Target Of Humoral Immune Response Research Articles

Recombinant Plasmodium vivax circumsporozoite surface protein allelic variants: antibody recognition by individuals from three communities in the Brazilian Amazon

Circumsporozoite protein (CSP) variants of P. vivax, besides having variations in the protein repetitive portion, can differ from each other in aspects such as geographical distribution, intensity of transmission, vectorial competence and immune response. Such aspects must be considered to P. vivax vaccine development. Therefore, we evaluated the immunogenicity of novel recombinant proteins corresponding to each of the three P. vivax allelic variants (VK210, VK247 and P. vivax-like) and of the C-terminal region (shared by all PvCSP variants) in naturally malaria-exposed populations of Brazilian Amazon. Our results demonstrated that PvCSP-VK210 was the major target of humoral immune response in studied population, presenting higher frequency and magnitude of IgG response. The IgG subclass profile showed a prevalence of cytophilic antibodies (IgG1 and IgG3), that seem to have an essential role in protective immune response. Differently of PvCSP allelic variants, antibodies elicited against C-terminal region of protein did not correlate with epidemiological parameters, bringing additional evidence that humoral response against this protein region is not essential to protective immunity. Taken together, these findings increase the knowledge on serological response to distinct PvCSP allelic variants and may contribute to the development of a global and effective P. vivax vaccine.

Characterization of HIV-1 CRF01_AE env Genes Derived from Recently Infected Indonesian Individuals

To eradicate human immunodeficiency virus type 1 (HIV-1) infection, a comprehensive strategy including preventive vaccine development is needed. Envelope glycoproteins (Env) play a central role in viral infection and are the major targets of humoral immune responses. Therefore, Env is a candidate vaccine antigen, and its characterization is necessary for vaccine development. The characterization of the transmitted/founder (T/F; i.e., recently infected) virus that is responsible for the establishment of infection and induction of primary anti-HIV-1 immune responses is important. We herein established HIV-1 env clones derived from recently infected Indonesian individuals. All env genes were classified into CRF01_AE. The immunological characterization of env clones was performed by neutralization tests using a series of broadly neutralizing antibodies. The present study is the first to immunologically characterize the CRF01_AE T/F virus circulating in Indonesia.

Analysis of HIV-1 envelope evolution suggests antibody-mediated selection of common epitopes among Chinese former plasma donors from a narrow-source outbreak

The HIV-1 envelope mutates rapidly to evade recognition and killing, and is a major target of humoral immune responses and vaccine development. Identification of common epitopes for vaccine development have been complicated by genetic variation on both virus and host levels. We studied HIV-1 envelope gp120 evolution in 12 Chinese former plasma donors infected with a purportedly single founder virus, with the aim of identifying common antibody epitopes under immune selection. We found five amino acid sites under significant positive selection in ≥50% of the study participants, and 22 sites consistent with antibody-mediated selection. Despite strong selection pressure, some sites housed a limited repertoire of amino acids. Structural modelling revealed that most of the variable amino acid sites were located on the exposed distal edge of the Gp120 trimer, whilst invariant sites clustered within the centre of the protein complex. Two sites, flanking the V3 hypervariable loop, represent novel antibody sites. Analysis of HIV-1 evolution in hosts infected with a narrow-source virus may provide insight and novel understanding of common epitopes under antibody-mediated selection. If verified in functional studies, such epitopes could be suitable as targets in vaccine development.

Identification of a Continuous Neutralizing Epitope within UL128 of Human Cytomegalovirus.

