants. All of six monoclonal antibodies developed against porcine ZP react with porcine but not with rabbit ZP. Only one of seven monoclonal antibodies developed against rabbit ZP cross-reacts with porcine ZP. None of these antibodies recognized antigens associated with other tissues tested. High-resolution, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by immunoblotting was used to demonstrate that the cross-reactive antibody recognizes an antigenic determinant which is associated with the major low molecular weight glycoprotein of both the pig and rabbit ZP. Since this antibody recognizes all charge species of this glycoprotein, it is apparent that the antigenic determinant recognized by this antibody involves protein. Further studies demonstrate that proteolytic digestion of ZP will destroy the antigenic determinant while glycosidic digestion of ZP has no effect on antibody binding. Although polyclonal antibodies to this glycoprotein inhibit sperm from binding to the zona pellucida, this monoclonal antibody does not affect sperm binding. None of the species-specific antibodies recognize ZP glycoproteins following 2D-PAGE. This is a property typical of antibodies directed against conformational antigenic determinants. The presence of common as well as unique zona antigenic determinants could explain why ZP proteins induce heteroantibodies which result in infertility while alloimmunization has no effect on fertility.     

Influence of the H-2u haplotype on immune function in F1 hybrid mice. I. Antigen presentation

Previously, we reported that myelin basic protein (MBP) peptide 1-37 contains an encephalitogenic epitope for PL/J mice, and MBP peptide 89-169 is encephalitogenic for SJL/J mice. (SJPL)F1 hybrid mice do not respond to immunization with these peptides in a co-dominant manner because the encephalitogenic response to peptide 1-37 dominates. To examine this phenomenon more closely, we tested the ability of MBP-primed parental or F1 T cells to respond to MBP or MBP peptides in the context of PL, SJL, or F1 antigen-presenting cells (APC). It was found that the F1 T cells responded to either the protein or the peptides when these were presented in the context of F1 or PL APC. However, F1 T cells would not respond to MBP in the context of SJL APC, although the latter cells were functionally intact. This effect was not antigen-specific because SJL APC would not present ovalbumin or PPD to primed F1 T cells. F1 T cells from mice immune to the strongly antigenic bacterium Listeria monocytogenes responded to bacterial antigens presented by SJL APC, although at a significantly lower level compared to the results obtained when these antigens were presented by F1 or PL APC. This finding implied that unbalanced antigen presentation was a quantitative rather than a qualitative phenomenon. When F1 hybrid mice from other strain combinations were tested, a similar effect was observed whenever one of the parental strains was PL/J. This effect was mapped to the MHC in MHC-congenic B10 mice.     

Mapping of B cell epitopes on the zona pellucida 2 protein of a marsupial, the brushtail possum (Trichosurus vulpecula)

Immunocontraceptive vaccines against zona pellucida (ZP) proteins are being developed for brushtail possum (Trichosurus vulpecula) management in New Zealand. Mapping of B cell epitopes on the ZP2 protein of possums was undertaken in this study to define the antigenic regions that may be crucial to sperm-egg binding. The amino acid sequence of the full-length possum ZP2 protein (712 amino acids) was used to synthesize a complete set of 71 (15-mer) biotinylated peptides with an offset of five amino acids. The peptides were used in a modified enzyme-linked immunosorbent assay (ELISA) to identify continuous epitopes recognized by antibodies in the sera of possums immunized with recombinant possum ZP2 (rZP2) constructs. Seventeen continuous epitopes were located on possum ZP2 protein. Comparisons of the peptide binding pattern of antibodies in individual sera with the fertility status of the same immunized possums revealed three significant infertility-relevant peptide epitopes (amino acids 111-125, 301-315, and 431-445). One of these (amino acids 431-445) bound to possum spermatozoa from the caudal epididymis. The implications of these findings for developing immunocontraceptive vaccines for possum control are discussed.     

