Characterization of epitope regions of thyrotropin beta-subunit recognized by the monoclonal antibodies mAb279 and mAb299: a chimeric peptide approach.

This investigation describes the design, synthesis and evaluation of chimeric peptides related to the bovine thyrotropin beta-subunit, bTSHbeta. The structures of these chimeric peptides were derived from investigations with linear peptides and sequence alignment studies, in association with a homology model of TSHbeta developed from the hCG X-ray crystallographic structure. The structures of these chimeric peptides comprised beta-turn regions of loop L1 [bTSHbeta(14-20)] and loop L3 [bTSHbeta(65-72)] held in close proximity by a bis-beta-alanine linker and the disulfide bond bTSHbeta[Cys16-Cys67]. Linear and cyclic chimeric peptides were evaluated in immunochemical assays for their ability to inhibit the binding of radio-iodinated bTSHbeta [125I-bTSHbeta] to the monoclonal antibodies, mAb279 and mAb299. Previously, mAb279 and mAb299 have been shown to recognize epitopes accessible on the surface of TSHbeta that lie in close proximity to the TSH receptor-binding site. The results indicate that these chimeric peptides can specifically inhibit in a dose-dependent manner the binding of 125I-bTSHbeta to mAb299, while having a lesser effect on the binding with mAb279. Based on these results, it can be concluded that the bTSHbeta-epitope recognized by mAb299 involves contributions from amino residues from the beta-turn regions of the L1 and L3 loops of TSHbeta, and that these loop regions flank part of the receptor binding site of the bTSH beta-subunit.     

Use of synthetic peptides to delineate discontinuous sequence regions involved in epitope sites of the thyrotropin β-subunit

Summary In this investigation, an overlapping set of synthetic peptides spanning the entire primary structures of the β-subunit of bovine and human thyrotropin, bTSHβ and hTSHβ respectively, have been prepared to aid the delineation of the amino acid sequence regions involved in two spatially related epitopes of bTSH. These peptides were then evaluated for their ability to inhibit the binding of two anti-hTSH monoclonal antibodies, designated mAb279 and mAb299, to radiolabeled I¹²⁵-bTSHβ using competitive radioimmunoassay procedures. Synthetic peptides related to the sequence region b/hTSHβ[56–68] were found to specifically inhibit the binding of I¹²⁵-bTSHβ to mAb299, whilst having no effect on the binding of mAb279. In previous studies we have shown that mAb279 and mAb299 recognise epitopic sites located within the receptor-binding site of the TSH β-subunit. This investigation has therefore permitted identification of a contribution to the receptor binding site from the TSHβ[56–68] sequence, which forms part of theL3 loop region of the TSH β-subunit that is held in close proximity to theL1 loop region and the C-terminus of the TSH β-subunit by the disulphide bonds TSHβ[Cys¹⁶-Cys⁶⁷] and TSHβ[Cys¹⁹-Cys¹⁰⁵]. This finding is in agreement with previous investigations which have shown that TSHβ[Tyr⁵⁹] and TSHβ[Tyr⁷⁴] are also associated with the mAb299 epitope site, as well as contributing to the receptor binding region of the TSH β-subunit.     

Evaluation of expanded bed adsorption chromatography for extraction of prothrombin complex from Cohn Supernatant I.

This study evaluated the feasibility of substituting expanded bed adsorption (EBA) chromatography for an existing chromatographic purification process for the isolation of prothrombin complex concentrate (PCC) from Cohn Supernatant I. The EBA chromatography (Streamline) resins were compared to the current DEAE-cellulose resin for the extraction of PCC from Cohn SNI. EBA chromatography resins efficiently bound PCC from Cohn SNI at a significantly higher flow rate of up to 300 cm/h compared to 30 cm/h for the current DEAE-cellulose process. Composition and yield of the recovered PCC reflected the elution conditions used. The results indicate that EBA chromatography could be used to efficiently produce PCC comparable to existing products.     

Problems of interpreting integumental D-glucose fluxes by the integument of Schistosoma

1. The parasitic trematode Schistosoma mansoni can take up glucose across its surface at a rate three times higher than the glucose uptake by the mucosal border of the rabbit ileum. 2. Washout of tritiated polyethylene glycol (M Wt 4000) indicated that it was being lost through the integument and that the gut contribution is very small. 3. There is a peripheral diffusion resistance that will profoundly influence any transepidermal solute transfer.     

Molecular architecture and biorecognition processes of the cystine knot protein superfamily: part I. The glycoprotein hormones

In this review article, the reader is introduced to recent advances in our knowledge on a subset of the cystine knot superfamily of homo- and hetero-dimeric proteins, from the perspective of the endocrine glycoprotein hormone family of proteins: follitropin (FSH), Iutropin (LH), thyrotropin. (TSH) and chorionic gonadotropin (CG). Subsequent papers will address the structure-function behaviour of other members of this increasingly significant family of proteins, including various members of the transforming growth factor-beta (TGF-beta) family of proteins, the activins, inhibins, bone morphogenic growth factor, platelet derived growth factor-beta, nerve growth factor and more than 35 other proteins with similar topological features. In the present review article, specific emphasis has been placed on advances with the glycoprotein hormones (GPHs) that have facilitated greater insight into their physiological functions, molecular structures and most importantly the basis of the molecular recognition events that lead to the formation of hetero-dimeric structures as well as their specific and selective recognition by their corresponding receptors and antibodies. Thus, this review article focuses on the structural motifs involved in receptor recognition and the current techniques available to identify these regions, including the role of immunological methodology, peptide fragment design and synthesis and mutagenesis to delineate their structure-function relationships and molecular recognition behaviour.     

