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PubMed1991

The influenza virus-infected host cell, a target for the immune response.

The exposure of some viral antigens at the surface of infected host cells was studied as an essential stimulus for the immune response during influenza virus infections. Only antibodies directed against the HA1 of the haemagglutinin were bound to the cell surface, anti-HA2 antibodies did not gain access to the membrane. Attempts to purify the cell-associated haemagglutinin (formerly called "viromicrosomes" by, R. Rott) indicated the formation of a special form of truncated haemagglutinin. This antigen and the nucleoprotein was purified from infected chorioallantoic membranes by immunoaffinity chromatography. NP-specific epitopes could not be defined on the native NP molecule which seems to be in solution in a discoordinate and partially polymeric array. Three non-overlapping epitopes were assigned to proteolytic peptides of the NP. Purified NP could induce cross-reactive cytotoxic T-cells in mice, but these animals were not protected against a challenge infection. CTL induced by exogenous stimulation or intracellular synthesis of the NP were restricted by MHC class I in both cases. NP could be incorporated into ISCOM after amphiphatic modification of the molecule. NP-ISCOM conferred some protection to experimental mice, but did not induce CTL demonstrable in vitro.

Animals↗
PubMed1993

High yield production of an inactivated coxsackie B3 adjuvant vaccine with protective effect against experimental myocarditis.

Dilated cardiomyopathy, perhaps chronic postviral fatigue syndrome as well as juvenile diabetes could be triggered by enteroviral infections. The frequency of sudden death after myocarditis and its relationship to enteroviral infections is disputed. Neonatal enteroviral disease is rare, but can be severe. It is also possible that enteroviruses pose a threat to immunocompromised patients, like bone marrow transplant recipients. Consequently, the emergence of chronic enteroviral diseases as a concept, prompted our attempts to produce an enteroviral vaccine. 1. Live attenuated enterovirus strains were previously in some cases shown to be suitable as vaccine candidates. We obtained neutralizing antibody titres ranging from 40-2560 against Coxsackie B3 virus (RD strain). Animals were protected to 90% against challenge infection. 2. Inactivated whole vaccine. We used beta-propiolactone to inactive Coxsackie B3 virus. 74% of the animals survived if the vaccine was prepared with Quil A matrix as adjuvant. The neutralisation antibody titres varied from < 5 to 320. By comparison aluminium hydroxide (p = 0.06) and Freund's adjuvant were inferior (p < 0.01). 3. Subunit vaccines. We have previously used the ISCOM (immunostimulatory complex) technology to produce a Coxsackie B3 subunit vaccine. High levels of neutralizing antibodies were obtained (512)-comparable to natural infection. All animals survived challenge infection after two booster doses with 16 nanogram of the ISCOM preparation. Limiting for this technique was the availability to include sufficient amount of antigenic protein material. In addition to neutralizing antibodies a cellular response might be obtainable. In conclusion we have shown that vaccine can be made against Coxsackie B3 virus with good protective effect and significant neutralisation antibody titre.(ABSTRACT TRUNCATED AT 250 WORDS)

Adjuvants, Immunologic↗
PubMed1992

Immunopotentiating reconstituted influenza virosomes (IRIVs) and other adjuvants for improved presentation of small antigens.

Synthetic peptides, purified subunits or inactivated small virus particles require immunopotentiation if they are to be effective vaccines. A large range of procedures to enhance immunogenicity has evolved over the last decades: aluminium salts, proteosomes, immunostimulating complexes (ISCOMs), liposomes, conjugation with bacterial products or derivatives, combination with surface-active agents or application of cytokines have been the most described classes of adjuvants. We describe here the design of an inactivated hepatitis A vaccine adjuvanted with immunopotentiating reconstituted influenza virosomes (IRIVs). The formalin-inactivated hepatitis A particles are attached to reconstituted protein-lipid complexes consisting of a mixture of phospholipids and influenza virus glycoproteins. With this new vaccine design we combined different immunostimulating effects: immunopotentiation by phospholipid vesicles, recognition of the haemagglutinin (HA) epitopes by the immune system, binding capacity of HA to sialic acid-containing receptors of macrophages and immunocompetent cells and mediation of entry into the cytoplasm of macrophages by a membrane-fusion event triggered by HA. Hepatitis A seronegative human volunteers received one intramuscular injection with this new vaccine. There were only few mild local reactions and 14 days after vaccination 100% of the subjects were seropositive. Among the individuals (control group) who received an alum-adsorbed vaccine, 88% developed local reactions. The seroconversion rate was 44%. We conclude from these results that the IRIVs provide a new approach to the future design of adjuvanted vaccines.

