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The IgG response to HIV-1 pl7 gag protein was studied for up to 6 years in 12 HIV-1-infected patients with haemophilia, who had seroconverted between 1982 and 1985. To assess any prognostic value, p17 IgG titres were compared with p24 IgG titres, CD4 cell counts and p24 antigenaemia. P17 IgG avidity index was also examined. A strong similarity was found between the IgG titre to HIV-1 pl7 and that to p24. In patients who developed AIDS the decline in p17 IgG titres could precede by several years the drop in CD4 cells to under 200 cells/μl; whereas some long-term asymptomatic patients (CDCII) had increasing p17 IgG titres and stable CD4 cell counts. Declining p17 and p24 IgG titres were not always associated with an increase in p24 antigenaemia. IgG titres were found to be better predictors of disease progression than CD4 cell counts or p24 antigenaemia. Patients who developed AIDS during the study were also characterized by a lower p17 IgG avidity than patients who remained asymptomatic . This result suggests that IgG avidity could have prognostic relevance and be of importance for host resistance to AIDS onset. [ABSTRACT FROM AUTHOR]

Plasmid DNA vectors have been constructed with minigenes encoding a single cytotoxic T lymphocyte (CTL) epitope from either the M2 protein of respiratory syncytial virus (RSV) or from the nucleoprotein of measles virus (MV) with or without a signal sequence (also called secretory of leader sequence). Following intradermal immunization, plasmids in which the CTL epitopes were expressed in-frame with the signal sequence were more effective at inducing peptide- and virus-specific CTL responses than plasmids expressing CTL epitopes without the signal sequence. This immunization resulted in protection against MV-induced encephalitis and a significant reduction in viral load following RSV challenge. The reduction of viral load following RSV challenge was abrogated by prior injection with anti-IFN-γ antibodies. These results highlight the ability of epitope-based DNA immunization to induce protective immune responses to well-defined epitopes and indicate the potential of this approach for the development of vaccines against infectious diseases. [ABSTRACT FROM PUBLISHER]

We describe the engineering of a human immunodeficiency virus-1 (HIV-1) p24-immunoglobulin A (IgA) antigen-antibody fusion molecule for therapeutic purposes and its enhancing effect on fused antigen expression in tobacco plants. Although many recombinant proteins have been expressed in transgenic plants as vaccine candidates, low levels of expression are a recurring problem. In this paper, using the HIV p24 core antigen as a model vaccine target, we describe a strategy for increasing the yield of a recombinant protein in plants. HIV p24 antigen was expressed as a genetic fusion with the alpha2 and alpha3 constant region sequences from human Ig alpha-chain and targeted to the endomembrane system. The expression of this fusion protein was detected at levels approximately 13-fold higher than HIV p24 expressed alone, and a difference in the behaviour of the two recombinant proteins during trafficking in the plant secretory pathway has been identified. Expressing the antigen within the context of alpha-chain Ig sequences resulted in the formation of homodimers and the antigen was correctly recognized by specific antibodies. Furthermore, the HIV p24 elicited T-cell and antibody responses in immunized mice. The use of Ig fusion partners is proposed as a generic platform technology for up-regulating the expression of antigens in plants, and may represent the first step in a strategy to design new vaccines with enhanced immunological properties.

Vaccine development has been hampered by difficulties in developing new and safe adjuvants, so alternative technologies that offer new avenues forward are urgently needed. The goal of this study was to express a monoclonal recombinant immune complex in a transgenic plant. A recombinant protein consisting of a tetanus toxin C fragment-specific monoclonal antibody fused with the tetanus toxin C fragment was designed and expressed. Immune complex formation occurred between individual fusion proteins to form immune complex-like aggregates that bound C1q and FcgammaRIIa receptor and could be targeted to antigen-presenting cells. Unlike antigen alone, the recombinant immune fusion complexes were highly immunogenic in mice and did not require coadministration of an adjuvant (when injected subcutaneously). Indeed, these complexes elicited antibody titers that were more than 10,000 times higher than those observed in animals immunized with the antigen alone. Furthermore, animals immunized with only 1 mug of recombinant immune complex without adjuvant were fully protected against lethal challenge. This the first report on the use of a genetic fusion between antigen and antibody to ensure an optimal expression ratio between the two moieties and to obtain fully functional recombinant immune complexes as a new vaccine model.

