A protein particle vaccine containing multiple malaria epitopes.

Ty virus-like particles consist of a single protein species that can be produced in yeast. Recombinant Ty-VLPs carrying a string of up to 15 defined cytotoxic T lymphocyte (CTL) epitopes from Plasmodium species prime protective CTL responses in mice following a single administration without adjuvant. Effective processing of epitopes from the string was demonstrated in vitro and in vivo and was not affected by flanking sequences.

Second-generation monoclonal antibodies to intestinal MUC2 peptide reactive with colon cancer.

BACKGROUND: Antitumor antibodies have traditionally been made to whole tumors or tumor extract. The use of defined synthetic antigens would be desirable for producing monoclonal antibodies. PURPOSE: Our purpose was to determine if antipeptide antibody to MUC2 had antitumor activity and specificity. METHODS: A 29-amino-acid peptide to MUC2 was synthesized and monoclonal antibodies were produced after immunizing BALB/c mice with peptide-keyhole-limpet hemocyanin in complete Freund's adjuvant, and the monoclonal antibodies were tested on peptides and human tissues. RESULTS: CCP31, CCP37, and CCP58 monoclonal antibodies were produced using MUC2 MI-29 (KYPTTTPISTTTMVTPTPTPTGTQTPTTT) containing one repeat unit of 23 amino acids and part of the next repeat of four amino acids. These antibodies reacted with the MUC2-derived peptide but not with MUC1- or MUC3-derived peptides. One of the monoclonal antibodies, CCP58, reacted strongly with human colon cancer and normal intestine in both fresh and formalin-fixed tissues; two other antibodies, CCP37 and CCP31, reacted only with fresh human tissues of normal colon and malignant colon tumors by immunoperoxidase staining. In addition, CCP37 and CCP58 reacted strongly with human gastric cancer; all antibodies reacted weakly with human salivary gland, and none reacted with tissues from normal human lung, kidney, stomach, pancreas, or endometrium. By analysis of mucin molecules by Western blotting, the antigen detected by monoclonal antibodies CCP37 and CCP58 was found to be of a high relative molecular mass (520 kd). CONCLUSIONS: Anti-MUC2 peptide antibodies appear to be relatively tissue specific and represent a new method of producing antitumor antibodies.

The breast tumor-associated epitope defined by monoclonal antibody 3E1.2 is an O-linked mucin carbohydrate containing N-glycolylneuraminic acid.

The breast cancer-associated epitope (mammary serum antigen or MSA) defined by monoclonal antibody (Mab) 3E1.2 is a neuraminidase-sensitive carbohydrate expressed on MUC-1-encoded molecules. However, the reactivity of Mab 3E1.2 is also reduced by protease treatment of the mucin, which suggests that 3E1.2 binds to multimers of the sialylated carbohydrate in a protein conformation-dependent manner. The common N-acetyl derivative of neuraminic acid (5-acetylneuraminic acid) is not involved in the epitope, since lectins specific for 5-acetylneuraminic acid (linked to GalNAc or Gal) are nonreactive with MSA-positive molecules. However, the N-glycolyl derivative, 5-glycolylneuraminic acid (Neu5Gc), forms a major part of the epitope since both free Neu5Gc and porcine stomach mucin (greater than 90% neuraminic acid as Neu5Gc) inhibit the binding of Mab 3E1.2, while bovine or ovine submaxillary mucins, fetuin, bovine gangliosides, and other carbohydrates do not. Indeed, the presence of Neu5Gc on human tumor mucin was confirmed by electrospray mass spectrometry. Neu5Gc is attached to an O-linked carbohydrate, since the expression of MSA by MCF-7 breast cancer cells is inhibited by the O-glycosylation inhibitor phenyl-N-acetyl-alpha-D-galactosaminide, but not by the N-glycosylation inhibitor tunicamycin, and the epitope is removed by treatment with O-glycanase but not N-glycanase F, endoglycosidase F, or endoglycosidase H, which are specific for N-linked glycans. This is likely to be a core glycan since 3E1.2 reacts after treatment of the mucin with trifluoromethanesulfonic acid, which removes most backbone and peripheral carbohydrates. Treatment with galactosidase or N-acetyl glucosaminidase enhances the binding of Mab 3E1.2, indicating that the Neu5Gc is not attached to galactose or N-acetyl galactosamine. Furthermore, the susceptibility of MSA to treatment with Arthrobacter urea-faciens neuraminidase [which is specific for alpha (2-6)-linked NeuNAc] and the loss in reactivity of GalNAc-specific lectins after periodate oxidation [alpha (2-3)-linked but not alpha (2-6)-linked NeuNAc protects GalNAc from periodate oxidation] indicate that the Neu5Gc may be attached alpha (2-6) to peptide-linked GalNAc. These results show that MSA is a Neu5Gc-containing O-linked core glycan, which represents a unique tumor-associated epitope not previously identified on human mucins.