As human cytomegalovirus (HCMV) is the most common infectious cause of fetal anomalies during pregnancy, development of a vaccine that prevents HCMV infection is considered a global health priority. Although HCMV immune correlates of protection are only poorly defined, neutralizing antibodies (NAb) targeting the envelope pentamer complex (PC) composed of the subunits gH, gL, UL128, UL130, and UL131A are thought to contribute to the prevention of HCMV infection. Here, we describe a continuous target sequence within UL128 that is recognized by a previously isolated potent PC-specific NAb termed 13B5. By using peptide-based scanning procedures, we identified a 13-amino-acid-long target sequence at the UL128 C terminus that binds the 13B5 antibody with an affinity similar to that of the purified PC. In addition, the 13B5 binding site is universally conserved in HCMV, contains a previously described UL128/gL interaction site, and interferes with the 13B5 neutralizing function, indicating that the 13B5 epitope sequence is located within the PC at a site of critical importance for HCMV neutralization. Vaccination of mice with peptides containing the 13B5 target sequence resulted in the robust stimulation of binding antibodies and, in a subset of immunized animals, in the induction of detectable NAb, supporting that the identified 13B5 target sequence constitutes a PC-specific neutralizing epitope. These findings provide evidence for the discovery of a continuous neutralizing epitope within the UL128 subunit of the PC that could be an important target of humoral immune responses that are involved in protection against congenital HCMV infection.IMPORTANCE Neutralizing antibodies (NAb) targeting the human cytomegalovirus (HCMV) envelope pentamer complex (PC) are thought to be important for preventing HCMV transmission from the mother to the fetus, thereby mitigating severe developmental disabilities in newborns. However, the epitope sequences within the PC that are recognized by these potentially protective antibody responses are only poorly defined. Here, we provide evidence for the existence of a highly conserved, continuous, PC-specific epitope sequence that appears to be located within the PC at a subunit interaction site of critical importance for HCMV neutralization. These discoveries provide insights into a continuous PC-specific neutralizing epitope, which could be an important target for a vaccine formulation to interfere with congenital HCMV infection.

CD4+ T-cell recovery with suppressive ART-induced rapid sequence evolution in hepatitis C virus envelope but not NS3.

CD4 T-cell depletion from HIV infection leads to a global decline in anti-hepatitis C virus (HCV) envelope neutralizing antibody (nAb) response, which may play a role in accelerating liver fibrosis. An increase in anti-HCV nAb titers has been reported during antiretroviral therapy (ART) but its impact on HCV remains poorly understood. The objective of this study is to determine the effects of ART on long-term HCV evolution. We examined HCV quasispecies structure and long-term evolution in HIV/HCV coinfected patients with ART-induced CD4 T-cell recovery, and compared with patients with CD4 T-cell depletion from delayed ART. We applied a single-variant sequencing (SVS) method to construct authentic viral quasispecies and compared sequence evolution in HCV envelope, the primary target for humoral immune responses, and NS3, a target for cellular immunity, between the two cohorts. The SVS method corrected biases known to skew the proportions of viral variants, revealing authentic HCV quasispeices structures. We observed higher rates of HCV envelope sequence evolution in patients with ART-induced CD4 T-cell recovery, compared with patients with CD4 T-cell depletion from delayed ART (P = 0.03). Evolutionary rates for NS3 were considerably lower than the rates for envelope (P < 0.01), with no significant difference observed between the two groups. ART-induced CD4 T-cell recovery results in rapid sequence evolution in HCV envelope, but not in NS3. These results suggest that suppressive ART disproportionally enhances HCV-specific humoral responses more than cellular responses, resulting in rapid sequence evolution in HCV envelope but not NS3.

A Borrelia burgdorferi Surface-Exposed Transmembrane Protein Lacking Detectable Immune Responses Supports Pathogen Persistence and Constitutes a Vaccine Target.

Borrelia burgdorferi harbors a limited set of transmembrane surface proteins, most of which constitute key targets of humoral immune responses. Here we show that BB0405, a conserved membrane-spanning protein of unknown function, fails to evoke detectable antibody responses despite its extracellular exposure. bb0405 is a member of an operon and ubiquitously expressed throughout the rodent-tick infection cycle. The gene product serves an essential function in vivo, as bb0405-deletion mutants are unable to transmit from ticks and establish infection in mammalian hosts. Despite the lack of BB0405-specific immunoglobulin M or immunoglobulin G antibodies during natural infection, mice immunized with a recombinant version of the protein elicited high-titer and remarkably long-lasting antibody responses, conferring significant host protection against tick-borne infection. Taken together, these studies highlight the essential role of an apparently immune-invisible borrelial transmembrane protein in facilitating infection and its usefulness as a target of protective host immunity blocking the transmission of B. burgdorferi.

