B-cell Epitope Mapping Research Articles

Antigenic properties of the Leishmania infantum GRP94 and mapping of linear B-cell epitopes

In this study, we show that the glucose-regulated protein 94 (GRP94) from Leishmania infantum is a major target for humoral immune response during both visceral (VL) and mucocutaneous leishmaniasis (MCL). Also, the time-course of appearance of anti-GRP94 antibodies along the infection was analysed in hamsters ( Mesocricetus auratus) experimentally infected with L. infantum. Remarkably, the reactivity against the Leishmania GRP94 was observed very soon after challenge, at the time of appearance of a humoral response against Leishmania total proteins, and long before that the animals develop clinical symptoms of disease. Therefore, at least for golden hamsters, the reactivity against Leishmania GRP94 is an early marker of infection. Using a collection of synthetic peptides covering the complete sequence of the L. infantum GRP94, we have determined the main linear antigenic determinants recognised by sera from humans, dogs, and hamsters suffering from VL. Four synthetic peptides, located in highly divergent regions of the protein, were found to be immunodominants and recognised by VL sera of these three different origins. We discuss that the prominent antigenicity of Leishmania GRP94 may be related to recent findings involving GRP94, and other heat shock proteins, in relevant immune functions such as tumour immunogenicity and antigen presentation.

B-Cell epitope mapping of the VapA protein of Rhodococcus equi: implications for early detection of R. equi disease in foals.

Linear B-cell epitopes of the Rhodococcus equi virulence-associated protein (VapA) were mapped using a synthetic peptide bank in this study. The peptides were screened in an enzyme-linked immunosorbent assay (ELISA) with a total of 70 sera from foals with current R. equi disease (51 sera), as well as from foals that had either recovered from R. equi infection 10 months previously (3 sera) or that had no known history of R. equi disease (16 sera). An epitope with the sequence NLQKDEPNGRA was identified and was universally recognized by all 51 sera from foals with R. equi disease and was not recognized by any of the other sera. There was poor reactivity between all sera and peptides relating to other areas of the VapA protein. It is proposed that an ELISA based upon a defined peptide epitope may be used in an improved serological diagnostic test for R. equi infection in foals.

Mapping of B-cell epitopes on human Parvovirus B19 non-structural and structural proteins

In the present study, the immune reactivity to Parvovirus B19 (B19) proteins and variations in antigenic reactivity in different clinical manifestations were investigated. Sera from healthy B19 IgG positive individuals were evaluated for antibody reactivity against linear peptides. Three antigenic regions (amino acid number 191–206, 271–286, 371–386) on the B19 non-structural (NS) protein 1 were identified. The highest seroreactivity against these peptides was found against amino acid number 271–286. Seroreactivity in this group of individuals was also investigated against peptides representing selected neutralising regions of the B19 capsid proteins viral protein (VP) 1/VP2. The antigenic NS1 and VP1/VP2 regions, thus defined, were further mapped by seroreactivity against peptides containing specific deletions. The frequencies of seroreactivity against the NS1 and VP1/VP2 peptides in healthy B19 IgG positive individuals were similar to those in HIV-seropositive and persistently B19 infected patients, except that the latter group showed a lower reactivity to the C-terminal end of VP1/VP2. The identification of antigenic regions and corresponding seroreactivity in asymptomatic and persistently B19-infected patients is important for the understanding of B19-pathogenesis and for the development of B19 vaccine candidates.

Mapping of B-cell epitopes in rabbits immunised with various gag antigens for the production of HIV-1 gag capture ELISA reagents