The use of transgenic plants for the production of recombinant proteins is not a universal solution for all proteins. The choice of this expression system depends very much on the type of protein and its applications. Many proteins will best be made by conventional microbial fermentation, similarly, we are already identifying proteins where plants represent the only practical option for one reason or another. It will be important to understand better the cellular mechanisms of protein folding, assembly and processing in plants, in order to maximise the potential of transgenic plants as a protein production system. One of the main advantages that plants offer is that they are higher eukaryotic organisms with an endomembrane system. Therefore, they fold and assemble recombinant proteins using protein chaperones that are homologous to those in mammalian cells, and they perform post-translational modifications. This allows, for example, the expression of monoclonal antibodies, first described in 1989, as well as a range of other types of immunoglobulin molecules and multimeric complexes.

Guy's 13 is a mouse monoclonal antibody that specifically recognizes the major cell-surface adhesion protein SA I/II of Streptococcus mutans, one of the major causative agents of dental caries. Passive immunization with Guy's 13 prevents bacterial colonization in humans. To help elucidate the mechanism of prevention of colonization conferred by this antibody, the SA I/II epitope recognized by Guy's 13 was investigated. It was previously established that the epitope is conformational, being assembled from two non-contiguous regions of SA I/II. In the current study, using recombinant fragments of SA I/II and, ultimately, synthetic peptides, the discontinuous epitope was localized to residues 170-218 and 956-969. This work describes the mapping of a novel discontinuous epitope that requires an interaction between each determinant in order for epitope assembly and recognition by antibody to take place. Guy's 13 binds to the assembled epitope but not to these individual epitope fragments. The assembled epitope results from the interaction between the individual antigenic determinants and can be formed by mixing together determinants present on separate polypeptide chains. The data are consistent with one of the epitope fragments adopting a polyproline II-like helical conformation.

Guy's 13 is a mouse monoclonal antibody which recognizes streptococcal antigen I/II (SA I/II), a major cell surface glycoprotein of Streptococcus mutans. In a number of clinical trials, this antibody has been shown to prevent colonization in the human oral cavity. The aim of this study was to identify the SA I/II epitope recognized by Guy's 13. The data suggest that the epitope is conformational, delimited by two noncontiguous regions of the antigen: residues 45 to 457, within the N-terminal half of SA I/II, and residues 816 to 983, within the C-terminal half. In fluid-phase immunoassays a strict requirement for the simultaneous presence of both regions was demonstrated for antibody binding. Furthermore, these two regions of SA I/II were shown to have the ability to interact with each other in the absence of Guy's 13 antibody, suggesting that the normal conformation of SA I/II might be determined by the interaction of these two regions.

The feasibility of using antibody expressing transgenic plants either to neutralize bioactive molecules in the rhizosphere, or to accumulate and concentrate the molecules in leaves has been demonstrated in a model system consisting of hydroponic Nicotiana plant cultures expressing a murine monoclonal IgG1. Two transgenic plant types were used; in the first, functional antibody was rhizosecreted and shown to bind with antigen in the surrounding medium to form an immune complex. In the second, a transmembrane sequence retained monoclonal antibody in the plants, on the plasma membrane. Antigen added to the nutrient medium around the roots of mIgG plants was transported within 24 h to the topmost leaves of the plant where it was sequestered as an immune complex by binding to antibody on the cell membrane. Concentration of immune complex in the leaf tissue remained constant over a 72 h period after removal of antigen from nutrient medium. Free antigen was not detected in the leaves of wild-type plants. The two strategies of rhizosecretion-mediated binding and sequestration in leaf tissue could potentially be used in the phytoremediation of any pollutant for which it is possible to generate a monoclonal antibody.