Mapping of an epitope recognized by a neutralizing monoclonal antibody specific to toxin Cn2 from the scorpion Centruroides noxius, using discontinuous synthetic peptides.

The Na+-channel-affecting toxin Cn2 represents the major and one of the most toxic components of the venom of the Mexican scorpion Centruroides noxius Hoffmann. A monoclonal antibody BCF2 raised against Cn2 has been shown previously to be able to neutralize the toxic effect of Cn2 and of the whole venom of C. noxius. In the present study the epitope was mapped to a surface region comprising the N- and C-terminal segments of Cn2, using continuous and discontinuous synthetic peptides, designed on the basis of the sequence and a three-dimensional model of Cn2. The study of peptides of varying length resulted in the identification of segments 5-14 and 56-65 containing residues essential for recognition by BCF2. The peptide (abbreviated SP7) with the highest affinity to BCF2 (IC50 = 5.1 microM) was a synthetic heterodimer comprising the amino acid sequence from position 3-15 (amidated) of Cn2, bridged by disulfide to peptide from position 54-66, acetylated and amidated. Similar affinity was found with peptide SP1 [heterodimer comprising residues 1-14 (amidated) of Cn2, bridged with synthetic peptide 52-66 (acetylated)]. SP1 and SP7 were used to induce anti-peptide antibodies in mouse and rabbit. Both peptides were highly immunogenic. The sera obtained were able to recognize Cn2 and to neutralize Cn2 in vitro. The most efficient protection (8.3 microgram Cn2 neutralized per mL of serum) was induced by rabbit anti-SP1 serum.     

Epitopes of peptide 43-88 of guinea pig myelin basic protein: localization with monoclonal antibodies

Two monoclonal antibodies, one raised by immunization with mouse myelin basic protein (MBP) and the second raised by immunization with peptide 68-88 of guinea pig MBP, were compared with respect to specificity. The former antibody (15.32) cross-reacted completely with rat, guinea pig, human, and bovine MBP. It also reacted with peptide 43-88 from each MBP. The latter antibody (22.17) was nonreactive with MBP, but cross-reacted with peptide 43-88 from rat, human, guinea pig, and bovine MBP. When tested with small peptides derived from peptide 43-88, antibody 22.17 reacted with an epitope in the C-terminal region. Antibody 15.32 reacted with an epitope in the N-terminal half of the peptide. The data show that 22.17 reacted with a unique epitope associated only with free peptide, whereas 15.32 recognized an epitope common to both peptide 43-88 and MBP.     

Polyvalent human immunodeficiency virus synthetic immunogen comprised of envelope gp120 T helper cell sites and B cell neutralization epitopes

In previous studies, we have used antisera raised to envelope (env)-gene-encoded synthetic peptides to identify a region of (HIV) glycoprotein (gp) 120 env protein designated SP10 that contains a type-specific neutralizing determinant. To develop a polyvalent, synthetic peptide inoculum that can evoke both neutralizing antibodies and T cell proliferative responses to more than one HIV isolate, synthetic peptides containing type-specific neutralizing determinants of gp120 from HIV isolates HTLV-IIIB (IIIB), HTLV-IIIMN (MN) and HTLV-IIIRF (RF) were coupled to a 16 amino acid T cell epitope (T1) of HIV-IIIB gp120 and used to immunize goats. Goat antisera to each T1-SP10 peptide derived from the SP10 region of gp120 of IIIB, MN, and RF neutralized HIV isolates IIIB, MN and RF in a type-specific manner. Moreover, peripheral blood T cells from immunized goats also proliferated in a type-specific manner to peptides derived from gp120 of IIIB, MN, and RF. When combined in a trivalent inoculum, T1-SP10 peptides from HIV-1 isolates IIIB, MN, and RF evoked a high titered neutralizing antibody response to isolates IIIB, MN, and RF in goats and as well induced immune T cells to undergo blast transformation in the presence of peptides derived from gp120 of all three HIV isolates. The T1 portion of the T1-SP10 construct was shown to induce a B cell antibody response against determinants within the T1 peptide in addition to inducing T cell proliferative responses in immune goat T cells. Moreover, the SP10 portion of the T1-SP10 constructs not only induced B cell antibody production but also induced type-specific T cell proliferative responses localized to the C-terminal variable sequences of the SP10 peptides. Finally, the T1-SP10 peptide construct induced memory T cell proliferative responses to native gp120 env protein. Thus, combinations of homologous SP10 region synthetic peptides containing type-specific neutralizing determinants and T cell epitopes of HIV gp120 may be useful in man to elicit high titered neutralizing B cell responses and, as well, T cell responses to more than one HIV isolate.     