An HIV-1 p24 capture enzyme linked immunosorbent assay (ELISA) was developed and used in a study of B-cell epitopes in rabbits immunised with different gag p24 antigens. Rabbits were immunised with virion HIV-1/Lai, baculovirus recombinant p24, Escherichia coli recombinant p24-15 and a mixture of synthetic peptides representing sequences of HIV-1 gag p24 protein, respectively. Five out of nine rabbits developed antibodies that could be used for an antigen capture ELISA. No significant differences in IgG titers to the whole gag protein were seen when comparing rabbits immunised with four different antigens. Three major common linear epitope regions were mapped in the rabbits immunised with virion HIV-1/Lai and baculovirus recombinant p24. The rabbit immunised with HIV-1 gag peptides had the broadest linear epitope reactive responses whereas animals immunised with E. coli recombinant antigen had the most restricted linear epitope response. The capture ELISA method thus developed using the different rabbit anti-p24 IgG preparations was shown to capture isolates from HIV-1 subtypes or clades A to G. Only rabbits immunised with virion HIV-1/Lai and baculovirus recombinant p24 developed IgG that was capable of efficiently capturing HIV-1 p24 in ELISA, indicating the importance of preparing antibodies able to recognise native or discontinuous and linear antigen configurations.

B-Cell epitope mapping of DNA topoisomerase I defines epitopes strongly associated with pulmonary fibrosis in systemic sclerosis.

We hypothesized that B-cell epitope mapping of DNA Topoisomerase I (type-I topoisomerase, or Topo I) may define epitopes strongly associated with pulmonary interstitial fibrosis (PIF) in systemic sclerosis (SSc). B-cell epitope mapping of Topo I was performed using 63 20-mer peptides overlapping by eight residues and spanning the entire length of the Topo I sequence. These peptides, coupled to polystyrene pins, were tested for antibody binding by enzyme-linked immunosorbent assays (ELISAs) using immunoglobulin G fractions from anti-Topo I, anticentromere, anti-U3RNP-positive, and normal sera. Four major epitopes were recognized by anti-Topo I sera, but not from the control sera: WWEEERYPEGIKWKFLEHKG (205-224, epitope I), RIANFKIEPPGLFRGRGNHP (349-368, epitope II), PGHKWKEVRHDNKVTWLVSW (397-416, epitope III), and ELDGQEYVVEFDFLGKDSIR (517-536, epitope IV). Peptide-epitopes were then synthesized in their soluble forms and ELISA systems were developed. Epitopes II to IV are localized at highly exposed sites of the Topo I tertiary structure, whereas epitope I is localized at a less accessible site. In a cohort of 81 patients with SSc with clinical data on the evolution of their disease, patients with antibodies in their sera recognizing at least three of the four epitopes had 3.1 times (P = 0.02) the hazard of developing PIF compared with patients whose sera recognized no epitopes or only one or two of the four epitopes. The discrimination was much stronger than that achieved by the simple determination of Topo I antibodies by counterimmunoelectrophoresis and immunoblot (hazard ratio 1.7, P = 0.30) in the same patients. B-cell epitope mapping of the anti-Topo I response has identified four major epitopes which cumulatively show a strong association with the development of PIF in SSc.

Mapping of immunodominant B-cell epitopes and the human serum albumin-binding site in natural hepatitis B virus surface antigen of defined genosubtype

Twelve MAbs were generated by immunization of BALB/c mice with plasma-derived hepatitis B virus surface spherical antigen particles subtype ayw2 (HBsAg/ayw2 genotype D). Their epitopes were mapped by analysis of reactivity with plasma-derived HBsAg/ayw2 and HBsAg/adw2 (genotype A) in enzyme immunoassays and blots. Mapping was supported by nested sets of truncated preS2 proteins and preS2 peptides. Five antibodies were S domain-specific, seven were preS2-specific and 11 had a preference for genotype D. According to our data, group I of the three known epitope groups of preS2 has to be divided into IA and IB. Three preS2-specific MAbs forming the new group IA reacted with genotype D residues 3-15 which have not yet been described as an epitope region. IA antibodies strongly inhibited the binding of polymerized human serum albumin. Two antibodies (group II) reacted with the glycosylated N-terminal region of preS2 in plasma-derived HBsAg, but not with a preparation from transfected murine cells. One group III antibody was subtype-specific and reacted with the highly variable preS2 sequence 38-48. Only one antibody (group IB) mapped to the region (old group I) which was believed to be immunodominant and genotype-independent. Geno(sub)type-specific epitopes of preS2 are obviously the immunodominant components of natural HBsAg in BALB/c mice, but these epitopes may be masked by serum albumins in humans. The data may explain why it is difficult to detect anti-preS2 antibodies in human recipients of preS2-containing vaccines, in spite of the preS2 immunodominance in mice.

