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Vaccination of harbour seals (Phoca vitulina) against phocid distemper with two different inactivated canine distemper virus (CDV) vaccines.

Two inactivated canine distemper virus (CDV) vaccines--an adjuvanted whole inactivated virus and a subunit ISCOM preparation--were tested for their ability to induce protective immunity in harbour seals (Phoca vitulina) against phocid distemper, a disease that recently killed greater than 17,000 harbour seals in the North and Baltic seas, and was shown to be caused by infection with a newly discovered morbillivirus, which is antigenically closely related to CDV. Four CDV seronegative harbour seals were vaccinated three times with the whole-virus vaccine, two with the ISCOM subunit vaccine and two were sham-vaccinated with an antigen-free preparation. Ten days after the last vaccination, when all six vaccinated animals had developed CDV neutralizing antibody titres ranging from 300 to 3000, all eight animals were challenged by the oculonasal and the peritoneal routes, with an organ suspension from dead seals. None of the six vaccinated animals developed clinical signs. The two sham-vaccinated seals died on days 14 and 18, respectively, after having shown a body temperature rise, respiratory symptoms and weight loss. In organs from both dead animals morbillivirus antigen was demonstrated with an enzyme-linked immunosorbent assay and an immunofluorescence assay. One of these two animals had developed a low titre of CDV-specific antibodies just before death. These data clearly indicate that seals can be protected from fatal challenge with the phocid distemper virus (PDV), by vaccination with certain inactivated CDV vaccines. They also reconfirm that infection with PDV should be considered the primary cause of the recent epizootic in seals.

Animals↗

Vaccination with immunodominant peptides encapsulated in Quil A-containing liposomes induces peptide-specific primary CD8+ cytotoxic T cells.

Immunostimulating complexes (ISCOMs), containing lipids, the saponin Quil A, and proteinaceous antigens, have been proven to vaccinate effectively CD8+ cytolytic T cells in vivo. However, conventional ISCOM technology is restricted to hydrophobic proteins or fatty acid-derivatized proteins or peptides. We therefore analysed whether Quil A-containing liposomes are an effective vehicle to shuttle hydrophilic proteins or peptides into the MHC class I pathway of antigen presentation resulting in the in vivo induction of antigen-specific cytolytic T cells (CTL). Liposomes were formed by a lipid dry-down method followed by resuspension with an aqueous solution containing protein/peptide and Quil A and then an extrusion step. Quil A-containing liposomes are an effective means to elicit a CD8+ CTL response to peptide antigen in vivo. CTL could be raised in C57B1/6 mice against ovalbumin (OVA) peptide 257-264 and vesicular stomatitis virus nucleoprotein 52-59, as well as in Balb/c mice against listeriolysin peptide 91-99 and cytomegalovirus pp89 168-176, demonstrating the versatility of this approach. The elicited response was peptide-specific, peptide dose-dependent and Quil A was necessary. Vaccination with liposomes entrapping the whole ovalbumin molecule or an extended (OVA) peptide 254-276 also yielded a CTL responsive to the immunodominant OVA peptide 256-264, implying cellular internalization and correct processing. Thus Quil A-containing liposomes appear to be a versatile vehicle to vaccinate CD8+ T cells in vivo; in addition, they could rapidly enhance the understanding of subunit vaccines and rules of antigen processing and peptide-MHC class I binding.

Adjuvants, Immunologic↗

Dendritic cells and immune responses to orally administered antigens.

The intestinal immune system discriminates between invasive pathogens and antigens that are harmless, such as food proteins and commensal bacteria. The latter groups of antigens normally induce tolerance and the nature of the intestinal immune response depends on how antigen is presented to CD4+ T cells by dendritic cells (DC). Expanding the numbers of DC in vivo using the cytokine flt3 ligand (flt3L) enhances the susceptibility of mice to the induction of oral tolerance. The relevant DC may be in Peyer's patches (PP), mesenteric lymph node (MLN) or the lamina propria (LP)of the villus mucosa. All these tissues contain a number of distinctive DC subsets, including some that can preferentially induce the differentiation of regulatory T cells. However, we find that the largest proportion of orally administered protein is taken up by DC in the lamina propria. Intestinal DC are not inherently tolerogenic. Under physiological conditions they are "quiescent", capable of presenting antigen and inducing tolerance, but being sufficiently responsive to inflammatory stimuli to allow T cell priming and protective immunity when necessary. Targeting local DC may be a useful means of inducing either tolerance or active immunity. We have achieved this using immune stimulating complexes (ISCOMS) containing Quil A, which are highly immunogenic by the oral route and appear to act by targeting DC preferentially. ISCOMS or saponins such as Quil A may prove useful adjuvants to incorporate in plant derived, mucosally active vaccines.