Previous studies have shown that the production of recombinant antibodies in plants is highly efficient and presents numerous therapeutic applications. It is, however, known that plant glycoproteins display different glycosylation patterns to those exhibited by mammalian glycoproteins. Thus, it is important to know if these plant recombinant antibodies could induce undesirable immune responses in mammals; and to date no report has documented the potential immunogenicity of parenterally administered plant recombinant antibodies in animals. In order to answer this question, mice were immunised subcutaneously with a recombinant mouse monoclonal antibody produced in tobacco plants, together with alum as adjuvant. Two control groups were immunised in the same way with either the original murine monoclonal antibody or horseradish peroxidase (a plant glycoprotein). Analyses by direct immunoassay, competition immunoassay and real-time surface plasmon resonance, showed undetectable levels of antibody directed against both the protein and the glycan part of the plant recombinant antibody. These results have a direct relevance for the application of plant recombinant proteins as therapeutic agents and vaccines in humans.

A functional comparison was made between a monoclonal secretory antibody generated in transgenic plants and its parent murine IgG antibody.The affinity constants of both antibodies for a Streptococcus mutans adhesion protein were similar. However the secretory antibody had a higher functional affinity due to its dimeric structure. In the human oral cavity, the secretory antibody survived for up to three days, compared with one day for the IgG antibody. The plant secretory antibody afforded specific protection in humans against oral streptococcal colonization for at least four months. We demonstrate that transgenic plants can be used to produce high affinity, monoclonal secretory antibodies that can prevent specific microbial colonization in humans. These findings could be extended to the immunotherapeutic prevention of other mucosal infections in humans and animals.

Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis and pneumonia in infants and young children worldwide. As yet, there is no effective vaccine against RSV infection, and previous attempts to develop a formalin-inactivated vaccine resulted in exacerbated disease in recipients subsequently exposed to the virus. In the work described here, a combinatorial solid-phase peptide library was screened with a protective monoclonal antibody (MAb 19) to identify peptide mimics (mimotopes) of a conserved and conformationally-determined epitope of RSV fusion (F) protein. Two sequences identified (S1 [HWYISKPQ] and S2 [HWYDAEVL]) reacted specifically with MAb 19 when they were presented as solid-phase peptides. Furthermore, after amino acid substitution analyses, three sequences derived from S1 (S1S [HWSISKPQ], S1K [KWYISKPQ], and S1P [HPYISKPQ]), presented as multiple antigen peptides (MAPs), also showed strong reactivity with MAb 19. The affinity constants of the binding of MAb 19, determined by surface plasmon resonance analyses, were 1.19 x 10(9) and 4.93 x 10(9) M(-1) for S1 and S1S, respectively. Immunization of BALB/c mice with these mimotopes, presented as MAPs, resulted in the induction of anti-peptide antibodies that inhibited the binding of MAb 19 to RSV and neutralized viral infection in vitro, with titers equivalent to those in sera from RSV-infected animals. Following RSV challenge of S1S mimotope-immunized mice, a 98.7% reduction in the titer of virus in the lungs was observed. Furthermore, there was a greatly reduced cell infiltration in the lungs of immunized mice compared to that in controls. These results indicate the potential of peptide mimotopes to protect against RSV infection without exacerbating pulmonary pathology.