Protein sequence analysis and mapping of IgE and IgG epitopes of an allergenic 98-kDa Dermatophagoides farinae paramyosin, Der f 11.

A 98-kDa mite paramyosin (Der f 11) from Dermatophagoides farinae (Df) is highly allergenic, and its cDNA (Df642) has been cloned. This paper describes the sequence characteristics and the mapping of the immunodominant human IgE and IgG epitopes of Der f 11. The protein sequence analysis was performed with a combination of FASTA, GCG, and CLUSTAL W computing packages. The whole cDNA insert and its PCR-derived DNA fragments were generated and expressed in E. coli. These overlapping recombinant peptides (F1 to F5) were used for B-cell epitope mapping with 18 mite-allergic sera by dot immunoassays. Df642 cDNA encodes a partial sequence that contains the 2nd to 26th 28-residue repeats and lacks the N-terminus and the C-terminus. The sequence identity of Der f 11 with other known paramyosins is 34-60%. The dominant IgE epitopes are located in peptides F1 and F4, whereas the dominant IgG epitopes are located in peptides F1 and F2. These peptides are more reactive than whole rDf642. Mite paramyosin is very similar to other known paramyosins. The human IgE and IgG epitopes are scattered throughout the entire molecule. Data also indicate the presence of unique IgE and IgG epitopes in Der f 11.

Immunological characterization of alpha antigen of Mycobacterium kansasii: B-cell epitope mapping.

Mapping of B-cell epitopes on alpha antigen of Mycobacterium kansasii (K-alpha) was carried out by using recombinant truncated K-alpha fusion peptides. We observed that two immunodominant B-cell epitopes (amino acids 222-268 and 267-306) and one minor epitope (amino acid 249-286) were located in the C-terminal region of K-alpha. The other three minor B-cell epitopes were mapped in N-terminal (amino acids 80-98 and 99-166) and central (amino acid 174-204) regions of K-alpha. All defined epitopes were common to Mycobacterium tuberculosis and M. kansasii. Besides these common epitopes, a region in K-alpha (amino acid 290-319) revealed different reactivities between antibodies against K-alpha and alpha antigen of M. tuberculosis. These findings may provide a basis for development of serodiagnosis that can distinguish between M. kansasii and M. tuberculosis infections.

Development of two monoclonal antibodies against Plasmodium falciparum sporozoite surface protein 2 and mapping of B-cell epitopes.

The Plasmodium yoelii sporozoite surface protein 2 (PySSP2) is the target of protective cellular immunity. Cytotoxic T cells specific for the Plasmodium falciparum analog PfSSP2, also known as thrombospondin-related anonymous protein (TRAP), are induced in human volunteers immunized with irradiated sporozoites. PfSSP2 is an important candidate antigen for a multicomponent malaria vaccine. We generated and characterized three monoclonal antibodies (MAbs) specific for PfSSP2/TRAP. The MAbs PfSSP2.1 (immunoglobulin G1 [IgG1]), PfSSP2.2 (IgG2a), and PfSSP2.3 (IgM) were species specific and identified three distinct B-cell epitopes containing sequences DRYI, CHPSDGKC, and TRPHGR, respectively. PfSSP2.1 partially inhibited P. falciparum liver-stage parasite development in human hepatocyte cultures (42 and 86% in two experiments at 100 microg/ml). Mice immunized with vaccinia virus expressing full-length PfSSP2 protein produced antibodies to (DRYIPYSP)3, and humans living in malaria-endemic areas (Indonesia and Kenya), who have lifelong exposure and partial clinical immunity to malaria, had antibodies to both (DRYIPYSP)3 and (CHPSDGKCN)2. Mice immunized with multiple antigen peptides MAP4 (DRYIPYSP)3P2P30 and MAP4 (CHPSDGKCN)3P2P30 in TiterMax developed antibodies to sporozoites that partially inhibited sporozoite invasion of human hepatoma cells (39 to 71% at a serum dilution of 1:50 in three different experiments). The modest inhibitory activities of the MAbs and the polyclonal antibodies to PfSSP2/TRAP epitopes do not suggest that a single-component vaccine designed to induce antibodies against PfSSP2/TRAP will be protective. Nonetheless, the MAbs directed against PfSSP2, and the peptides recognized by these MAbs, will be essential reagents in the development of PfSSP2/TRAP as a component of a multivalent P. falciparum human malaria vaccine.