The PGRS Domain from PE_PGRS33 of Mycobacterium tuberculosis is Target of Humoral Immune Response in Mice and Humans.

The PE_PGRS33 protein is a member of the PE family, which encompasses the PE and the PE_PGRS subfamilies. Among PE_PGRS’s, this protein is one of the most studied antigens and its immunomodulatory properties are influence by both PE and PGRS domains. However, the contribution of these domains to the host immune recognition of the PE_PGRS33 protein and their potential role in latent tuberculosis infection in humans is still unknown. In this study, the immunogenic properties of the complete PE_PGRS33 protein and each domain separately were evaluated in BALB/c mice and latent tuberculosis infected (LTBI) humans. In mice, PE_PGRS33 and its domains induced similar antibody production and secretion of IFN-γ. PE_PGRS33 and the PE domain stimulated higher CD4+ and CD8+ T-cell proliferation compared to the PGRS domain. This demonstrated that the principal difference in the immune recognition of the domains is the higher activation of T-cell subpopulations involved in the control of tuberculosis. In humans, the secretion of IFN-γ in response to PE_PGRS33 was detected in both LTBI and in non-infected vaccinated individuals. The same was observed for antibody response, which targets epitopes located in the PGRS domain but not in the PE domain. These observations suggest that T and B cell responses to PE_PGRS33 are induced by BCG vaccination and can be maintained for many years in non-infected individuals. This also indicates that the IFN-γ response detected might not be associated with latent tuberculosis infection. These results contribute to the elucidation of the role of the PE_PGRS33 protein and its PE and PGRS domains in the immune response against Mycobacterium tuberculosis.

Active and Passive Immunization Protects against Lethal, Extreme Drug Resistant-Acinetobacter baumannii Infection

Extreme-drug-resistant (XDR) Acinetobacter baumannii is a rapidly emerging pathogen causing infections with unacceptably high mortality rates due to inadequate available treatment. New methods to prevent and treat such infections are a critical unmet medical need. To conduct a rational vaccine discovery program, OmpA was identified as the primary target of humoral immune response after intravenous infection by A. baumannii in mice. OmpA was >99% conserved at the amino acid level across clinical isolates harvested between 1951 and 2009 from cerebrospinal fluid, blood, lung, and wound infections, including carbapenem-resistant isolates, and was ≥89% conserved among other sequenced strains, but had minimal homology to the human proteome. Vaccination of diabetic mice with recombinant OmpA (rOmpA) with aluminum hydroxide adjuvant markedly improved survival and reduced tissue bacterial burden in mice infected intravenously. Vaccination induced high titers of anti-OmpA antibodies, the levels of which correlated with survival in mice. Passive transfer with immune sera recapitulated protection. Immune sera did not enhance complement-mediated killing but did enhance opsonophagocytic killing of A. baumannii. These results define active and passive immunization strategies to prevent and treat highly lethal, XDR A. baumannii infections.