Factors influencing the immunogenicity of the haptenic drug chlorhexidine in mice--Part I. Molecular requirements for the induction of IgE and IgG anti-hapten antibodies.

The immunogenicity of a novel hapten, the anti-microbial agent, chlorhexidine (1,1'-hexamethylene bis [5-(p-chlorophenyl)biguanide]) was assessed in mice, using alum as adjuvant. The i.p. injection of electrostatically-linked chlorhexidine + keyhole limpet haemocyanin (KLH) mixtures induced low level primary IgG anti-chlorhexidine antibody synthesis. In contrast, covalently-linked chlorhexidine-KLH complexes induced both IgE and IgG anti-chlorhexidine antibody synthesis. Covalent binding was facilitated by the N-chlorination of secondary amide groups on the biguanide moeities of the chlorhexidine molecule. The immunogenicity of such complexes was related to the degree of conjugation of chlorhexidine to KLH; which, in turn, was related to the molarity of chlorine used to effect N-chlorination. Immune responses to covalently-linked complexes could be enhanced by carrier priming. The induction of low levels of IgG anti-hapten antibodies by electrostatically-linked complexes may reflect T cell-independent specific B cell activation, either by chlorhexidine itself or by a "pseudo-plurivalent" chlorhexidine + KLH antigen.

A cell ELISA technique. The direct detection and semi-quantitation of immunoglobulin positive cells in 7 day lymphocyte cultures using the microtitre culture plates as the solid phase.

A sensitive ELISA is described which allows the detection and quantitation of immunoglobulin positive Peyer's patch and spleen lymphocytes in tissue culture microtitre plates. A methanol fixation step is used to fix the cells to the microtitre plate. The effects of various mitogens and an antigen on the number of immunoglobulin positive cells present after a 7 day culture were studied. Results were expressed either in optical densities or as percentages of the unstimulated control. The method proves a useful addition to existing techniques for studying the effects of mitogens and other agents on in vitro immunoglobulin synthesis by lymphocytes, and reflects immunoglobulin levels in the culture supernatants.

The quantitation of IgG4 antibodies to three common food allergens by ELISA with monoclonal anti-IgG4.

Enzyme-linked immunosorbent assays, capable of detecting nanogram quantities of IgG4 to 3 common food allergens, have been developed with use of a monoclonal anti-human IgG4. Calibrated positive human sera were used to construct standard curves. The allergens employed, major protein constituents of egg (ovalbumin), milk (beta-lactoglobulin) and-wheat (gluten), were highly effective in coating microtitre plate wells; and the assays proved to be sensitive, reproducible and specific, in terms of both allergen and IgG subclass. On testing sera from a group of non-allergic individuals a high proportion were found to contain IgG4 antibodies to the 3 allergens. A pool of 60 sera obtained from blood donors also contained IgG4 antibodies to all 3 allergens. The results illustrate that IgG4 antibodies against common food constituents are frequently encountered in sera from normal individuals; and that this needs to be taken into consideration in assessing the possible role of such antibodies in immediate hypersensitivity reactions.

Sequence variation in the early genes E1E4, E6 and E7 of human papilloma virus type 6.