Adjuvants, Immunologic↗
PubMed1993

Adjuvants--a balance between toxicity and adjuvanticity.

Adjuvants have been used to augment the immune response in experimental immunology as well as in practical vaccination for more than 60 years. The chemical nature of adjuvants, their mode of action and the profile of their side effects are highly variable. Some of the side effects can be ascribed to an unintentional stimulation of different mechanisms of the immune system whereas others may reflect general adverse pharmacological reactions. The most common adjuvants for human use today are still aluminium hydroxide, aluminium phosphate and calcium phosphate although oil emulsions, products from bacteria and their synthetic derivatives as well as liposomes have also been tested or used in humans. In recent years monophosphoryl lipid A, ISCOMs with Quil-A and Syntex adjuvant formulation (SAF) containing the threonyl derivative of muramyl dipeptide have been under consideration for use as adjuvants in humans. At present the choice of adjuvants for human vaccination reflects a compromise between a requirement for adjuvanticity and an acceptable low level of side effects.

Adjuvants, Immunologic↗
PubMed2005

Partial protection against tissue cysts formation in pigs vaccinated with crude rhoptry proteins of Toxoplasma gondii.

A vaccine containing crude Toxoplasma gondii rhoptry proteins incorporated in the immunostimulating complexes (ISCOM) adjuvant was tested in pigs for protecting against tissue cyst formation. For this, 38 mixed breed pigs were divided into four groups, G1 (vaccinated challenged, n=10) received two doses (100 microg/dose) of the rhoptry vaccine at days 0 and 21, G2 (vaccinated challenged, n=10) received viable tachyzoites (7 x 10(7)) of the RH strain at day 0, G3 (unvaccinated challenged, n=10) and G4 (unvaccinated unchallenged, n=8). Pigs were challenged with 4 x 10(4) VEG strain oocysts 57 days later. The G1 pigs produced high IgG antibody levels in the indirect enzyme-linked immunosorbent assay (ELISA) after the second dose of rhoptry vaccine, but were not clinically protected against a high dose oocyst challenge. Partial protection was observed in G1 at the chronic phase of infection, when compared with G3. Pigs in group 2 developed high antibody levels and were protected against clinic signs. T. gondii was not detected in two (G1) and three (G2) pigs by mouse bioassay. The results indicate partial protection in pigs vaccinated with a rhoptry vaccine.

Animals↗
PubMed2005

HPLC purification of recombinant NcGRA6 antigen improves enzyme-linked immunosorbent assay for serodiagnosis of bovine neosporosis.

The gene for a dense granule protein (NcGRA6) of Neospora caninum was expressed in Escherichia coli as a His-tag fusion protein and purified by NiNTA affinity chromatography. In a preliminary study, high binding of antibodies from N. caninum-negative cows was observed in enzyme-linked immunosorbent assay (ELISA) using NiNTA-purified NcGRA6. Analysis of NiNTA eluates revealed a significant number of E. coli proteins that co-purified with recombinant NcGRA6. In an attempt to improve the relative sensitivity and specificity of the NcGRA6-based ELISA, the rNcGRA6 eluates were subjected to a secondary purification using reverse phase-high performance liquid chromatography (RP-HPLC). Analysis of RP-HPLC eluates by SDS-PAGE/silver staining revealed the purification of recombinant NcGRA6 from contaminating E. coli proteins. ELISAs using the RP-HPLC purified NcGRA6 (dELISA) or singly purified NcGRA6 (sELISA) for identifying seropositive and seronegative cows in a beef herd experiencing an epidemic outbreak of neosporosis were compared to standard assays based on native tachyzoite protein-immunofluorescence antibody test, immunoblot assay, and ISCOM-ELISA. The relative sensitivity, specificity, and kappa value of the NcGRA6d-ELISA were greatly improved over the NcGRA6s-ELISA when compared to the three native antigen immunoassays. These results indicate that removal of contaminating E. coli proteins improves the performance of recombinant NcGRA6 ELISA in diagnosing bovine neosporosis, and may have applicability to the use of recombinant proteins in diagnosing other infectious agents.