Species-specific B-cell epitope on the C-terminal region of the α antigen fromMycobacterium scrofulaceum

In the amino acid (AA) sequences of α antigen from mycobacteria, C-terminal regions were variable among a variety of mycobacterial species though the N-terminal regions were relatively conserved. These regions may possess some species-specific antigenic determinants of the α antigen fromMycobacterium scrofulaceum(S-α). AAs288–300 of S-α fused to β-galactosidase was reactive with the antisera raised against S-α. The same fused peptide did not react with the antisera raised against the α antigen fromMycobacterium avium(A-α) andMycobacterium bovisBCG (B-α). B-cell epitope mapping then was performed focusing on the C-terminal region of S-α using the synthetic peptides. Their reactivities with antisera raised against the α antigens of three different mycobacterial species were assessed by ELISA. AAs279–286 were a cross-reactive common immunodominant region among three mycobacterial species. This region may be one of the cross-reactive common epitopes in mycobacterial species. And AAs291–300 were reactive only with the antisera raised against S-α. This region may possess a species-specific epitope.

Mapping of IgE-binding epitopes on the recombinant major group I allergen of velvet grass pollen, rHol I 1

New and more successful approaches to diagnosis and therapy of allergic diseases require a more subtle understanding of the structure and the epitopes on the allergen molecule. This study was done to obtain more information on the structure and the IgE-binding epitopes of a major allergen of velvet grass pollen, Hol l 1. We cloned Hol l 1 from a complementary DNA library and performed B-cell epitope mapping with 21 recombinant fragments expressed as fusion proteins in Escherichia coli. The fragments were analyzed by Western blotting with sera from 50 different patients. The patients' sera individually recognized at least four different IgE-binding regions (amino acids 1 to 27, 61 to 76, 84 to 105, and 158 to 240). According to their binding patterns with these epitopes, they were divided into five groups. Most sera (92%) bound to the C-terminal peptide (158 to 240), which consists of more than 80 amino acids, whereas there was virtually no binding to smaller fragments covering this region. In contrast to the C-terminal peptide, the IgE-binding peptides on the N terminus and on the middle region of the molecule were of a smaller size (15 to 30 amino acids). The major group I allergen of velvet grass bears at least four different IgE-binding epitopes, which were individually recognized by sera from different patients. The C terminus represents the major IgE-binding region and contains at least one discontinuous IgE-binding epitope, whereas the N terminus and middle region of Hol l 1 seem to contain continuous IgE-binding epitopes.

Differential Recognition of the 52–Kd Ro(Ss–A) Antigen by Sera From Patients With Primary Biliary Cirrhosis and Primary Sjogren's Syndrome

Antibodies against the 52-kd Ro(SS-A) protein are significantly associated with the primary Sjogren's syndrome (pSS). A small proportion of patients suffering from primary biliary cirrhosis (PBC) with secondary Sjogren's syndrome (PBC/SS) who are serologically characterized by antimitochondrial type 2 antibodies also express anti-52-kd Ro(SS-A) antibodies. The primary B-cell-derived antigenic responses by autoimmune sera were analyzed in both entities using truncated recombinant proteins to examine whether different epitopes are associated with these diseases. Sera were collected from 25 patients with pSS and 9 anti-52-kd Ro(SS-A)-positive patients suffering from PBC/SS. B-cell epitope mapping was performed using different 52-kd Ro(SS-A) fusion proteins in enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Sera from patients with pSS showed the broadest reactivity against antigenic epitopes at AA 153-245 compared to the significantly limited reactivity against AA 228-245 of sera from patients with PBC. B-cell epitopes within AA 190-245 represent immunodominant epitopes recognized by pSS sera in a significantly higher degree than by PBC sera, which react predominantly with AA 228-245 (P < .0001). Anti-La(SS-B) antibodies were significantly associated with anti-52-kd Ro(SS-A) in sera from patients with pSS compared with PBC patients (P < .025). Thus, the antibody response to 52-kd Ro(SS-A) in PBC appears to be induced differently than in pSS. Although a limited immune response to 52-kd Ro(SS-A) occurs in PBC/SS patients, a more extended epitope spreading is evident in patients with pSS.