The majority of condylomata acuminata (anogenital warts) are caused by infection with Human Papilloma Virus type 6 (HPV-6). We have sequenced the HPV-6 early genes, E1-E4, E6 and E7 from wart biopsy DNA samples sourced from the UK and USA and derived a consensus sequence for these genes and the proteins they encode. When compared to the prototype HPV-6b sequence, published over 12 years ago, the E1-E4 consensus sequence showed 3/91 (3.3%) amino acid changes, the E6 consensus sequence showed 1/150 (0.7%) changes and the E7 consensus sequence showed 1/98 (1.0%) changes. Since many of the early gene sequences from biopsy material were more similar to the HPV-6a subtype than HPV-6b, this data supports the use of HPV-6a as the HPV-6 prototype.

Hybrid Ty virus-like particles.

Vaccines need to activate antigen presenting cells, overcome genetic restriction in T-cell responses and elicit both T and B memory cells. In order to produce recombinant vaccines which can do this, considerable effort has been put into developing particulate antigen presentation systems to generate polyvalent, high molecular weight antigens which should maximally stimulate the immune system. One such antigen-carrier system is based on the ability of a protein encoded by the yeast retrotransposon, Ty, to self-assemble into virus-like particles (VLPs). Ty-fusion proteins retain this ability to form particles and a range of hybrid VLPs carrying a variety of heterologous antigens have been produced and shown to elicit potent immune responses. Hybrid VLPs carrying human immunodeficiency virus (HIV) antigens stimulate the three main components of the immune system, namely antibody synthesis, T-cell proliferative responses and cytotoxic T-lymphocyte (CTL) responses.

Yeast retrotransposon particles as antigen delivery systems.

The development of technologies to produce recombinant proteins for use in the pharmaceutical industry has made substantial advances, in particular in the area of generating antigens containing multiple copies of important immunological regions. One such antigen-carrier system is based on the ability of a protein encoded by the yeast retrotransposon, Ty, to self-assemble into virus-like particles. Ty-fusion proteins retain this ability to form particles, and a range of hybrid VLPs carrying a variety of heterologous antigens have been produced and shown to induce potent immune responses. In particular, hybrid VLPs carrying the core protein p24 of HIV (p24-VLPs) have been shown to induce antibody and T-cell proliferative responses in both experimental animals and human volunteers, and immunization of rabbits with VLPs carrying the principal neutralizing determinant of HIV (V3-VLPs) resulted in the induction of neutralizing antibody responses and T-cell proliferation. Further studies with V3-VLPs have shown that this particulate antigen stimulates enhanced V3-specific lymphoproliferative responses as compared to whole recombinant gp120 or to V3 peptide conjugated to albumin. The V3-VLPs also induce potent CTL responses following immunization of mice in the absence of adjuvant. These responses are MHC class I restricted and are mediated by CD8-positive cells. These observations therefore demonstrate that hybrid Ty-VLPs induce both humoral and cellular immune responses against HIV and suggest that these immunogens may be important in combatting AIDS and other infections.

The effects of oral and combined parenteral/oral immunization against an experimental Escherichia coli urinary tract infection in mice.

A double oral immunization (PO/PO) with an outer membrane protein (OMP) from a human uropathogenic strain of Escherichia coli, resulted in the partial protection of mice infected per urethrally with the same strain. Complete protection was achieved by immunizing with OMP in Freund's complete adjuvant (FCA), intramuscularly (i.m.), followed by an oral boost (i.m./PO). The PO/PO protocol stimulated mainly local urinary antibody synthesis, particularly IgA, whilst the i.m./PO regimen resulted in the appearance of both serum and urine antibodies. A single dose of OMP, 6 days after infection, rendered the mice resistant to reinfection, in contrast to non-immunized mice, and led to a significant increase in urine, serum and bile IgA anti-OMP levels. Our results confirm previous reports that the urinary tract forms part of the common mucosal immune system and provides further evidence for immunological memory in mucosal immunity. These results also demonstrate that our OMP preparation is a highly effective immunizing antigen, and that such preparations may be suitable as oral vaccines against urinary tract infection in humans.

The optimum conditions for antibody production in vitro by Peyer's patch lymphocytes and a comparison between total immunoglobulins and antigen (TNP)-specific antibody synthesis following mitogen stimulation.