Trypanosoma cruzi: cellular and antibody response against the parasite in mice immunized with a 19-amino acid synthetic peptide

Several monoclonal antibodies were prepared against the flagellar fraction of Trypanosoma cruzi epimastigotes (Tulahuén strain, stock Tul 2). One of them, FCH-F8-4, has previously shown biologic activity against the parasite (complement-mediated lysis and neutralization of the trypomastigote infectivity). Immunopurified antigens using this monoclonal antibody elicited a protective immune response in mice. Two recombinant cDNA clones were detected with this anti-flagellar fraction monoclonal antibody on a lambda gt11 expression library prepared from T. cruzi epimastigote mRNA. The insert of one of these cDNA clones, lambda(FCH-F8-4)1 (150 bp) coded for a 19-amino acid peptide (PAFLGCSSRFSGSFSGVEP). This insert hybridized with a 5.0-kb mRNA from epimastigotes. The beta-galactosidase fusion protein was produced in lysogenic bacteria. The monoclonal antibody recognized the epitope present in the fusion protein after western blotting of the crude lysate. A synthetic peptide (SP4) containing the complete sequence of lambda(FCH-F8-4)1 was constructed on solid phase. This peptide was able to inhibit the ELISA reactivity (in a range from 13 to 52%) of flagellar fraction immunized mouse sera and when administered (coupled to KLH or alone) to BALB/c mice with Bordetella pertussis as adjuvant, it induced a humoral and cellular immune response which was detected by ELISA, immunofluorescence, blotting, and DTH reactions against T. cruzi antigens. The immune response obtained indicates that this synthetic peptide resembles the parasite antigen conformation and could be useful for diagnosis purposes or be able to elicit immunoprotection against T. cruzi infection.     

Archaeosomes as adjuvants for combination vaccines

The present study evaluated the potential of archaesomes, prepared from the total polar lipids extracted from Methanobrevibacter smithii, as adjuvants for combination (multivalent) vaccines. Groups of Balb/c mice were immunized subcutaneously at day 0 and 21 with one of the following vaccines: trivalent vaccine formulated by the simultaneous co-encapsulation of bovine serum albumine (BSA), ovalbumin (OVA) and hen egg lysozyme (HEL) into archaeosomes (CEC vaccine); an univalent archaeosome vaccine (UVE vaccine) containing either BSA, OVA or HEL; or an admixture vaccine (AMC vaccine) consisting of the three UVE vaccines. Serum specific antibody (IgG + M) responses were determined at day 32, 112 and 203, and specific IgG1 and IgG2a responses were determined at day 112. Mice immunized with the CEC of AMC vaccine developed strong and sustained specific antibody responses to all three antigens at a magnitude similar to those seen in control mice immunized with UVE vaccines. Moreover, the serum BSA-, OVA-, and HEL-specific IgG1 and IgG2a levels in the CEC and AMC immunized mice were overall comparable to those of the UVE immunized control mice. Boosting CEC and AMC vaccinated mice with antigens alone at day 203 elicited strong antibody memory responses, comparable to those in the UVE vaccinated groups. These results show that archaeosomes could be used as adjuvants in developing combination vaccines.     

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.