Differential recognition of the 52-kd Ro(SS-A) antigen by sera from patients with primary biliary cirrhosis and primary Sjogren's syndrome

Antibodies against the 52-kd Ro(SS-A) protein are significantly associated with the primary Sjogren's syndrome (pSS). A small proportion of patients suffering from primary biliary cirrhosis (PBC) with secondary Sjogren's syndrome (PBC/SS) who are serologically characterized by antimitochondrial type 2 antibodies also express anti-52-kd Ro(SS-A) antibodies. The primary B-cell-derived antigenic responses by autoimmune sera were analyzed in both entities using truncated recombinant proteins to examine whether different epitopes are associated with these diseases. Sera were collected from 25 patients with pSS and 9 anti-52-kd Ro(SS-A)-positive patients suffering from PBC/SS. B-cell epitope mapping was performed using different 52-kd Ro(SS-A) fusion proteins in enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Sera from patients with pSS showed the broadest reactivity against antigenic epitopes at AA 153-245 compared to the significantly limited reactivity against AA 228-245 of sera from patients with PBC. B-cell epitopes within AA 190-245 represent immunodominant epitopes recognized by pSS sera in a significantly higher degree than by PBC sera, which react predominantly with AA 228- 245 (P < .0001). Anti-La(SS-B) antibodies were significantly associated with anti-52-kd Ro(SS-A) in sera from patients with pSS compared with PBC patients (P < .025). Thus, the antibody response to 52-kd Ro(SS-A) in PBC appears to be induced differently than in pSS. Although a limited immune response to 52-kd Ro(SS-A) occurs in PBC/SS patients, a more extended epitope spreading is evident in patients with pSS. (Hepatology 1996 Dec;24(6):1404-7)

B-cell epitope mapping within the MA16 antigenic sequence found in Eimeria acervulina merozoites and sporozoites

Overlapping heptapeptides derived from the MA16 Eimeria acervulina antigenic sequence (Castle et al., 1991) were synthesised on polypropylene pins (‘pepskan’ technique, Cambridge Research Biochemicals, UK). Binding of antibodies from chickens and rabbits infected and immunised respectively with various species of Eimeria oocysts ( E. acervulina, E. tenella, E praecox, E. necatrix and E. maxima), was examined using the coated pins as the solid phase of an enzyme immunoassay (EIA). Antigenicity of the overlapping synthetic heptapeptides was then analysed using a number of algorithms based on the amino acid sequence to predict secondary protein structure, hydrophilicity, acrophilicity and chain flexibility profiles. The antigenicity of this sequence appears to be quite different from that found for the E. tenella GX3264 antigenic sequence (Bhogal et al., 1992) whose profile was similarly examined (Talebi and Mulcahy, 1994) using the same rabbit and chicken anti- Eimeria oocyst sera.

Mapping epitopes on antigens by immunodiffusion in gel.

The mapping of B-cell epitopes on antigen molecules is best done with monoclonal antibodies (MAbs). Most methods require physical chemical procedures for purifying, binding, or labeling the antigen or the MAb. These procedures require some technical proficiency, may present methodological problems, and are based on much trial and error, but, worst of all, these manipulations, including the simple adsorption of molecules to surfaces, may induce the unfolding of the protein antigen, and therefore the loss of native epitopes with the exposure of new determinants (“unfoldons”) (). Thus, when a mapping technique is successfully set up, there is often the nagging question: “Is the antigen still in its native conformation?” Our immunodiffusion method avoids many of these problems since in most instances, both the antigen and the antibody can be used without any manipulation. The method is based on the fact that a single MAb generates soluble immune complexes with an antigen, but two MAbs to different epitopes, when tested in appropriate conditions, generate insoluble complexes that are detected as a precipitin line.