Molecular Evolution of HIV-1 CRF01_AE Env in Thai Patients

BackgroundThe envelope glycoproteins (Env), gp120 and gp41, are the most variable proteins of human immunodeficiency virus type 1 (HIV-1), and are the major targets of humoral immune responses against HIV-1. A circulating recombinant form of HIV-1, CRF01_AE, is prevalent throughout Southeast Asia; however, only limited information regarding the immunological characteristics of CRF01_AE Env is currently available. In this study, we attempted to examine the evolutionary pattern of CRF01_AE Env under the selection pressure of host immune responses.Methodology/Principal FindingsPeripheral blood samples were collected periodically over 3 years from 15 HIV-1-infected individuals residing in northern Thailand, and amplified env genes from the samples were subjected to computational analysis. The V5 region of gp120 showed highest variability in several samples over 3 years, whereas the V1/V2 and/or V4 regions of gp120 also showed high variability in many samples. In addition, the N-terminal part of the C3 region of gp120 showed highest amino acid diversity among the conserved regions of gp120. Chronological changes in the numbers of amino acid residues in gp120 variable regions and potential N-linked glycosylation (PNLG) sites are involved in increasing the variability of Env gp120. Furthermore, the C3 region contained several amino acid residues potentially under positive selection, and APOBEC3 family protein-mediated G to A mutations were frequently detected in such residues.Conclusions/SignificanceSeveral factors, including amino acid substitutions particularly in gp120 C3 and V5 regions as well as changes in the number of PNLG sites and in the length of gp120 variable regions, were revealed to be involved in the molecular evolution of CRF01_AE Env. In addition, a similar tendency was observed between CRF01_AE and subtype C Env with regard to the amino acid variation of gp120 V3 and C3 regions. These results may provide important information for understanding the immunological characteristics of CRF01_AE Env.

HLA-DP antibodies before and after renal transplantation

Human leukocyte antigen (HLA)-DP is considered a target for humoral immune response in clinical transplantation. This study analyses the incidence of HLA-DP antibodies in renal patients. Development and epitope specificity of donor-specific antibodies (DSA) and non-DSA (NDSA) were examined. Pre- and posttransplant sera of 338 patients were screened for HLA-DP antibodies using the luminex single antigen assay. Positive patients, partners and/or kidney donors were HLA-DP typed by sequence-specific oligonucleotides. Potential epitopes were mapped by comparing the amino acid sequences of HLA-DP hypervariable regions (HVR) A-F of recipient, partner and/or donor. Specificities in the sera were aligned to deduce the HVR motif responsible for the antibodies. HLA-DP antibodies were detected in 14% of the patients (48/338). Before transplantation, the antibodies were shown in 23% (10 females and 1 male) and 77% were found after transplantation (30 in patients after the first, 7 after the second graft). Specificities were never restricted to individual mismatched antigens; broad HLA-DP sensitization was found as a rule. A single HVR mismatch was present in 80% of the DSA and in 79% of the NDSA. No HLA-DPA specific antibodies were found. Our findings confirm that HLA-DP antibodies are specific for epitopes shared by different HLA-DP antigens, indicating that only a restricted number of mismatched epitopes are recognized by the recipients immune system. Matching for immunogenic HLA-DP epitopes for renal transplantation seems to be functionally more relevant than classical matching at the allelic level.

Population genetic analysis of large sequence polymorphisms in Plasmodium falciparum blood-stage antigens

Plasmodium falciparum, the causative agent of human malaria, invades host erythrocytes using several proteins on the surface of the invasive merozoite, which have been proposed as potential vaccine candidates. Members of the multi-gene PfRh family are surface antigens that have been shown to play a central role in directing merozoites to alternative erythrocyte receptors for invasion. Recently, we identified a large structural polymorphism, a 0.58Kb deletion, in the C-terminal region of the PfRh2b gene, present at a high frequency in parasite populations from Senegal. We hypothesize that this region is a target of humoral immunity. Here, by analyzing 371 P. falciparum isolates we show that this major allele is present at varying frequencies in different populations within Senegal, Africa, and throughout the world. For allelic dimorphisms in the asexual stage antigens, Msp-2 and EBA-175, we find minimal geographic differentiation among parasite populations from Senegal and other African localities, suggesting extensive gene flow among these populations and/or immune-mediated frequency-dependent balancing selection. In contrast, we observe a higher level of inter-population divergence (as measured by F(st)) for the PfRh2b deletion, similar to that observed for SNPs from the sexual stage Pfs45/48 loci, which is postulated to be under directional selection. We confirm that the region containing the PfRh2b polymorphism is a target of humoral immune responses by demonstrating antibody reactivity of endemic sera. Our analysis of inter-population divergence suggests that in contrast to the large allelic dimorphisms in EBA-175 and Msp-2, the presence or absence of the large PfRh2b deletion may not elicit frequency-dependent immune selection, but may be under positive immune selection, having important implications for the development of these proteins as vaccine candidates.