Using optimised conditions in terms of cell concentration (2 X 10(6)/ml), duration of culture (7 days) and pH (7.0-7.1) and the highly sensitive enzyme-linked immunosorbent assay for antibody quantitation, we assessed the effects of lipopolysaccharide (LPS) and concanavalin A (Con A) on the levels of both total immunoglobulins and TNP-specific antibodies and compared the proportion of IgA anti-TNP to total IgA synthesised following the in vivo priming of mice, 5 days prior to culture, with trinitrophenylated sheep red blood cells (TNP-SRBC). LPS stimulation resulted in an increase in total IgA (ninefold), IgM (sixfold) and IgG (eightfold) synthesis over unstimulated cultures and similar increases were seen in IgA anti-TNP (eightfold), IgM anti-TNP (fivefold) and IgG anti-TNP (twelvefold). Con A stimulation resulted in a slight increase in total IgA (threefold), IgG (twofold) but a decrease in IgM (twofold) but did not appear to affect TNP-specific antibody levels. In unstimulated cultures, IgA anti-TNP comprised 7.8% of the total IgA synthesised. In LPS and Con A stimulated cultures the proportion was 6.6% and 3.2%, respectively. Inclusion of 10% fetal calf serum in the culture medium resulted in a substantial increase in the synthesis of IgG, IgM and IgA in unstimulated cultures but increased IgA levels only in LPS stimulated cultures. Our results emphasise the importance of optimising culture conditions when studying in vitro antibody synthesis and demonstrate that Peyer's patches respond quite substantially to parenterally administered antigen and that these responses can subsequently be monitored in vitro.

Serum factors affecting the specificity of anticardiolipin antibodies.

The effects were investigated of two pretreatments of human serum and plasma test samples on their subsequent reactivity in the anticardiolipin antibody enzyme-linked immunosorbent assay (ACA-ELISA). The first treatment involved heat inactivation of test samples at 56 degrees C for 30 min, a process sometimes used to inactivate samples from suspected human immunodeficiency virus positive individuals. Such treatment significantly increased the IgG ACA unit/mL values of normal sera, but when this effect was examined further, it was found that the increase in binding occurred on both cardiolipin-coated and uncoated wells and was therefore non-specific. Heat inactivation of sera prior to ACA testing should therefore be avoided. The second treatment involved diluting immunoglobulin (Ig)G and IgM ACA-positive sera in normal human serum (NHS) or newborn calf serum (NCS); sera diluted in NHS showed a significant increase in titre, particularly IgM ACA-positive sera. This phenomenon was found to be due to a serum cardiolipin-binding cofactor which enhances antibody recognition. The cofactor is heat stable and is present in normal sera (male and female) and also in IgG ACA-positive sera. The binding of a human IgM monoclonal antibody to cardiolipin was not affected by the cofactor. The cardiolipin/cofactor complex may represent the optimal autoantigen/autoimmunogen and a re-appraisal, therefore, of the clinical relevance of antibodies to cardiolipin and other negatively charged molecules is warranted.

Factors influencing the immunogenicity of the haptenic drug chlorhexidine in mice. II. The role of the carrier and adjuvants in the induction of IgE and IgG anti-hapten responses.

The roles of carrier and adjuvant in the induction of primary antibody responses to the haptenic drug chlorhexidine (which interacts only electrostatically with proteins) and its N-chlorinated derivative (which binds covalently to proteins) were investigated. N-chloro chlorhexidine, covalently linked to either ovalbumin, KLH, thaumatin, LPS-associated protein or human serum protein, but not autologous mouse serum protein or LPS itself, induced both IgE and IgG anti-chlorhexidine antibody synthesis when injected, with alum adjuvant, into BALB/c mice. Bordetella pertussis (BP) could function as both carrier and adjuvant, but no response was obtained by injection of N-chloro chlorhexidine alone or with alum adjuvant. The immunogenicity of N-chlorinated chlorhexidine was directly related to the degree of its substitution onto the carrier which, in turn, was proportional to the level of chlorine (mM) employed. Chlorine also affected the immunogenicity of the various carriers. In the absence of chlorine, chlorhexidine induced only low level IgG antibody synthesis, but only if presented in a 'pseudoplurivalent' form, as a chlorhexidine-mediated protein precipitate (with alum) or electrostatically bound to a particulate carrier such as BP.