Transport of exogenous organic substances by invertebrate integuments: the field revisited

The notion that some marine invertebrates can use integumental uptake of organic compounds as a nutritional supplement dates back to the late 19th and early 20th centuries. This article provides a brief overview of more than a century's research, as it relates to significant stages in the development of experimental methods and concepts of general physiology. Emphasis is placed on changing paradigms and on the interplay between this specialized field of investigation and the mainstream of physiological thought. The present status of the field is summarized. The general consensus is challenged on the basis of previously published and new data from the author's laboratory. Particular emphasis is placed on data pointing toward an ultra-rapid turnover of amino acids in part of the epidermal space of the polychaete worm Nereis diversicolor. It is suggested that intra-epidermal L-alanine is compartmentalized metabolically or physically, and the consequences of this proposition are discussed in view of the general concepts of secondary active transport and intracellular isosmotic regulation. Future studies in this area of comparative physiology should concentrate not only on the molecular characteristics of the transporter proteins, but also on the way their function is integrated in the cellular physiology of the transporting cells. J. Exp. Zool. 289:254-265, 2001.     

Comparison of Heterologous Prime-Boost Strategies against Human Immunodeficiency Virus Type 1 Gag Using Negative Stranded RNA Viruses

This study analyzed a heterologous prime-boost vaccine approach against HIV-1 using three different antigenically unrelated negative-stranded viruses (NSV) expressing HIV-1 Gag as vaccine vectors: rabies virus (RABV), vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV). We hypothesized that this approach would result in more robust cellular immune responses than those achieved with the use of any of the vaccines alone in a homologous prime-boost regimen. To this end, we primed BALB/c mice with each of the NSV-based vectors. Primed mice were rested for thirty-five days after which we administered a second immunization with the same or heterologous NSV-Gag viruses. The magnitude and quality of the Gag-specific CD8⁺ T cells in response to these vectors post boost were measured. In addition, we performed challenge experiments using vaccinia virus expressing HIV-1 Gag (VV-Gag) thirty-three days after the boost inoculation. Our results showed that the choice of the vaccine used for priming was important for the detected Gag-specific CD8⁺ T cell recall responses post boost and that NDV-Gag appeared to result in a more robust recall of CD8⁺ T cell responses independent of the prime vaccine used. However, the different prime-boost strategies were not distinct for the parameters studied in the challenge experiments using VV-Gag but did indicate some benefits compared to single immunizations. Taken together, our data show that NSV vectors can individually stimulate HIV-Gag specific CD8⁺ T cells that are effectively recalled by other NSV vectors in a heterologous prime-boost approach. These results provide evidence that RABV, VSV and NDV can be used in combination to develop vaccines needing prime-boost regimens to stimulate effective immune responses.     

Uniform ionophore A23187 distribution and cytoplasmic calcium buffering in intact human red cells

The divalent cation-selective ionophore A23187 has been used to characterize cytoplasmic Ca and Mg buffering, Ca2+-pump parameters and the properties of a Ca2+-activated K+-channel in intact red cells. A critical assumption in these studies has been that the ionophore causes a uniform increase in divalent cation-permeability in all the cells. This has now been tested directly in ATP-depleted human red cells by analysing the kinetics of ionophore-induced 45Ca-tracer and net Ca2+ fluxes. The experimental curves were all adequately fitted by single-exponentials at all ionophore concentrations tested. Moreover, statistical analysis of 61 individual tracer influx curves and of pooled data showed no trend towards fast second exponential components. These results demonstrate uniformity of ionophore distribution, ionophore-induced Ca2+-permeability, and cytoplasmic Ca-buffering among all the cells. Experiments involving mixing of cell suspensions with high and low original ionophore content, and involving ionophore extraction by albumin, demonstrate a rapid redistribution of ionophore among the cells, indicating that homogeneity of ionophoric effects is achieved through dynamic ionophore redistribution.     