A comparative study of HIV-1 clade C env evolution in a Zambian infant with an infected rhesus macaque during disease progression

To evaluate whether HIV-1 clade C (HIV-C) envelope variations that arise during disease progression in rhesus macaque model reflect changes that occur naturally in human infection. An infant macaque was infected with SHIV-1157i, an R5 tropic clade C SHIV, that expresses a primary HIV-C envelope derived from an infected human infant and monitored over a 5-year period. Genetic variation of the V1-V5 envelope region, which is the main target for humoral immune responses, derived from the infected macaque and infant was examined. The V1-V5 envelope region was cloned and sequenced from longitudinal peripheral blood mononuclear cell samples collected from the infected macaque and infant. Phylogenetic analysis [phylogenetic tree, diversity, divergence, ratio of nonsynonymous (dN) and synonymous substitution (dS) and dN distribution] was performed. Plasma RNA viral load, CD4(+) T-cell count, changes in the length of V1-V5 region, putative N-linked glycosylation site number and distribution were also measured. Phylogenetic analysis revealed that changes in the macaque closely reflected those of the infant during disease progression. Similar distribution patterns of dN and hot spots were observed between the macaque and infant. Analysis of putative N-linked glycosylation sites revealed several common variations between the virus populations in the two host species. These variations correlate with decline of CD4 T-cell count in the macaque and might be linked with disease progression. SHIV-C infection of macaque is a relevant animal model for studying variation of primary HIV-C envelope during disease progression and could be used to analyze the selection pressures that are associated with those changes.

Novel protein targets of the humoral immune response to Listeria monocytogenes infection in rabbits

The role of the humoral immune response in protective immunity against listerial infection has been overlooked and is essentially unknown. This study aimed to discover the protein targets of Listeria monocytogenes that elicit an antibody response following infection in a rabbit model. A genomic expression library for L. monocytogenes was constructed and differentially screened to identify genes encoding proteins that reacted with antiserum from rabbits infected with live L. monocytogenes serotype 4b (RalphaL), but not with that from animals immunized with heat-killed bacteria (RalphaK). Thirty-one clones expressing proteins that reacted exclusively with RalphaL were identified and sequenced. Sequence analysis, together with Western blot analysis of the proteins expressed from positive clones, led to the identification of eight L. monocytogenes proteins as targets of humoral immune responses during listerial infection: three internalin members (InlA, InlD and InlC2) and five novel proteins of unknown function (designated IspA, IspB, IspC, IspD and IspE, respectively). Exhibition of humoral immune responses to these proteins in actively infected rabbits but not in animals receiving heat-killed L. monocytogenes suggested that they were induced or significantly upregulated in vivo during infection and thus are important in Listeria pathogenesis. With the exception of antibodies to InlA, this is the first demonstration of antibodies to the other seven proteins in infected hosts. These immunogenic proteins may be useful candidates for elucidation of the role of antibodies in protective immunity in the context of listerial infection, as well as potential targets for serodiagnostic reagents and vaccine and drug development.

