Karin Lövgren Bengtsson

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.

ISCOM technology-based Matrix M™ adjuvant: success in future vaccines relies on formulation

ISCOM technology-based Matrix M (TM) adjuvant : success in future vaccines relies on formulation

Adjuvant activity of iscoms ; effect of ratio and co-incorporation of antigen and adjuvant

We have studied the importance of co-incorporation of antigen and adjuvant in iscoms and the effects of different ratios of adjuvant and antigen in the iscom particles. Immune responses to influenza virus antigens (flu-Ag) in iscoms were compared to those induced by flu-Ag mixed with iscom-matrix, i.e. antigen and adjuvant delivered in separate packages. Higher doses of Quil A were required with iscom-matrix to induce strong immune responses compared to iscoms containing the same amount of antigen. The immunogenic properties of iscoms were affected by the ratio between antigen and adjuvant in the particles. Both iscoms and flu-Ag mixed with iscom-matrix induced antigen-specific antibodies with similar IgG subclass distribution and activated spleen cells producing high levels of IL-2 and IFN-gamma in vitro.

Systemic and intestinal antibody secreting cell responses and protection in gnotobiotic pigs immunized orally with attenuated Wa human rotavirus and Wa 2/6-rotavirus-like-particles associated with immunostimulating complexes

The undesirable side effects and variable efficacy of some oral live rotavirus vaccines in infants have necessitated alternative vaccine approaches. We evaluated a recombinant RFVP2/WaVP6 rotavirus-like-particle (2/6VLP) oral vaccine, using an immunostimulating complex (ISCOM) matrix as adjuvant, in a gnotobiotic (Gn) pig model of human rotavirus (HRV) disease. The 2/6VLPs adhered to the ISCOM-matrix (2/6VLP-ISCOM ) and were antigenic, but they failed to induce protection. However, when combined with attenuated (Att) HRV for oral priming, the 2/6VLP-ISCOM vaccine was effective as a booster and induced partial protection against virulent Wa HRV. The 250 microg 2/6VLP dose was more effective than 100 microg. The highest mean numbers of IgA antibody secreting cells evaluated by ELISPOT in intestinal lymphoid tissues were in pigs receiving AttHRV+2/6VLP-ISCOM or three doses of AttHRV and were associated with the highest protection rates.

Immune enhancing properties of the novel Matrix-M™ adjuvant leads to potentiated immune responses to an influenza vaccine in mice.

The novel saponin based adjuvant Matrix-M™ was recently used in a Phase I study of seasonal influenza in elderly. The present study is a pre-clinical evaluation of the efficacy and mode-of-action of Matrix-M™ formulated influenza vaccine in mice. A manuscript on safety profile and immunogenicity in elderly humans is under preparation. We have previously shown that subcutaneous injections of Matrix-M™, without coformulated antigen, results in a dose-dependent recruitment of leukocytes to draining lymph nodes (dLNs). Herein we compared the mode of action of Matrix-M™ with Alum, FCA and AS03 alone or formulated with influenza split virion antigen injected intramuscularly. The elicited responses in dLNs and spleen were investigated 48h later. Matrix-M™ was particularly efficient in activation of central innate immune cells such as neutrophils, DCs and macrophages compared to the other adjuvants analyzed. Moreover, the adjuvant influence on the recall immune response to influenza antigen was studied by in vitro re-stimulation of splenocytes from mice immunized with influenza antigen adjuvanted with Matrix-M™, Alum or AS03. Splenocytes from mice immunized with influenza antigen and Matrix-M™ produced both Th1 and Th2 cytokines upon re-stimulation. This response was significantly stronger than that induced by the other adjuvants studied. Interestingly, increased levels of the neutrophil chemoattractant KC were produced by antigen stimulated splenocytes from mice immunized with Matrix-M™ adjuvanted vaccine, which is in agreement with the increase of neutrophils into dLNs and spleen after Matrix-M™ injection. Furthermore, influenza antigen adjuvanted with Matrix-M™ induced significantly higher antigen-specific IgG1 and IgG2a responses compared to antigen alone. In conclusion, adjuvant Matrix-M™ activates the innate immune system without antigen present. This activation may explain the enhanced immunity to influenza seen with Matrix-M™ adjuvant. Despite this potent immune activation mediated by Matrix-M™, GLP-toxicity studies and clinical data suggest that Matrix-M™ adjuvant has a mild to moderate safety profile.

Antibody and T-cell responses to a virosomal adjuvanted H9N2 avian influenza vaccine: impact of distinct additional adjuvants.

A highly efficacious vaccine is required to counteract a threat of an avian influenza pandemic. Increasing the potency of vaccines by adjuvation is essential not only to overcome generally low immunogenicity of pandemic strains, but also to allow dose sparing and as such to make it feasible to satisfy huge global production demands. In this study we evaluated the ability of four distinct adjuvants to further increase immune responses to a virosomal adjuvanted avian H9N2 influenza vaccine in mice. Currently registered adjuvants aluminium phosphate, aluminium hydroxide and MF59, as well as a novel promising adjuvant MATRIX-M were included in the study. Our results demonstrate that all adjuvants significantly increased the H9N2 haemagglutinin (HA) inhibition and ELISA antibody titers induced with the virosomal adjuvanted vaccine. The adjuvants exhibited different effect on the isotype of virus specific antibodies, with MATRIX-M inducing the most pronounced skewing to IgG2a, i.e. towards Th1 type of response. While the virosomal adjuvanted pandemic influenza vaccine efficiently induced CD4(+) T-cell response, with no further increase upon adjuvation, the CD8(+) T-cell responses induced with virosomal adjuvanted vaccine could be significantly improved upon additional adjuvation with MATRIX-M or MF59. All adjuvants demonstrated a dose sparing effect, i.e. in combination with the virosomal adjuvanted pandemic influenza vaccine they increased immune responses to comparable level independent of the tested vaccine dose. In conclusion, our results demonstrate that immune responses to a virosomal adjuvanted pandemic influenza vaccine can be further enhanced by add-on adjuvants, with MATRIX-M being overall the most potent adjuvant in combination with virosomes, followed by MF59 and finally aluminium-based adjuvants.