Administration, Oral↗

Immune responses to bovine norovirus-like particles with various adjuvants and analysis of protection in gnotobiotic calves.

We evaluated serum and fecal antibody responses to bovine norovirus (BoNoV) virus-like particles (VLP) and protection against virulent BoNoV in gnotobiotic calves. Calves were vaccinated with two or three doses of VLPs (250 microg/dose) coadministered with oil, mutant E. coli heat-labile toxin (R192G) (mLT) or immunostimulating complexes (ISCOM). Fecal IgA antibody was detected only in calves vaccinated intranasally with VLP+mLT and after challenge, partial protection with delayed, shortened diarrhea (1-2 days) was observed only in these calves. Diarrhea presented from 2 to 6 days in calves vaccinated with VLP with oil or ISCOM and 8 to 9 days in controls. Virus shedding was detected post-challenge by ELISA in all vaccinated calves. All calves recovered from BoNoV infection had high titers of fecal IgA antibodies and were completed protection against challenge. We conclude that only BoNoV VLP+mLT given intranasally stimulated both serum and fecal IgA antibodies and partial protection.

Adjuvants, Immunologic↗

Seroprevalences of Toxoplasma gondii and Neospora sp. infections in Swedish horses.

Sera from 414 Swedish horses were investigated for the presence of antibodies to Toxoplasma gondii and Neospora sp. by the T. gondii direct agglutination test (DAT), and an Neospora caninum iscom-ELISA. Five sera (1%) had a titre >1:40 in DAT, but when analysed by immunoblotting against T. gondii antigens only two of them were positive, giving a seroprevalence of 0.5%. Since the Neospora iscom ELISA had not been validated for equine sera it was used for an initial screening, and all sera with an optical density exceeding 0.200 absorbance units were selected for further investigation by immunoblot analysis. Of the 39 sera tested by immunoblotting, four reacted with at least two of the immunodominant Neospora antigens recognized by the positive control sera and were judged as positive, resulting in a seroprevalence of 1%. This is the first evidence of Neospora infection in Swedish horses. The study illustrates the necessity of critically evaluating results of serological analyses performed by methods that are not validated for the animal species under investigation.

Agglutination Tests↗

Longevity of neutralizing antibody levels in macaques vaccinated with Quil A-adjuvanted measles vaccine candidates.

Quil A-based candidate measles vaccines have been shown to be immunogenic and protective in cotton rats and macaques. Here we studied the longevity of protective VN antibody levels induced in macaques with one dose of measles virus (MV) iscom. Inactivated MV adjuvanted with iscom-matrix or with purified Quillaja saponin QA-22 were also tested. All animals developed high levels of VN antibody and MV-specific IFNgamma-producing cells. Especially the high VN antibody levels induced by the latter two preparations showed virtually no decrease during the 2-year follow-up. These highly promising candidate MV vaccines should now be tested in infant macaques in the presence or absence of passively transferred and/or maternally derived VN antibodies. In addition, the immunopathological safety of the constructs should be evaluated in the atypical measles model in rhesus macaques.

Adjuvants, Immunologic↗

A recombinant prime, peptide boost vaccination strategy can focus the immune response on to more than one epitope even though these may not be immunodominant in the complex immunogen.

Rhesus monkeys were successfully vaccinated using a strategy of priming with a candidate envelope subunit vaccine and boosting with synthetic peptides. Priming was carried out with recombinant HIV-1 SF2 envelope glycoprotein incorporated into ISCOMs, following the attachment of a lipid tail. Peptides, covalently linked to ISCOMs, representing linear sequences with the V2 and V3 regions, were used to boost functional antibodies-to neutralizing epitopes in both of these regions. Injections with these peptide formulations substantially increased the titre of serum neutralizing antibodies from low or undetectable levels. In addition to completely neutralizing the homologous HIV-1 SF2 strain, these sera also neutralized the escape variant, HIV-1 SF13. However, no antibodies were boosted which could compete with human, neutralizing monoclonal antibodies recognising conformational epitopes. The peptides also boosted antibodies to a peptide whose sequence lies close to the V2 region neutralizing epitope but does not overlap with it. Importantly, the level of antibodies to an unrelated epitope associated with enhancement of HIV-1 SF13 continued to fall after the peptide boost. Successful protection against challenge with chimeric simian immunodeficiency virus expressing HIV-1 SF13 envelope glycoproteins (SHIV SF13) may be due to an increase in the ratio of neutralizing to enhancing antibodies by selectively boosting with peptides to critical neutralizing epitopes.