Animals↗
PubMed2006

Saponin-adjuvanted particulate vaccines for clinical use.

Saponins are well recognised as potent immune stimulators, but their applicability as vaccine adjuvants have been limited due to associated toxicity. Formulation of saponin adjuvant with cholesterol and phospholipid produces the particulate ISCOMATRIX adjuvant, and when antigen is also contained within the particle, an ISCOM vaccine is produced. These particulate vaccines retain the adjuvant activity of the saponin component but without toxicity. Saponin-adjuvanted particulate vaccines have significant potential as a novel strategy in vaccine development. This review discusses (i) recent methodologies which have attempted to increase the flexibility and applicability of this technology by modifying either the vaccine composition or the mode of formulation; (ii) recent evaluations of these technologies for inducing protection against infectious diseases and as cancer immunotherapeutics.

Adjuvants, Immunologic↗
PubMed2000

Use of cotton rats for preclinical evaluation of measles vaccines.

The continued prevalence and medical impact of measles worldwide has created interest in the development of new generations of measles vaccines. Monkeys can be used for preclinical testing of these vaccines. However, a more practical and less expensive animal model is highly desirable, particularly for initial vaccine development and evaluation. Cotton rats have been shown to support the replication of different strains of measles virus (MV), and thus may be useful for these purposes. To test this concept, the immunogenicity and protective efficacy of two standard (Moraten and trivalent measles, mumps, rubella) and four experimental (two recombinant ALVAC, one ISCOM subunit and live attenuated Edmonston-Zagreb) MV vaccines were evaluated in naïve cotton rats, and cotton rats with passively acquired MV-specific neutralizing serum antibodies. All of the test vaccines were immunogenic and protected naíve animals from pulmonary infection and viral dissemination. However, under the conditions utilized, only the Edmonston-Zagreb vaccine provided such protection to animals with significant levels of passively acquired MV-specific neutralizing antibodies. The results of these tests and the potential of using cotton rats as an animal model for preliminary testing of MV vaccines are discussed.

Animals↗
PubMed1998

Vaccination with recombinant Parasite Surface Antigen 2 from Leishmania major induces a Th1 type of immune response but does not protect against infection.

Vaccination with the native Parasite Surface Antigen 2 of Leishmania major with Corynebacterium parvum as adjuvant protects mice from leishmaniasis through a Th1 mediated response. Here we show that vaccination with a recombinant form of this protein, purified from Escherichia coli and administered in iscoms or with C. parvum as adjuvant, does not induce protective immunity despite the induction of Th1 responses. The results suggest that protective immunity depends on the ability of the vaccinating antigen to induce Th1-like T cells with ability to be recalled by infection. Therefore, the conformation of antigens may play a more major role for the induction of T cell mediated immunity than originally considered.

Animals↗
PubMed2000

Preparation and characterisation of quillaja saponin with less heterogeneity than Quil-A.

Immunisation against pathogens remains one of the most effective ways of preventing or reducing losses due to infectious diseases in animal husbandry. When inactivated vaccines are used, adjuvants are most often required to obtain satisfactory immune responses. One such type of adjuvant is saponin derived from the bark of Quillaja saponaria Molina, a tree of the rose family. A few different commercial sources exist, but due to the structural complexity and heterogeneity of these saponin preparations, it has been difficult to establish exactly which components are responsible for the adjuvant activity. By carefully selecting the bark source, we have succeeded in preparing a much less heterogeneous preparation of quillaja saponin. In this report we describe the preparation, in terms of structural complexity, hemolytic activity, adjuvant activity, and its ability to form ISCOM matrix. This new preparation could have implications for use per se, or as starting material for more effective preparation of pure substances.

Adjuvants, Immunologic↗
PubMed2001

Recent developments in adjuvants for vaccines against infectious diseases.