A novel surface autolysin of Listeria monocytogenes serotype 4b, IspC, contains a 23-residue N-terminal signal peptide being processed in E. coli

The 86-kDa protein IspC of 774 amino acids in Listeria monocytogenes serotype 4b has been recently identified as the target of humoral immune response to listerial infection and as a novel surface autolysin. A signal peptide is predicted at the N-terminal end of IspC, but no biochemical data has been shown to confirm the presence of the cleavage site of a signal peptidase. To address this and prepare sufficient amount of the protein for biochemical and structural characterization, we present a strategy for efficient expression and purification of IspC and analyze the purified protein by N-terminal sequencing and mass spectrometry. Expression of IspC in Escherichia coli using a pET30a-based expression construct was efficiently improved by incubating the culture at 37°C for 2h followed by 4°C for 16–18h. The recombinant product rIspC remained as a soluble form in the cellular extract and was purified to electrophorectic homogeneity by the combination of metal chelate affinity chromatography with cation-exchange chromatography. The IspC was shown to contain a 23-residue N-terminal signal peptide being processed between Thr 23 and Thr 24 in E. coli, resulting in an 84-kDa mature protein. The highly purified form of rIspC from this study, exhibiting both peptidoglycan hydrolase activity and immunogenicity as previously reported, would facilitate further biochemical, structural, and functional studies of this autolysin.

Identification of IspC, an 86-Kilodalton Protein Target of Humoral Immune Response to Infection with Listeria monocytogenes Serotype 4b, as a Novel Surface Autolysin

We identified and biochemically characterized a novel surface-localized autolysin from Listeria monocytogenes serotype 4b, an 86-kDa protein consisting of 774 amino acids and known from our previous studies as the target (designated IspC) of the humoral immune response to listerial infection. Recombinant IspC, expressed in Escherichia coli, was purified and used to raise specific rabbit polyclonal antibodies for protein characterization. The native IspC was detected in all growth phases at a relatively stable low level during a 22-h in vitro culture, although its gene was transiently transcribed only in the early exponential growth phase. This and our previous findings suggest that IspC is upregulated in vivo during infection. The protein was unevenly distributed in clusters on the cell surface, as shown by immunofluorescence and immunogold electron microscopy. The recombinant IspC was capable of hydrolyzing not only the cell walls of the gram-positive bacterium Micrococcus lysodeikticus and the gram-negative bacterium E. coli but also that of the IspC-producing strain of L. monocytogenes serotype 4b, indicating that it was an autolysin. The IspC autolysin exhibited peptidoglycan hydrolase activity over a broad pH range of between 3 and 9, with a pH optimum of 7.5 to 9. Analysis of various truncated forms of IspC for cell wall-hydrolyzing or -binding activity has defined two separate functional domains: the N-terminal catalytic domain (amino acids [aa] 1 to 197) responsible for the hydrolytic activity and the C-terminal domain (aa 198 to 774) made up of seven GW modules responsible for anchoring the protein to the cell wall. In contrast to the full-length IspC, the N-terminal catalytic domain showed hydrolytic activity at acidic pHs, with a pH optimum of between 4 and 6 and negligible activity at alkaline pHs. This suggests that the cell wall binding domain may be of importance in modulating the activity of the N-terminal hydrolase domain. Elucidation of the biochemical properties of IspC may have provided new insights into its biological function(s) and its role in pathogenesis.

A high-throughput proteo-genomics method to identify antibody targets associated with malignant disease

Identification of antibody targets associated with malignant disease is indispensable to developing passive and active antibody-based therapeutics or diagnostic agents. We have developed a novel technique combining Western blotting, genetic profiling, and mass spectroscopy that allows for the rapid and unambiguous identification of such antigens in a high-throughput manner. Herein, we demonstrate this technique, designated Ab SCAN, by deducing the known target of a monoclonal antibody and by identifying a new antigen that was observed to be the frequent target of humoral immune responses in prostate cancer patients. In both instances, a specific antigen emerged as the sole protein candidate. The newly identified antigen, mannose-6-phosphate/IGF II receptor, may be an important naturally immunogenic antigen involved in prostate cancer. The Ab SCAN technique is uniquely suited to the analysis of longitudinal serum samples from clinical studies and could be a powerful tool to correlate humoral immune responses directed against discreet antigens with clinical events.

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.

HSP60, HSP70 and HSP90 from Trichinella spiralis as targets of humoral immune response in rats.