AIDS Vaccines↗

Vaccine strategies to overcome maternal antibody mediated inhibition of measles vaccine.

A vaccine that is effective in the presence of maternally derived virus neutralizing antibodies and can be administered successfully at an early age, would be favoured over the presently used live attenuated measles vaccines. With the advent of new molecular and immunological techniques, several options for the development of new generation vaccines, fulfilling these criteria, have arisen. We have recently evaluated the efficacy of recombinant vaccinia virus- and iscom-based candidate vaccines, presenting the F and H proteins of measles virus, in macaques with passively transferred virus neutralizing macaque antibodies. The data indicate that the further exploration of the potential of iscom based measles vaccines should be encouraged.

Animals↗

An indirect enzyme-linked immunoassay (ELISA) for demonstration of antibodies to Neospora caninum in serum and milk of cattle.

An indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Neospora caninum in serum from cattle is described. Extracted tachyzoite proteins incorporated into immunostimulating complexes (iscoms) were used as coating antigen and a mouse monoclonal antibody to bovine immunoglobulin G1 as conjugate. Western blot analysis of the iscom preparation revealed a restricted number of antigens compared with whole parasite homogenates. When probed with a serum from an experimentally infected calf, heavily stained antigens with apparent molecular masses of 28, 35, 45 and 78 kDa were seen. The sensitivity and specificity of the ELISA was 100% and 96%, respectively, against an indirect fluorescent antibody test as indicator of true status. The applicability of the ELISA for demonstration of antibodies in milk was evaluated and the agreement between serum and milk ELISA was 95%.

Animals↗

Induction of cell death by saponin and antigen delivery.

PURPOSE: Saponin is the major component in the formation of immune stimulating complex (ISCOM), a potent adjuvant able to induce both humoral and cellular immune reactions. The immunogenicity induced by saponin, however, has been unclear. The objective of this study was to investigate the apoptotic and necrotic effects induced by saponin in ELA mouse lymphoma cells, expected to be a possible mechanism of the cytotoxic T-lymphocyte (CTL) effect elicited by the ISCOM. METHODS: EL4 cells were treated with saponin, and viability of the cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase release assays. Fluorescence microscopy was used to detect the morphological changes by staining saponin-treated cells with Hoechst 33342. Extent of apoptosis and necrosis was determined by Annexin V-FITC/propidium iodide staining, followed by flow cytometric analysis. Dendritic cells were cultured with either saponin-protein complexes or saponin-treated cells and analyzed by flow cytometry. RESULTS: Treatment of EL4 cells with saponin resulted in concentration-dependent cytotoxicity and the appearance of the hypodiploid DNA peak. Cells treated with saponin showed highly condensed chromatin when stained with fluorescent DNA-binding dye Hoechst 33342. Analysis of EL4 cells by flow cytometry after Annexin V/propidium iodide staining demonstrated that saponin induced both apoptosis and necrosis. Pretreatment of EL4 cells with zVAD-fmk, a broad-range caspase inhibitor, did not prevent cell death induced by saponin, indicating the non-caspase-dependent cell death. Dendritic cells were shown to phagocytose both the antigen-saponin complexes and the saponin-induced dead cells. CONCLUSIONS: Results obtained in this study demonstrated that saponin induced both apoptosis and necrosis in ELA cells. These events are critical for antigen processing and presentation.

Adjuvants, Immunologic↗

Intranasal immunization of mice with herpes simplex virus type 2 recombinant gD2: the effect of adjuvants on mucosal and serum antibody responses.

Mucosal immunization offers the potential for inducing IgA antibody responses in the vagina, the site of infection for many viruses, including herpes simplex type 2 (HSV-2). To investigate this possibility, mice were immunized intranasally with 10 micrograms glycoprotein D2 (gD2) from HSV combined with a series of adjuvants of proven efficacy; the oil in water emulsion MF59, poly(D,L-lactide-co-glycolide) microparticles (PLG) (encapsulated or co-administered), immune-stimulating complexes (iscoms) (incorporated or co-administered with iscomatrix) and the genetically detoxified enterotoxin from Escherichia coli, LT-K63. Encapsulation of gD2 into PLG microparticles, incorporation of gD2 into iscoms and co-administration of gD2 with LT-K63 induced mucosal IgA antibody responses (nasal wash, saliva and vaginal wash) which were greater than those induced by intramuscular administration of gD2 with MF59. Intranasal immunization with these formulations also induced substantial levels of serum IgG and neutralizing antibodies. These studies demonstrated that intranasal immunization with potent adjuvants is an effective means to induce mucosal antibody responses, even in the lower genital tract.