The specificity of murine polyclonal and monoclonal antibodies to the haptenic drug chlorhexidine induced by chlorine-generated chlorhexidine-protein conjugates.

Polyclonal and monoclonal antibodies to the antibacterial agent chlorhexidine (1,1'-hexamethylene bis [5-(p-chlorophenyl)]biguanide, mol. wt = 505) were raised using a chlorine-generated N-chloro chlorhexidine-keyhole limpet haemocyanin (NCC-KLH) conjugate as the immunogen. Antibodies were detected by ELISA, using a semi-chlorhexidine derivative conjugated to human serum albumin (SC-HSA) as the antigen. Free chlorhexidine could completely inhibit both polyclonal and monoclonal antibody binding to SC-HSA. Direct binding and inhibition ELISA studies revealed that the N-chlorination of chlorhexidine does not significantly alter its specificity as an immunogen or antigen and that chlorhexidine has two identical epitopes. Each epitope consists of the p-chlorophenyl biguanide structure of which the terminal p-chlorophenyl group appears to be immunodominant. Chlorhexidine is, therefore, a symmetrical divalent hapten and this implies that it may be capable of eliciting immediate hypersensitivity reactions by divalent interaction with antibodies induced by chlorine-generated N-chloro-chlorhexidine-protein immunogens. The clinical significance of these findings is discussed.

The incidence of IgE and IgG antibodies to chlorhexidine.

IgE antibodies to the antiseptic agent chlorhexidine have recently been detected in the majority of sera from a small group of predominantly Japanese individuals showing anaphylactic-type adverse reactions towards chlorhexidine. In this study the prevalence of IgE and IgG antibodies with specificity for chlorhexidine was investigated in groups of Japanese and British individuals. The RAST data, using a better defined semi-chlorhexidine-HSA antigen than previously employed, revealed that chlorhexidine-specific IgE was only detected in Japanese individuals who had experienced anaphylactic-type reactions and was not detected in groups of Japanese nurses and patients, or in groups of British nurses and hospital staff, all in regular contact with chlorhexidine. A group of British blood donors was also negative. In contrast, IgG antibodies were detected not only in sera from chlorhexidine-sensitive Japanese patients, but also in several sera from Japanese nurses, non-sensitive Japanese patients and several British individuals. The possible reasons for these observations are discussed.

The kinetics of in vivo sensitization of rat peritoneal and lung mast cells: temporal dissociation from circulating levels of IgE.

Following i.p. injection of ovalbumin (OVA) plus Bordetella pertussis vaccine into Hooded Lister rats, the time-course of sensitization of peritoneal and lung mast cells (MC) did not parallel kinetic changes in the levels of circulating OVA-specific and total IgE. OVA-induced secretion of 5-hydroxytryptamine from isolated peritoneal and lung MC and the presence of OVA-specific IgE in serum were first demonstrated at day 14 post-immunization. However, subsequent to day 14, the responsiveness of both types of MC to OVA declined, while circulating levels of OVA-specific IgE continued to rise. Peritoneal MC, but not lung MC, showed increased responsiveness to challenge with anti-IgE on day 7 post-immunization, whereas circulating levels of total IgE were not elevated until day 14, thus demonstrating that nonantigen-specific IgE was acquired by peritoneal MC before it entered the circulation. Lung MC generally showed decreased reactivity to both OVA and anti-IgE, compared with peritoneal MC; no significant correlations were demonstrated between the responses of MC from these two tissue sites.

Enhanced proliferative cellular responses to HIV-1 V3 peptide and gp120 following immunization with V3:Ty virus-like particles.