New generation vaccines, particularly those based on recombinant proteins and DNA, are likely to be less reactogenic than traditional vaccines, but are also less immunogenic. Therefore, there is an urgent need for the development of new and improved vaccine adjuvants. Adjuvants can be broadly separated into two classes, based on their principal mechanisms of action; vaccine delivery systems and 'immunostimulatory adjuvants'. Vaccine delivery systems are generally particulate e.g. emulsions, microparticles, iscoms and liposomes, and mainly function to target associated antigens into antigen presenting cells (APC). In contrast, immunostimulatory adjuvants are predominantly derived from pathogens and often represent pathogen associated molecular patterns (PAMP) e.g. LPS, MPL, CpG DNA, which activate cells of the innate immune system. Once activated, cells of innate immunity drive and focus the acquired immune response. In some studies, delivery systems and immunostimulatory agents have been combined to prepare adjuvant delivery systems, which are designed for more effective delivery of the immunostimulatory adjuvant into APC. Recent progress in innate immunity is beginning to yield insight into the initiation of immune responses and the ways in which immunostimulatory adjuvants may enhance this process. However, a rational approach to the development of new and more effective vaccine adjuvants will require much further work to better define the mechanisms of action of existing adjuvants. The discovery of more potent adjuvants may allow the development of vaccines against infectious agents such as HIV which do not naturally elicit protective immunity. New adjuvants may also allow vaccines to be delivered mucosally.

Adjuvants, Immunologic↗
PubMed1993

Immunogenicity of a hybrid Plasmodium falciparum malaria antigen.

A recombinant baculovirus-expressed hybrid protein containing epitopes for the C-terminal fragment of the Plasmodium falciparum precursor to the major merozoite surface antigens (PMMSA) and the tetrapeptide repeats of the circumsporozoite protein (CSP) was assessed for its immunogenicity. Murine MHC-II restriction of the antibody response to the CSP repeats was not overcome by the PMMSA component, the response to which showed no restriction. In an adjuvant trial the highest antibody titres in rabbits to both components of the hybrid were obtained using Freund's adjuvant. Lack of a boosting antibody response to the CSP repeats appeared to be linked to the conformation of the PMMSA component. Formulation of the hybrid protein into Iscoms gave antibody titres of only short duration to both components.

Adjuvants, Immunologic↗
PubMed1997

Antigen-specific chemotaxis of B cells.

The chemoattractant effect of soluble protein antigens for B cells from immunized mice was examined. Mice were immunized either via the footpad with ovalbumin (OVA) in complete Freund's adjuvant (CFA) or with CFA alone; or intraperitoneally with OVA incorporated in immune-stimulating complexes (OVA-ISCOM) or with saline. After 8-14 days B cells were purified from the spleens or the draining popliteal nodes and tested in vitro for locomotor responses to antigen using polarization (shape-change) and filter assays. Cells obtained by both routes of immunization, but not cells from control mice, gave locomotor responses to OVA. Responses were seen in B cells directly after preparation (5-8% of cells responding) but were enhanced if the cells were cultured overnight in the presence of interleukin-4 (IL-4) before testing (10-12% of cells responding). Checkerboard filter assays suggested that the response to OVA was chemotactic. The response was antigen specific since cells from OVA-immunized mice did not respond to bovine serum albumin (BSA), and cells from BSA-immunized mice responded to BSA but not to OVA. The response to OVA was inhibited by preincubation of OVA with anti-OVA but not with anti-BSA. Many of the cells that polarized in response to antigen were larger than any B cells found in control populations suggesting that the responsive cells are those that had been stimulated to enter cell cycle following immunization.

Animals↗
PubMed2000

Monoclonal antibodies to native HIV type 1 reverse transcriptase and their interaction with enzymes from different subtypes.

Recombinant reverse transcriptase (RT) from HIV-1 subtype B was used to produce mouse anti-RT monoclonal antibodies (MAbs). Immunization was done by mixing RT with the ISCOM matrix-forming adjuvant saponin (Quil A). Two different assays, both based on the interaction of native RT and antibodies, were used to monitor the immune response in mice and for screening, selection, and characterization of the MAbs. The first assay measures the capacity of antibodies to inhibit the polymerase activity of the RT and the second assay measures the ability of antibodies to capture enzymatically active RT. Twelve clones with the capacity to inhibit at least 50% of the RT activity and 34 clones with high RT-capturing capacity were found. The MAb panel was utilized to evaluate the immunological properties of 18 different RTs representing 9 different HIV1 subtypes. The RT-inhibitory MAbs could be divided into two groups based on their pattern of cross-reactivity toward the different HIV-1 RTs. The degree of diversity recorded among MAbs with RT-capturing capacity was larger. At least seven groups of MAbs with distinct cross-reactivity patterns were identified. Thus, the degree of isoenzyme specificity varied greatly, from MAbs that were quite specific for subtype B RT to one MAb that was able to capture the RTs from all HIV-1 isolates tested except one of the two group O isolates. In conclusion, our study revealed that there exist surprisingly large immunological differences between RTs from different HIV-1 subtypes as well as from the same subtype.