In the work described here, the effect of intranasal immunization of BALB/c mice with synthetic chimeric peptides consisting of a cytotoxic T-cell epitope (amino acids 81-95) from the M2 protein of respiratory syncytial virus (RSV) and a fusion peptide (amino acids 113-131) from the F1 protein of measles virus on response to challenge with RSV has been assessed. Three intranasal immunizations with the chimeric peptides without adjuvant induce peptide- and RSV-specific cytotoxic T-cell responses (CTL) at 1 or 3 weeks after the third immunization. The CTL responses significantly declined at 6 weeks after immunization. Furthermore, viral load in the lungs following challenge with RSV was significantly reduced in mice immunized with the F/M2:81-95 chimeric peptide compared to control animals at 1 or 3 weeks after immunization and no reduction of RSV titers was detectable 6 weeks after immunization. The CTL activity induced by F/M2:81-95 was therefore short-lived (less than 6 weeks) but was significantly correlated with the reduction in viral load in the lungs.

Objective: To study the humoral immune response to HIV-1 p17 and p24 Gag proteins longitudinally and assess any prognostic value., Design and Methods: Fifteen HIV-1 infected patients with haemophilia were asymptomatic at entry to the study in 1986. Each patient was monitored at 6- to 12-month intervals for up to 7 years for p17 and p24 immunoglobulin (Ig)G titres, p17 IgG avidity, total IgG, p24 antigenaemia and CD4 cell counts. Results were correlated with the clinical course., Results: Between 1986 and 1993, six of the patients developed CD4 cell counts below 200 x 10(6)/l (AIDS patients) while nine retained counts above this level (asymptomatic patients). All AIDS patients were characterized by declining IgG titres to p17 and p24 from 1986-1987 onwards. A reduction in specific p17 and p24 IgG preceded, by at least 3-4 years, the onset of CD4 cell depletion (< 200 x 10(6)/l). These six patients also had significantly lower p17 IgG avidity than the asymptomatic patients throughout the study., Conclusion: Titres of p17 and p24 IgG appeared to alter in the same manner. A progressive reduction in IgG titres and a low p17 IgG avidity were earlier predictors of disease progression than CD4 cell counts or p24 antigenaemia.

A two-site sandwich fluorescent-ELISA was optimized for the detection of HIV-1 p24 antigen produced by lymphoid cells infected with HIV-1 in vitro. To improve the sensitivity of the ELISA, a combination of streptavidin-beta-galactosidase and a fluorogenic substrate (4-methylumbelliferyl-beta-D-galactopyranoside) was employed for the enzymatic detection stage. Using recombinant p24 as standard antigen, a two-step assay detected as little as 0.7 pg/ml (3.10(-14) M) with an upper limit of 10,000 pg/ml. This detection range (approx. 50-70-times greater than ELISAs using a chromogenic detection) permitted an accurate and straightforward quantitation of p24 in culture supernatants. Overall, the fluorescent-ELISA had increased detectability, sensitivity and efficiency over existing ELISAs for HIV-1 p24.

CTL and antibody responses to HIV-1 p17 and p24 antigens were monitored from 1986-1991, in 4 hemophiliacs. The patients had been infected with HIV-1 between 1980 and 1984. Two patients have remained asymptomatic while two progressed to AIDS in 1990. CTL were boosted by culturing with peptides from p17 aa 86-115, or p24 aa 265-279; and aa 270-373 or PHA. Lysis was measured on autologous or allogeneic targets pulsed with peptides or infected with recombinant vaccinia virus carrying HIV-1 gag or influenza A matrix genes. Antibodies to p17 and p24 were tested by ELISA using peptides and by Western blotting. High levels of CTL activity to p17 and p24 antigens could be generated only with lymphocytes from the two asymptomatic patients between 1986 and 1989, but these responses were absent in 1990 and 1991. Antibodies to p17 peptides disappeared in parallel with CTL activity. Antibodies to some p24 peptides also declined but most patients retained activity to others. In all patients a > or = 3-fold increase in CD8+ cell numbers occurred over time and accompanied the decline of CTL and antibody responses. The loss of CTL and p17 antibodies occurred irrespective of whether patients remained asymptomatic or progressed to AIDS in the intervening two years.