T-cell, adhesion, and B-cell epitopes of the cell surface Streptococcus mutans protein antigen I/II

The T-cell and antibody responses to a cell surface streptococcal antigen (SA I/II) were investigated in naturally sensitized humans. Serum antibody responses were directed predominantly to the N-terminal (residues 39 to 481) and central (residues 816 to 1213) regions of SA I/II which may be involved in bacterial adhesion to salivary receptors. T-cell responses were also directed predominantly towards the central region. The linear peptide relationship of the immunodominant and minor T- and B-cell as well as adhesion epitopes was mapped within residues 816 to 1213. Immunodominant T-cell and B-cell epitopes were identified within residues 803 to 853, which were separated in linear sequence from the adhesion epitopes (residues 1005 to 1044). Adhesion epitopes overlapped with minor B- and T-cell epitopes (residues 1005 to 1054 and 1085 to 1134). An immunodominant promiscuous T-cell epitope (residues 985 to 1004) was adjacent to an adhesion epitope (residues 1005 to 1024). The limited B-cell response to adhesion epitopes is consistent with the success of Streptococcus mutans in colonizing the oral cavity. The strategy of T-cell, adhesion, and B-cell epitope mapping has revealed a general approach for identifying components of subunit vaccines which may focus responses to critical functional determinants. Such epitopes of SA I/II may constitute the components of a subunit vaccine against dental caries.

Mapping of B-Cell Epitopes Recognized by Antibodies to Histones in Subsets of Juvenile Chronic Arthritis

The sera of 138 patients with juvenile chronic arthritis (JCA) were tested in ELISA with the five individual histories, 34 histone peptides covering the full length of the four core histones, and two peptides corresponding to the N- and C-terminal domains of H1. The occurrence of IgG antibodies was examined regarding the different subsets of JCA (pauciarticular, polyarticular, and systemic onset) and regarding clinical features (chronic anterior uveitis, CAU) and other serological features (antinuclear antibodies, ANA). Seventy-two percent of the 138 sera reacted with at least 1 histone peptide. The peptides 204-218 of H1, 1-25 of H2B, and 111-130 of H3 were recognized by 22-28% of JCA sera, and 42% of JCA sera reacted with one or both peptides 1-25 of H2B and 111-130 of H3. The frequency of occurrence of anti-histone antibodies (AHA) regarding the type of histone fraction (H1, H2A, H2B, H3 and H4) or the regions of histones was not significantly different in the three subsets of JCA. No obvious association was found between IgG antibodies to histone peptides and uveitis. In the subset of pauciarticular JCA, 13/31 patients (41.9%) with CAU against 14/41 patients (34.1%) without CAU possessed IgG antibodies reacting with peptides of the C-terminal domain 83-135 of H3. This difference is not statistically significant. Finally, the presence of antibodies to histones and histone peptides cannot completely explain ANA reactivity found in patients' sera. Although antibodies to histone peptides occur frequently in the serum of children with JCA, the antibody profiles seem to be highly individual and do not correlate with disease subtype or activity. Identification of AHA present not only in the circulation but also in tissue deposits may provide better insight into the identification of pathogenic antibodies.

High-resolution mapping of B-cell epitopes within an antigenic sequence from Eimeria tenella.