Animals↗
PubMedDate not supplied

The role of type-1 and type-2 T-helper immune responses in HIV-1 vaccine protection.

The dichotomy of type-1 and type-2 T-helper (Th) immune responses is thought to be an obstacle to develop Human immunodeficiency virus-type- (HIV-1) vaccines capable of inducing effective cellular as well as humoral immune responses. Macaca mulatta were immunized using two different HIV-1sf2 envelope vaccine strategies, based on either immune-stimulating complexes (ISCOM) or chimeric Fowlpox (FP) vaccines. One month following the third immunization all animals were heterologously challenged with simian/human immunodeficiency virus (SHIVsf13). Vaccinated monkeys, which were protected had the highest levels of both type-1 and type-2 HIV-1 specific T-helper cell (Th) responses in addition to the highest homologous and heterogenous virus neutralizing antibodies. To determine how long Th responses persisted and if they correlated with protection, animals were rechallenged after waiting for four months without re-boosting. Macaques which maintained the highest gp120-specific type-1 (IFN-gamma) responses were protected, while there was evidence of viral clearance in two others. These findings demonstrate the importance of both or mixed type-1 and type-2 Th responses in HIV-1 vaccine induced immunity while suggesting a possible role of persistent type-1 responses in maintaining protective immunity over time.

AIDS Vaccines↗
PubMed1998

Different vaccine strategies used to protect against Theileria annulata.

SPAG-1, a sporozoite surface antigen of T. annulata, has previously been shown to elicit partial protection when used, as an hepatitis B core antigen fusion, to immunize cattle. The objective of this study was to try and improve the protective capacity of this antigen by enlisting different vaccine strategies. Cattle were immunized with SPAG-1, as a fusion protein with a His6 tag, either incorporated into ISCOMs, with or without the merozoite antigens TAMS 1-1 and 1-2, or with RWL as adjuvant three times at monthly intervals. Another group of cattle were immunized with p67, the T. parva sporozoite antigen, in RWL to assess whether any cross-protection could be induced. The animals were then challenged with an estimated LD50 of T. annulata sporozoites, and their ability to resist the infection was investigated. Serum responses and T-cell proliferative responses were analyzed throughout the trial. Post-challenge analyses included lymph node biopsies and blood smears to check for the presence of parasites, routine hematological parameters, and observation for clinical manifestations of the disease. The results of this trial will be discussed.

Animals↗
PubMed2006

Achieving directed immunostimulating complexes incorporation.

In recent years, several studies have been reported with the common aim of generating general expression systems for straightforward production and subsequent coupling of expressed antigens to an adjuvant system. Here, we describe a series of such efforts with a common theme of using gene fusion technology for association of recombinant antigens to immunostimulating complexes (iscoms). In the early stages of vaccine development, uniform antigen preparations are crucial to allow the comparison of immune responses to different antigens, or even subdomains thereof, and we believe that the described systems constitute an important development in this context.

Animals↗
PubMed2002

Vaccination with recombinant alphavirus or immune-stimulating complex antigen against respiratory syncytial virus.

Respiratory syncytial virus (RSV) causes severe respiratory diseases in infants and young children. Inappropriate immunity to the virus can lead to disease enhancement upon subsequent infection. In this study, we have characterized the antiviral immunity elicited by the recombinant Semliki Forest virus (SFV) encoding the RSV fusion (F) and attachment (G) protein, and compared with that induced by the immune-stimulating complex (ISCOM)-incorporated FG proteins. Antiviral immunity against RSV elicited nasally or parentally by either of the immunogen having divergent profiles could reduce lung RSV titers upon challenge. However, resistance to RSV without disease enhancement was only observed in those vaccinated with SFV recombinants via nasal route. Presence of postvaccination pulmonary IFN-gamma response to the H-2K(d)-restricted T cell epitope (F(85-93); KYKNAVTEL) was found to be associated with absence of enhanced pulmonary disease and goblet cell hyperplasia as well as reduced Th2-cytokine expression. This result demonstrates that the SFV recombinants can result in enhanced clearance of RSV without enhancing the RSV-associated disease, and underlines the importance in priming pulmonary MHC class I-restricted T cells when RSV FG-based vaccines are used.

Administration, Intranasal↗