Adjuvants, Immunologic↗

Quillaja saponin formulations that stimulate proinflammatory cytokines elicit a potent acquired cell-mediated immunity.

We examined the ability of various Quillaja saponins in iscom-matrix formulations to induce proinflammatory cytokines, such as interleukin (IL)-1alpha and IL-6, and to stimulate acquired immune responses to influenza virus envelope proteins. The A-fraction of Quillaja saponins (QH-A) was shown to stimulate antigen-presenting cells (APC) to produce proinflammatory cytokines, and elicited a high primary antigen-specific antibody response and potent cell-mediated responses, as measured by T-cell proliferation, production of cytokines and cytotoxic T-lymphocyte (CTL) activity. The C-fraction of Quillaja saponins (QH-C) was shown to have a low capacity to stimulate proinflammatory cytokines and elicited low primary antibody and T-cell responses. However, the QH-C iscom-matrix mediated a potent booster effect, resulting in a high secondary antibody response. The ability of APC to discriminate and to respond to QH-A formulations more efficiently than to QH-C with release of proinflammatory cytokines, which precedes a potent acquired immune response, identifies an important mechanism through which some adjuvants may exert their immunoenhancing activities.

Animals↗

Field studies on equine influenza vaccination regimes in thoroughbred foals and yearlings.

SUMMARY: The purpose of these studies was to examine the response of Thoroughbred foals and yearlings to different influenza vaccines and vaccination regimes. The horses' antibody levels against haemagglutinin, an established correlate of protection were measured by haemagglutination inhibition. The first study investigated the extent to which maternal antibodies interfered with the humoral response to a subunit vaccine. The findings suggest that repeat vaccination in the face of maternal antibodies may induce tolerance as defined by serological testing. The second study compared the immune response elicited by a subunit immune stimulating complex (ISCOM) vaccine, an inactivated whole virus vaccine and the same product containing equine herpesviruses and equine reoviruses in addition to equine influenza virus. The monovalent vaccine induced a significantly better response than the ISCOM or the multivalent vaccine. The final study demonstrated that the inclusion of an additional booster vaccination, between the second and third vaccination recommended by the vaccine manufacturers and required under the rules of racing in certain countries, is of benefit to young horses. Since these studies were performed, several of the vaccines have been updated with more recent virus strains in line with WHO/OIE recommendations. However, the general principles investigated in the studies remain relevant to these vaccines.

Animals↗

Vaccine protection against HIV-2 infection in cynomolgus monkeys.

The aim of this study was to determine if protection against an infectious human immunodeficiency virus type 2 (HIV-2) challenge could be obtained in cynomolgus macaques by active immunization using whole killed virus vaccine. Four monkeys were immunized with killed HIV-2SBL-6669, two of them with five intramuscular (im) injections of viral preparation containing 100 or 300 micrograms protein emulsified in incomplete Freund's adjuvant (IFA) and the two remaining received four im injections of 25-50 micrograms viral protein in iscoms. Each of the four vaccinated cynomolgus monkeys, along with four unvaccinated controls, were challenged intravenously two weeks after the last booster with approximately 100 animal infectious doses (ID50) of live HIV-2SBL-6669. All four immunized monkeys developed antibodies to HIV-2 envelope and core proteins before challenge exposure to HIV-2, but only the two animals vaccinated with virus in IFA developed detectable neutralizing antibodies. The two monkeys immunized with killed virus in IFA have shown no evidence of infection nine months after challenge with live virus. When blood and lymph node cells from these animals were transfused into naive cynomolgus monkeys, the recipients remained free of infection. In contrast, virus was recovered repeatedly in all nonimmunized animals and in the two animals immunized with iscom-associated viral antigens, which had a low content of envelope gp125 antigen. The demonstration of vaccine-induced protection against HIV-2 in a nonhuman primate raises hope for effective immunization against HIV infections in humans as well.

Animals↗

Prevention of Epstein-Barr (EB) virus-induced lymphoma in cottontop tamarins by vaccination with the EB virus envelope glycoprotein gp340 incorporated into immune-stimulating complexes.