Overlapping hexapeptides representing part of an Eimeria tenella antigenic sequence, shown to induce partial immunity to homologous challenge in chickens, were synthesized on polypropylene pins (Pepskan technique; Cambridge Research Biochemicals, Cambridge, United Kingdom). The binding to these hexapeptides of antibodies from chickens infected and rabbits immunized with five species of Eimeria was studied, using the coated pins as the solid phase of an enzyme-linked immunoassay. Antibody binding to most regions of the sequence was demonstrated, with peak areas of antigenicity correlating with the most hydrophilic regions. A particularly hydrophilic and antigenic area towards the N terminus of the sequence consists of a peptide motif repeated five times in the native antigen. Homologous antisera (chicken and rabbit anti-E. tenella antisera) differed in their pattern of reactivity from heterologous sera raised against other Eimeria species. While the former bound to fewer of the hexapeptides than the latter, they did so very strongly, indicating affinity maturation of the antibody response to E. tenella-specific sequences. No antibody reactivity to two regions of the sequence was detected. These regions occur in relatively hydrophilic areas and so are unlikely to be situated in transmembrane domains or in the interior of globular proteins. Synthetic peptides, as used in these experiments, make possible analysis of the fine specificity of immune responses and thus have a role to play in the development of novel vaccines for the control of coccidiosis.

Cloning and B-cell-epitope mapping of MPT64 from Mycobacterium tuberculosis H37Rv.

The gene of the immunogenic protein MPT64 found in culture filtrates of Mycobacterium tuberculosis H37Rv was cloned and sequenced. A comparison showed mpt64 and the gene encoding MPB64 from Mycobacterium bovis BCG Tokyo to be identical except for one silent mutation. The regions encoding the promoter and the signal peptide were also well conserved for the two sequences. Southern blot experiments on genomic mycobacterial DNA showed the presence of mpt64 in the M. tuberculosis substrains H37Rv, H37Ra, and Erdman and in the M. bovis BCG substrains Tokyo, Moreau, and Russian, whereas the M. bovis BCG substrains Glaxo, Pasteur, Canadian, Tice, and Danish 1331 and Mycobacterium leprae lack the gene. Southern blot analyses revealed differences in the restriction enzyme patterns within the M. tuberculosis substrains as well as within the M. bovis BCG substrains, indicating either different chromosomal localization of mpt64 or that mutations have occurred at different locations on the chromosomes. N-terminal and C-terminal deletion mutants were constructed for the mapping of B-cell epitopes on MPT64 with five monoclonal antibodies, C24b1, C24b2, C24b3, L24b4, and L24b5. Western blot (immunoblot) analysis revealed that the murine antibodies bind to one linear and three conformational epitopes.

T- and B-cell functions and epitope expression in nonhuman primates immunized with simian immunodeficiency virus antigen by the rectal route.

Transmission of human immunodeficiency virus (HIV) in North America and Europe occurs most commonly through the rectal mucosa during homosexual intercourse. The simian immunodeficiency virus (SIV) macaque model has been used to investigate rectal immunization. The vaccine used was a recombinant SIV gag p27 expressed as hybrid Ty virus-like particles (Ty-VLP). Sequential ororectal (OR) mucosal immunization was compared with i.m. immunization. Whereas both routes of immunization induced serum IgA and IgG p27 antibodies, only OR immunization induced rectal secretory IgA antibodies. Specific CD4+ T-cell proliferative responses to stimulation with p27 were found after i.m. immunization only in the blood and spleen, but after OR immunization they were found in the internal iliac and inferior mesenteric lymph nodes in addition to the blood and spleen. T-cell epitope mapping of the proliferative responses of short-term cell lines (STCLs) grown from peripheral blood or lymphoid cells revealed a major epitope within the polypeptide 121-150 after either route of immunization. Two minor T-cell epitopes were found within peptide 41-80 in STCLs from splenic and circulating cells. B-cell epitope mapping of serum or biliary IgA and IgG antibodies revealed two overlapping or adjacent immunodominant epitopes to the T-cell epitopes within the polypeptides 121-170 and 51-90. The results suggest that rectal augmented by oral immunization with a recombinant particulate antigen in nonhuman primates elicits secretory IgA and to a lesser extent IgG responses in the draining lymph nodes and the rectal mucosa, whereas systemic immunization targets predominantly splenic and circulating T- and B-cell responses. These findings may have important implications in the strategy of designing vaccines in prevention of homosexual transmission of HIV infection.