The induction of CD4+ T-helper (Th) cell responses is likely to be an important requirement of vaccine candidates designed to prevent or moderate human immunodeficiency virus-1 (HIV-1) infection. We have investigated the ability of hybrid Ty virus-like particles carrying the V3 loop region of the HIV-1 IIIB envelope gp120 (V3:Ty-VLP) to elicit V3-specific proliferative responses. Significant proliferation in response to stimulation in vitro with homologous IIIB V3 peptide was observed following immunization of mice with V3:Ty-VLP either as an aluminium hydroxide precipitate or without adjuvant. Responses to MN V3 peptide were also observed in certain mouse haplotypes. To assess the effect of presenting the V3 loop in this particulate form, we compared the responses induced by V3:Ty-VLP with those obtained with two non-particulate immunogens, recombinant gp120 (rgp120) and V3 peptide conjugated to albumin. V3-specific responses to V3 peptide in vitro were reproducibly higher following immunization with V3:Ty-VLP than with either rgp120 or V3-albumin coagulate (V3-alb). The data indicate that immunization with the V3 loop as a hybrid Ty-VLP results in enhanced proliferative responses to V3 peptide and recognition of rgp120 in vitro. Some cross-reactivity of Th cells for V3 sequences from different isolates was also observed.

Induction of neutralizing antibody and T-cell responses to varicella-zoster virus (VZV) using Ty-virus-like particles carrying fragments of glycoprotein E (gE).

During infection with Varicella-zoster virus (VZV), the envelope proteins are highly immunogenic and glycoprotein E (gE) is one of the most abundant and antigenic. We have previously identified the immunodominant regions of gE and mapped the B-cell epitopes. In this study, we have evaluated the immunogenicity of recombinant hybrid Ty-virus-like particles (VLPs) carrying amino acids (1-134) or (101-161) of gE which contain the immunodominant sequences. VZV-specific antibodies were detected by ELISA in sera from mice and guinea pigs immunized with either gE(1-134)-VLPs or gE (101-161)-VLPs. The dominant B-cell epitopes, mapped by pepscan analysis of the sera, were found in peptides spanning amino acids 41-60, 56-75, 101-120, 116-135, 131-150 and 141-161. These sera also showed neutralizing activity against VZV in vitro. Epitopes recognized by neutralizing MAbs were mapped to both gE sequences (3B3 MAb recognizing amino acids 141-161 and IFB9 MAb recognizing amino acids 71-90). Lymphocyte proliferative responses to VZV were detected in four different mouse strains immunized with either gE(1-134)-VLPs or gE(101-134)-VLPs in alum. All mouse strains immunized with gE(1-134)-VLPs recognized epitopes in amino acids 11-30 and 71-90 and all those immunized with gE(101-161)-VLPs recognized epitopes in amino acids 91-110 and 106-125. These results indicate that VLPs carrying these gE sequences can prime potent humoral and cellular anti-VZV responses in small animals and warrant further investigation as potential vaccine candidates against varicella-zoster infections.

The effects of adjuvants on CTL induction by V3:Ty-virus-like particles (V3-VLPs) in mice.

We have previously described the generation of HIV-1 V3-specific cytotoxic T-lymphocytes (CTL) responses in BALB/c (H-2d) mice following immunization with Ty-virus-like particles carrying the V3 loop of gp120 (V3-VLPs) without adjuvant. In this study the effects of various adjuvants on CTL induction by V3-VLPs was examined. Mice immunized with V3-VLPs formulated in aqueous-based adjuvants, Detox, gamma-inulin, galactosaminylmuramyl dipeptide and Chemivax generated V3-specific CTL responses, although at reduced levels when compared to the no adjuvant group. V3-VLPs prepared in Alhydrogel, algamulin or as an oil emulsion in SAF-MF failed to generate V3-specific CTL responses. The mechanism whereby alum prevented the induction of a CTL response was investigated further. Immunization with V3-VLPs prepared in non-saturating doses of alum or alum plus EDTA primed for strong CTL responses, indicating that free VLPs do, but alum-bound VLPs do not enter the MHC class I processing pathway of antigen-presenting cells (APCs). Furthermore, V3-VLPs with very low doses of alum led to an enhancement of the CTL response. The formulation of hybrid Ty-VLPs in oil based or precipitating adjuvants, therefore, inhibits access to the MHC class I processing pathway of APCs. The intact particulate structure of hybrid VLPs is therefore strictly necessary for CTL induction.