Variable major proteins of Borrelia hermsii. Epitope mapping and partial sequence analysis of CNBr peptides.

The variable major proteins (VMP) of serotypes 7 and 21 of the relapsing fever agent Borrelia hermsii were isolated by detergent extraction and high performance liquid chromatography. Cyanogen bromide (CNBr) digestion of the isolated VMP yielded two peptides of apparent molecular weights 20,000 (20 K) and 16 K from VMP7, and three peptides of 14.5, 14, and 7 K mol wt from VMP21. Serotype-specific monoclonal antibodies bound in Western blots to one of each of the two or three CNBr fragments from the homologous VMP. A single monoclonal antibody bound to the whole cells, the isolated VMP, and a CNBr fragment of both serotype 7 and serotype 21. (This crossreactive antibody did not, however, bind to any of four other serotypes examined.) Regional conservation of structure between VMP7 and VMP21 was also shown by amino acid sequence analysis of the N-termini of the five CNBr fragments. One pair of aligned fragments from VMP7 and VMP21 had 80% amino acid homology in sequence; a second pair had 40% homology. The partial amino acid homologies between two VMP suggest that these proteins are products of members of a polygene family

Production of a T cell hybridoma that expresses the T cell receptor gamma/delta heterodimer.

We have produced a T cell hybridoma line by fusion of an IL-2-dependent, long-term T cell receptor (TCR) gamma/delta+ Thy-1+, bone marrow-derived, dendritic epidermal cell line to the BW5147 tumor line. The resultant hybridoma was rapidly growing, lymphokine independent, and expressed T3 in association with the TCR gamma/delta heterodimer. Several subclones of the hybridoma line produced easily detectable levels of IL-2 after stimulation by anti-T3 or Con A. The availability of these cloned cell lines should greatly facilitate further functional, biochemical, and molecular studies of the TCR delta chain

Mechanism of Protection Induced by Group A Streptococcus Vaccine Candidate J8-DT: Contribution of B and T-Cells Towards Protection

Vaccination with J8-DT, a leading GAS vaccine candidate, results in protective immunity in mice. Analysis of immunologic correlates of protection indicated a role of J8-specific antibodies that were induced post-immunization. In the present study, several independent experimental approaches were employed to investigate the protective immunological mechanisms involved in J8-DT-mediated immunity. These approaches included the passive transfer of mouse or rabbit immune serum/antibodies in addition to selective depletion of T-cell subsets prior to bacterial challenge. Passive transfer of J8-DT antiserum/antibodies from mice and rabbits conferred significant resistance against challenge to mice. To exclude the possibility of involvement of other host immune factors, the studies were repeated in SCID mice, which highlighted the need for an ongoing immune response for long-lived protection. Depletion of CD4+ and CD8+ T-cell subsets confirmed that an active de novo immune response, involving CD4+ T-helper cells, is required for continued synthesis of antibodies resulting in protection against GAS infection. Taken together these results indicate an involvement of CD4+ T-cells in J8-DT-mediated protection possibly via an ability to maintain antibody levels. These results have considerable relevance to the development of a broad spectrum passive immunotherapy for GAS disease

Identification of a Highly Conserved H1 Subtype-Specific Epitope with Diagnostic Potential in the Hemagglutinin Protein of Influenza A Virus

Subtype specificity of influenza A virus (IAV) is determined by its two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). For HA, 16 distinct subtypes (H1–H16) exist, while nine exist for NA. The epidemic strains of H1N1 IAV change frequently and cause annual seasonal epidemics as well as occasional pandemics, such as the notorious 1918 influenza pandemic. The recent introduction of pandemic A/H1N1 IAV (H1N1pdm virus) into humans re-emphasizes the public health concern about H1N1 IAV. Several studies have identified conserved epitopes within specific HA subtypes that can be used for diagnostics. However, immune specific epitopes in H1N1 IAV have not been completely assessed. In this study, linear epitopes on the H1N1pdm viral HA protein were identified by peptide scanning using libraries of overlapping peptides against convalescent sera from H1N1pdm patients. One epitope, P5 (aa 58–72) was found to be immunodominant in patients and to evoke high titer antibodies in mice. Multiple sequence alignments and in silico coverage analysis showed that this epitope is highly conserved in influenza H1 HA [with a coverage of 91.6% (9,860/10,767)] and almost completely absent in other subtypes [with a coverage of 3.3% (792/23,895)]. This previously unidentified linear epitope is located outside the five well-recognized antigenic sites in HA. A peptide ELISA method based on this epitope was developed and showed high correlation (χ2 = 51.81, P<0.01, Pearson correlation coefficient R = 0.741) with a hemagglutination inhibition test. The highly conserved H1 subtype-specific immunodominant epitope may form the basis for developing novel assays for sero-diagnosis and active surveillance against H1N1 IAVs

Identification of a Highly Conserved H1 Subtype-Specific Epitope with Diagnostic Potential in the Hemagglutinin Protein of Influenza A Virus