Characterization of a Single-Cycle Rabies Virus-Based Vaccine Vector

Recombinant rabies virus (RV)-based vectors have demonstrated their efficacy in generating long-term, antigen-specific immune responses in murine and monkey models. However, replication-competent viral vectors pose significant safety concerns due to vector pathogenicity. RV pathogenicity is largely attributed to its glycoprotein (RV-G), which facilitates the attachment and entry of RV into host cells. We have developed a live, single-cycle RV by deletion of the G gene from an RV vaccine vector expressing HIV-1 Gag (SPBN-ΔG-Gag). Passage of SPBN-ΔG-Gag on cells stably expressing RV-G allowed efficient propagation of the G-deleted RV. The in vivo immunogenicity data comparing single-cycle RV to a replication-competent control (BNSP-Gag) showed lower RV-specific antibodies; however, the overall isotype profiles (IgG2a/IgG1) were similar for the two vaccine vectors. Despite this difference, mice immunized with SPBN-ΔG-Gag and BNSP-Gag mounted similar levels of Gag-specific CD8⁺ T-cell responses as measured by major histocompatibility complex class I Gag-tetramer staining, gamma interferon-enzyme-linked immunospot assay, and cytotoxic T-cell assay. Moreover, these cellular responses were maintained equally at immunization titers as low as 10³ focus-forming units for both RV vaccine vectors. CD8⁺ T-cell responses were significantly enhanced by a boost with a single-cycle RV complemented with a heterologous vesicular stomatitis virus glycoprotein. These findings demonstrate that single-cycle RV is an effective alternative to replication-competent RV vectors for future development of vaccines for HIV-1 and other infectious diseases.The global spread of HIV-1 represents one of the most significant pandemics to afflict humans (22). Despite tremendous efforts to increase HIV awareness in the general population, UNAIDS reports that fewer than one in five people has access to HIV prevention strategies and many are subject to cultural stigmas thwarting such efforts (43). As such, an HIV vaccine is paramount for preventing disease transmission. It is not yet clear precisely what characteristics are critical for an effective HIV vaccine, yet evidence suggests one would need to induce both antibody and CD8⁺ T-cell-mediated immunity (reviewed in reference 25). Live viruses are at the forefront of HIV vaccine development (7) because they are powerful inducers of both of these arms of immunity. We previously demonstrated that replication-competent rabies virus (RV)-based vectors can induce long-lasting antigen-specific immune responses in both murine and monkey models, as well as protect rhesus macaques from an AIDS-like disease (23, 24, 26-29, 42). However, there are safety concerns with the use of any replication-competent virus for widespread immunization. To address this, we sought to develop and evaluate the immunogenicity of a safer alternative: a single-cycle RV expressing HIV-1 Gag as a model antigen.Single-cycle viral vectors are defective in certain viral components that are required for infectious particle assembly (reviewed in reference 12). As such, the virus undergoes one complete cycle of replication in the primary infected cell and produces progeny virions that are unable to spread to a second round of cells. The progeny are noninfectious and provide inert antigen that may or may not be immunogenic (12). In contrast, so-called replication-deficient viruses do not complete that initial round of replication. These two attenuation strategies have been adopted for use with many different viruses including, but not limited to, adenovirus (Ad), vaccinia virus (VV), canarypox virus (CPV), herpes simplex virus (HSV), vesicular stomatitis virus (VSV), and, more recently, RV (4, 6, 9, 18, 21, 33, 35, 36, 38). However, the results regarding the immunogenicity of such vectors are mixed. For example, both the replication-deficient Ad5 vector and modified vaccinia Ankara (MVA) showed reduced humoral and cellular immunogenicity compared to their replication-competent counterparts, but the use of higher titers and multiple immunizations did increase such responses (18, 33, 35). In the case of CPV, the replication-deficient vector provided poor HIV-specific cellular responses, causing the termination of phase II HIV-1 vaccine trials (38). In contrast, single-cycle VSV, a rhabdovirus closely related to RV, has been shown to induce HIV-1 Env-specific CD8⁺ T-cell responses equivalent to full-length VSV when administered intramuscularly (36). However, protection of rhesus macaques against highly pathogenic simian immunodeficiency virus (SIV) challenge by both replication-competent and single-cycle VSV needs to be shown.In the study described here, we generated a single-cycle RV vector expressing HIV-1 Gag (SPBN-ΔG-Gag) by deletion of the entire RV glycoprotein (RV-G) from the RV genome. RV-G was chosen due to its critical role in the attachment and entry of RV into host cells, which makes RV-G one of the most important determinants of viral pathogenicity (10, 11, 37). RV particles lacking G are unable to spread, as evidenced by intracranial infection with a G-deleted RV that remains restricted to the primary infected neurons (13, 44). It must be noted that in the absence of RV-G, virions are still capable of budding though at a 30-fold lower efficiency (32). These virions, however, are incapable of attachment and entry into a secondary host cell. Because of this, SPBN-ΔG-Gag was propagated on a trans-complementing cell line induced to express RV-G (or VSV-RV-G), effectively facilitating virus spread. To evaluate the immunogenicity of the single-cycle vector, we immunized mice and compared the humoral and cellular responses to responses generated by replication-competent RV. Our results indicate that single-cycle RV generates reduced vector-specific antibody responses but similar HIV-1 Gag-specific CD8⁺ T-cell responses. Moreover, these responses can be significantly enhanced by a heterologous boost with a single-cycle RV complemented with a VSV glycoprotein. Taken together, the results presented here show evidence that single-cycle RV is a promising platform for a safe, live viral vaccine for use against HIV-1 and other applications.