Heterotypic Protection against Influenza by Immunostimulating Complexes Is Associated with the Induction of Cross-Reactive Cytotoxic T Lymphocytes

Influenza immunostimulating complexes (flu-ISCOMs) and a monovalent subvirion vaccine prepared with an H1N1 strain of influenza virus were compared in mice for immunogenicity and protection against challenge with homologous and heterotypic influenza viruses. flu-ISCOMs but not subvirion vaccine fully protected mice against homologous virus challenge after one immunization as assessed by measurement of virus lung titers. The improved protection induced by flu-ISCOMs was associated with a 10-fold higher prechallenge serum hemagglutination inhibition titer. Furthermore, only flu-ISCOMs fully protected mice against mortality and reduced morbidity following challenge with an influenza virus of the serologically distinct H2N2 subtype. This cross-protection correlated with the induction of virus cross-reactive cytotoxic T lymphocytes that recognized a known major histocompatibility class I (H2-Kd)-restricted epitope within the hemagglutinin of influenza virus that is conserved among the H1 and H2 influenza virus subtypes. flu-ISCOMs may offer significant advantages over current commercial formulations as an improved influenza vaccine.

Functional aspects of iscoms

The iscom is a delivery system, designed for both parenteral and mucosal modes of administration, for both antigens and adjuvants, components which are interchangeable. By the parenteral route a prominent systemic Th1 type of response is evoked, but the mucosal immunoglobulin A (IgA) response was insignificant. Intranasal (i.n.) immunization with iscoms evoked potent mucosal IgA response and serum IgG which was much higher than that induced by i.n. administration of the B subunit of cholera toxin (rCTB), both to rCTB itself as well as to co‐administered antigen. The immunomodulatory effect on rCTB or co‐administered antigens imposed by the iscom was demonstrated by a potent mucosal IgA switch and an enhanced IgG2a serum response. The incorporation of a targeting molecule in the iscom enhanced the remote IgA response in the genital tract mucosa. The capacity to induce CD8‐restricted cytotoxic T lymphocytes (CTL) is unique for the iscom as a non‐replicating system, which is facilitated by the delivery of antigens to the cytosol. The immunomodulatory capacity of iscoms also paved the way to override the inhibitory effect of maternally derived antibodies and the relative unresponsiveness of an immature neonatal immune system.

Novel adjuvants and vaccine delivery systems

Abstract Conventionally the efficiency of an adjuvant is measured by the capacity to induce enhanced antibody serum titres and cell mediated immunity (CMI) to a given antigen. Nowadays the capacity of an adjuvant is also measured by the quality as well as the magnitude of the induced immune response, guided by the protective immune response required. Quality includes isotype and IgG subclass responses, T-helper cell responses characterized by the cytokine profile and cytotoxic T cells (CTL). In the early phase of immunization some adjuvants influence the antigen administration and uptake by a so- called depot effect exemplified by aluminium hydroxide gel and oil adjuvants, which possibly is not as desired as alledged. A modern depot is exerted by slow release formulations continuously releasing the antigen over a period of time or by pulses at intervals aiming at ‘single injection’ vaccine. Great efforts are made to formulate efficient delivery formulations targeting the antigens from the site of administration, to draining lymph nodes or distant lymphatic tissue or to mucosal surfaces by parenteral or mucosal administrations. Nowadays, non-replicating carriers besides replicating vaccines are formulated to induce mucosal immune responses encompassing secretory IgA and CMI. Efforts to evoke immune responses on mucosal membranes distant from the site of administration have resulted mostly in little success. For a long time it was considered that CTL under the restriction of MHC Class I only could be evoked by replicating viruses or intracellular parasites. However, novel adjuvant delivery systems readily induce CTL by delivering the antigen to the APC resulting in intracellular transport to the cytosol for the MHC Class I presentation system, as well as to the endosomal pathway for the MHC Class II presentation.

Kinetics, localization and cytokine profile of T cell responses to immune stimulating complexes (iscoms) containing human influenza virus envelope glycoproteins

Immune stimulating complexes (iscoms) are 40 nm particles combining adjuvant-active Quillaja saponins and multimeric presentation of antigens. The distribution in mice of influenza virus iscoms and the resulting T cell responses in the lymph nodes (LN) and spleen were characterized. After a single subcutaneous injection, iscoms were delivered to the draining LN where they induced a transient population of LN cells which responded with proliferation and secretion of interleukin-2 (IL-2), gamma-interferon (IFN-gamma) and interleukin-4 (IL-4) after restimulation. The response in the spleen developed more slowly, sustained for 12 weeks and was characterized by cells producing in particular IL-2 and IFN-gamma but also IL-4. A booster resulted in a dramatic enhancement of the production of IFN-gamma, indicating that iscoms efficiently recruit cells with Th1 properties. Comparisons of T cell responses to iscoms and to influenza virus antigen in Freunds complete adjuvant demonstrate that these adjuvants affect both the localization and cytokine profile of T cell responses.