Experimental induction of malignant lymphomas can be achieved in the cottontop tamarin by inoculation with Epstein-Barr (EB) virus. This system provides an animal model for assessing the efficacy of vaccine protection against the virus which is intended to reduce the incidence of human tumours associated with EB virus infection, namely endemic Burkitt's lymphoma and undifferentiated nasopharyngeal carcinoma. Cottontop tamarins have been vaccinated with the major envelope glycoprotein of EB virus, gp340, incorporated into immune-stimulating complexes (iscoms) and were thereby protected against a 100% lymphomagenic dose of virus. The gp340 iscoms are highly immunogenic, requiring only a few micrograms of immunogen to induce protective immunity and thus would be a strong candidate for further development as an EB virus vaccine for use in man.

Adjuvants, Immunologic↗

Protective immune response to 16 kDa immunoreactive recombinant protein encoding the C-terminal VP1 portion of Foot and Mouth Disease Virus type Asia 1.

Recombinant protein of Foot and Mouth Disease Virus (FMDV) type Asia 1 corresponding to the C-terminal half of VP1 was expressed in Escherichia coli. As an alternative to the synthetic peptide, this selected C-terminal region was used as a protein vaccine in guinea pigs in order to study the immune response with various adjuvant formulations: immune stimulatory complexes (ISCOMs), Montanide ISA 206, Freund's incomplete adjuvant (FIA), lipopolysaccharide (LPS) and cytokine mixture. A primary dose of 40 microg/animal followed by a booster of the same dose was injected after a 21-day interval. The sera were collected at intervals of 21, 42 and 63 days after the booster. The humoral response to vaccine was monitored by sandwich enzyme-linked immunosorbent assay (ELISA) and a serum neutralization test (SNT). The guinea pig sera showed high titers both in ELISA and SNT, which could be protective. Further, irrespective of the adjuvant preparation used, the vaccine conferred protection against the challenge virus 105 days post-vaccination in 13 of 15 animals (86%). The results indicated that a combination of recombinant protein ISCOMs and Montanide ISA 206 would be a better choice for achieving early protective titers and longer lasting immunity and that the C-terminal half of the VP1 protein may be tried as a safe vaccine for secondary immunization.

Animals↗

The serum antibody response distributed in subclasses and isotypes after intranasal and subcutaneous immunization with influenza virus immunostimulating complexes.

Immunostimulating complexes (iscoms) were prepared from influenza virus glycoproteins (A/PR8(H1N1]. Mice were inoculated with iscoms by local (oral or intranasal) as well as by subcutaneous administration. It was shown that one subcutaneous or intranasal inoculation induced serum antibody responses of similar magnitude. The intranasal administration, however, induced somewhat higher IgA and IgM titres than that induced subcutaneously. After a second intranasal or subcutaneous inoculation a prominent increase of the IgG isotypes was found. The secondary IgA and IgM responses were of similar magnitude as those induced after the primary inoculation. The antibody responses induced were evenly distributed within the IgG isotypes, although the highest titres recorded were of the IgG1 and IgG2 isotypes.

Adjuvants, Immunologic↗

Efforts to broaden HIV-1-specific immunity by boosting with heterologous peptides or envelope protein and the influence of prior exposure to virus.

In two previous studies, we have demonstrated the successful protection of human immunodeficiency virus type 1 (HIV-1)-vaccinated rhesus macaques from challenge with SHIV(SF13) with envelop immunogens derived from the closely related HIV-1(SF2) strain. Here we report on two follow-up studies in which we aimed to broaden immunity in order to elicit protection from a more diverse heterologous challenge with SHIV(SF33). In the first study, animals were boosted once with HIV-1(SF33) V2 and V3 peptides that were cross-linked to influenza immune-stimulating complexes (ISCOMs). In the second study, monkeys were boosted twice at 12-week intervals, using a heterologous recombinant gp120 derived from HIV-1(SF33) that was either incorporated into ISCOMs or mixed with the MF59 adjuvant. In both studies, the animals were challenged with 50 monkey infectious doses of SHIV(SF33) 4 weeks after the final boost. All controls became readily infected with the heterologous challenge virus SHIV(SF33). Neither boosting with heterologous SF33 peptides or gp120 afforded protection from infection to SF2-vaccinated animals that had previously resisted SHIV(SF13) challenge. These results demonstrate the importance of developing vaccine strategies that are capable of generating broad immune responses early in the immunization protocol. Furthermore, these findings may illustrate the potential pitfalls of early antigenic sin.

AIDS Vaccines↗