Subtype specificity of influenza A virus (IAV) is determined by its two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). For HA, 16 distinct subtypes (H1–H16) exist, while nine exist for NA. The epidemic strains of H1N1 IAV change frequently and cause annual seasonal epidemics as well as occasional pandemics, such as the notorious 1918 influenza pandemic. The recent introduction of pandemic A/H1N1 IAV (H1N1pdm virus) into humans re-emphasizes the public health concern about H1N1 IAV. Several studies have identified conserved epitopes within specific HA subtypes that can be used for diagnostics. However, immune specific epitopes in H1N1 IAV have not been completely assessed. In this study, linear epitopes on the H1N1pdm viral HA protein were identified by peptide scanning using libraries of overlapping peptides against convalescent sera from H1N1pdm patients. One epitope, P5 (aa 58–72) was found to be immunodominant in patients and to evoke high titer antibodies in mice. Multiple sequence alignments and in silico coverage analysis showed that this epitope is highly conserved in influenza H1 HA [with a coverage of 91.6% (9,860/10,767)] and almost completely absent in other subtypes [with a coverage of 3.3% (792/23,895)]. This previously unidentified linear epitope is located outside the five well-recognized antigenic sites in HA. A peptide ELISA method based on this epitope was developed and showed high correlation (χ2 = 51.81, P<0.01, Pearson correlation coefficient R = 0.741) with a hemagglutination inhibition test. The highly conserved H1 subtype-specific immunodominant epitope may form the basis for developing novel assays for sero-diagnosis and active surveillance against H1N1 IAVs

Stage-Specific Pathways of Leishmania infantum chagasi Entry and Phagosome Maturation in Macrophages

The life stages of Leishmania spp. include the infectious promastigote and the replicative intracellular amastigote. Each stage is phagocytosed by macrophages during the parasite life cycle. We previously showed that caveolae, a subset of cholesterol-rich membrane lipid rafts, facilitate uptake and intracellular survival of virulent promastigotes by macrophages, at least in part, by delaying parasitophorous vacuole (PV)-lysosome fusion. We hypothesized that amastigotes and promastigotes would differ in their route of macrophage entry and mechanism of PV maturation. Indeed, transient disruption of macrophage lipid rafts decreased the entry of promastigotes, but not amastigotes, into macrophages (P<0.001). Promastigote-containing PVs were positive for caveolin-1, and co-localized transiently with EEA-1 and Rab5 at 5 minutes. Amastigote-generated PVs lacked caveolin-1 but retained Rab5 and EEA-1 for at least 30 minutes or 2 hours, respectively. Coinciding with their conversion into amastigotes, the number of promastigote PVs positive for LAMP-1 increased from 20% at 1 hour, to 46% by 24 hours, (P<0.001, Chi square). In contrast, more than 80% of amastigote-initiated PVs were LAMP-1+ at both 1 and 24 hours. Furthermore, lipid raft disruption increased LAMP-1 recruitment to promastigote, but not to amastigote-containing compartments. Overall, our data showed that promastigotes enter macrophages through cholesterol-rich domains like caveolae to delay fusion with lysosomes. In contrast, amastigotes enter through a non-caveolae pathway, and their PVs rapidly fuse with late endosomes but prolong their association with early endosome markers. These results suggest a model in which promastigotes and amastigotes use different mechanisms to enter macrophages, modulate the kinetics of phagosome maturation, and facilitate their intracellular survival

Characterization of the Rabbit Neonatal Fc Receptor (FcRn) and Analyzing the Immunophenotype of the Transgenic Rabbits That Overexpresses FcRn

The neonatal Fc receptor (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes an active role in phagocytosis, and delivers antigen for presentation. We have previously shown that overexpression of FcRn in transgenic mice significantly improves the humoral immune response. Because rabbits are an important source of polyclonal and monoclonal antibodies, adaptation of our FcRn overexpression technology in this species would bring significant advantages. We cloned the full length cDNA of the rabbit FcRn alpha-chain and found that it is similar to its orthologous analyzed so far. The rabbit FcRn - IgG contact residues are highly conserved, and based on this we predicted pH dependent interaction, which we confirmed by analyzing the pH dependent binding of FcRn to rabbit IgG using yolk sac lysates of rabbit fetuses by Western blot. Using immunohistochemistry, we detected strong FcRn staining in the endodermal cells of the rabbit yolk sac membrane, while the placental trophoblast cells and amnion showed no FcRn staining. Then, using BAC transgenesis we generated transgenic rabbits carrying and overexpressing a 110 kb rabbit genomic fragment encoding the FcRn. These transgenic rabbits – having one extra copy of the FcRn when hemizygous and two extra copies when homozygous - showed improved IgG protection and an augmented humoral immune response when immunized with a variety of different antigens. Our results in these transgenic rabbits demonstrate an increased immune response, similar to what we described in mice, indicating that FcRn overexpression brings significant advantages for the production of polyclonal and monoclonal antibodies

Limited Transplantation of Antigen-Expressing Hematopoietic Stem Cells Induces Long-Lasting Cytotoxic T Cell Responses

Harnessing the ability of cytotoxic T lymphocytes (CTLs) to recognize and eradicate tumor or pathogen-infected cells is a critical goal of modern immune-based therapies. Although multiple immunization strategies efficiently induce high levels of antigen-specific CTLs, the initial increase is typically followed by a rapid contraction phase resulting in a sharp decline in the frequency of functional CTLs. We describe a novel approach to immunotherapy based on a transplantation of low numbers of antigen-expressing hematopoietic stem cells (HSCs) following nonmyeloablative or partially myeloablative conditioning. Continuous antigen presentation by a limited number of differentiated transgenic hematopoietic cells results in an induction and prolonged maintenance of fully functional effector T cell responses in a mouse model. Recipient animals display high levels of antigen-specific CTLs four months following transplantation in contrast to dendritic cell-immunized animals in which the response typically declines at 4–6 weeks post-immunization. Majority of HSC-induced antigen-specific CD8+ T cells display central memory phenotype, efficiently kill target cells in vivo, and protect recipients against tumor growth in a preventive setting. Furthermore, we confirm previously published observation that high level engraftment of antigen-expressing HSCs following myeloablative conditioning results in tolerance and an absence of specific cytotoxic activity in vivo. In conclusion, the data presented here supports potential application of immunization by limited transplantation of antigen-expressing HSCs for the prevention and treatment of cancer and therapeutic immunization of chronic infectious diseases such as HIV-1/AIDS