This study identifies three heat shock proteins (HSPs) using purified preparations from Trichinella spiralis larvae. The proteins: HSP60, HSP70 and HSP90 were found to be targets of the humoral immune response in rats. Three approaches were adopted to obtain T. spiralis HSP-enriched material and/or to purify HSPs to homogeneity. The former product was prepared using affinity chromatography on gelatin sepharose and elution with ATP. Pure 90 kDa-protein was isolated from parasite extract by sequential DEAE (A50) column chromatograpy and preparative electrophoresis. Immunoblot analysis using monoclonal antibodies to HSP60, HSP70 and HSP90 detected the HSP60 and HSP70 in the affinity-purified product and HSP90 in the product obtained by sequential anionic chromatography and preparative electrophoresis. Finally, the reactivy of preimmune, T. spiralis immune and irrelevant immune rat sera on immunoblots were also examined. Only sera taken from infected rats at time-points after day 7 following the first infection exhibited activity against 60, 70 and 90 kDa proteins on blots. The fact that the serum antibodies were anti-HSP was established by immunoadsorption of HSPs to microtiter plates coated with anti-HSP60, anti-HSP70, or anti-HSP90 and using rat sera, positive on blots, to also give positive scores by continued enzyme-linked immunosorbent assay.

A 23-kDa Recombinant Antigen of Cryptosporidium parvum Induces a Cellular Immune Response on in Vitro Stimulated Spleen and Mesenteric Lymph Node Cells from Infected Mice

Bonafonte, M.-T., Smith, L. M., and Mead, J. R. 2000. A 23-kDa recombinant antigen of Cryptosporidium parvum induces a cellular immune response on in vitro stimulated spleen and mesenteric lymph node cells from infected mice. Experimental Parasitology96, 32–41. In the present study, we focused on a 23-kDa antigen, Cp23, which has been shown to be a major target of humoral immune responses in Cryptosporidium parvum infections and is present in both the sporozoite and merozoite stages. Recombinant Cp23 antigen was shown to stimulate a specific proliferative response by splenocytes and mesenteric lymph node cells from infected interferon gamma knockout BALB/c mice. Cp23 stimulation also induced TNF-α, IL-2, and IL-5 mRNA production by spleen cells from infected animals. In contrast, IL-12 mRNA was decreased by Cp23 stimulation compared with unstimulated splenocytes. These data suggest that, as with humoral responses, Cp23 is an important target of cellular immune responses in experimental C. parvum infections. The potential role of this antigen in conferring protective immunity is also discussed.

The variable C-terminal region of the Mycobacterium leprae 70-kilodalton heat shock protein is the target for humoral immune responses.

The 70-kDa heat shock protein of Mycobacterium leprae has a high degree of homology with the human hsp70 protein, yet it still elicits T-lymphocyte responses in subjects infected with M. leprae or vaccinated with the related Mycobacterium bovis BCG. We examined the serological responses to this protein by using recombinant protein fragments expressed from mutants with deletions of the M. leprae p70 gene. Monoclonal antibodies raised against either M. bovis or M. leprae p70 reacted with the C-terminal fragments but not the N-terminal fragments in a solid-phase enzyme-linked immunosorbent assay and an immunoblot assay. Inhibition enzyme-linked immunosorbent assays confirmed that two separate epitopes were defined by these monoclonal antibodies. Murine polyclonal sera also showed stronger binding to the C-terminal fragments. Sera from 33 and 48% of lepromatous leprosy patients reacted with M. leprae and M. bovis p70. This reactivity was mycobacterium specific, since few sera from control subjects in the same leprosy-endemic region were seropositive. The levels of anti-mycobacterial hsp70 antibodies were higher in patients with lepromatous leprosy than in those with tuberculoid leprosy or tuberculosis. The reactivity of sera from patients with leprosy was maximal with the C-terminal fragments. Therefore the C-terminal portion of M. leprae hsp70, which includes the region of maximum divergence from human hsp70, is the major target for the humoral immune response to the protein.