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Hantaviruses are rodent-borne agents that cause severe human diseases. The coding sequences for the authentic and a His-tagged Puumala hantavirus (PUUV) nucleocapsid (N) protein were expressed in yeast ( Saccharomyces cerevisiae ). N-specific monoclonal antibodies demonstrated native antigenicity of the two proteins. All bank voles vaccinated with the His-tagged N protein in Freund’s adjuvant ( n =12) were defined as completely protected against subsequent virus challenge, based on the absence of viral N protein, RNA and G2-specific antibodies. In the group vaccinated with the yeast-expressed authentic N protein in Freund’s adjuvant, 2/6 animals were defined as completely protected and 4/6 as partially protected. Moreover, when animals were vaccinated with the His-tagged N protein in an adjuvant certified for human use (alum), all ( n =8) were at least partially protected (six completely, two partially). The general advantages of the yeast expression system make the described recombinant proteins promising candidate vaccines against hantavirus infection.

Puumala virus is a member of the hantavirus genus (family Bunyaviridae) and is one of the causative agents of hemorrhagic fever with renal syndrome (HFRS) in Europe. A genetic vaccination approach was conducted to investigate if the immune response could be modulated using different cellular secretion and/or localisation signals, and the immune responses were analysed in BALB/c mice and in a bank vole infectious model. Rodents vaccinated with DNA constructs encoding the antigen fused to an amino-terminal secretion signal raised significantly higher antibody levels when compared to using constructs lacking secretion signals. Furthermore, the ratios of the IgG subclasses (IgG2a/IgG1) were raised by the use of cellular localisation signals, indicating a more pronounced Th1-type of immune response. The majority of the mice, or bank voles, immunised with DNA encoding a secreted form of the antigen showed a positive lymphoproliferative response and were protected against challenge with Puumala virus (strain Kazan-wt).

Prophylactic DNA vaccination protects mice against infection with Leishmania major by inducing an exclusive Th1 immune response dominated by the production of IFN-gamma. Here we show that DNA vaccines, initially designed to prevent infection, can also have a significant therapeutic effect. In L. major infected mice, vaccination with DNA encoding the Parasite Surface Antigen/gp46/M2 causes reduction in lesion size and promotes healing in both genetically resistant C3H/He mice and susceptible BALB/c mice. The therapeutic effect is underpinned by a shift in the T cell-derived cytokine environment with an increase in the IFN-gamma producing Th1 type cells. Application of such immunotherapy in conjunction with antiparasite drugs may result in faster or more certain cure of the disease in humans.

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.

Hantaviruses are rodent-born agents which are pathogenic in humans causing haemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome. To induce a protective immunity against a European hantavirus (Puumala) we constructed chimaeric hepatitis B virus (HBV) core particles carrying defined fragments of the Puumala virus nucleocapsid protein. After immunisation of bank voles, the natural host of Puumala virus, with core particles possessing an insertion of the N-terminal part of Puumala virus nucleocapsid protein, four of five animals were protected against subsequent virus challenge. The results show that the major protective region of the nucleocapsid protein is located between amino acids 1 and 45 and that chimaeric HBV core-like particles are useful carries of foreign protective epitopes.

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 Freund's complete adjuvant demonstrate that these adjuvants affect both the localization and cytokine profile of T cell responses.

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-γ in vitro.

The immunogenicity in guinea-pigs of the human immunodeficiency virus type 1 envelope glycoprotein gp120 in immune stimulating complex (iscom) was compared to that of gp120 adjuvanted with QuilA-matrix (iscom without attached antigen), aluminium hydroxide (alum) and the Ribi adjuvant system. Gp120 was either incorporated into iscoms by covalent conjugation (iscom(c)) or by acid treatment of gp120 (iscom(a) and both these preparations induced high ELISA antibody titres to gp120. Virus neutralizing (VN) antibodies were most frequently induced by gp120 in iscom(c), iscom(a) or in alum and correlated to high titres to the V3-region of gp120. Further, antibodies induced by gp120-iscom(c) most efficiently inhibited binding of a VN monoclonal antibody GP13 to the CD4 binding region of gp120 whereas gp120-iscom(a) induced the highest mean titre of antibodies blocking the binding of [125I]gp120 to CD4. These results suggest that the gp120-iscom preparations efficiently induced high levels of gp120 specific antibodies and that the adjuvant formulation of gp120 affect the specificity and functional properties of elicited antibodies.

Immunostimulating complexes (ISCOMs) are spherical structures where immunogens are presented as multimers in a matrix of the adjuvant Quil A. ISCOMs have been shown to enhance the immunogenicity of several antigens important to both human and veterinary vaccine development. We have coupled a fusion protein, designated ZZ-M2, comprising eight copies of the C-terminal repeat subunit EENV of the Plasmodium falciparum blood-stage antigen Pf155/RESA and two IgG-binding domains of staphylococcal protein A (SpA), to preformed influenza virus envelope protein ISCOMs. Rabbits immunized with the conjugated ISCOMs produced high titres of antibodies even after the first injection. These antibodies reacted with the EENV repeat sequence in ELISA and with Pf155/RESA in immunofluorescence on infected erythrocytes. The antibody response, which was sustained for more than 20 weeks, was efficiently boosted and superior or equal to that obtained after immunization with ZZ-M2 in Freund's complete adjuvant. In contrast, the antibody response induced in rabbits immunized with ZZ-M2 in Syntex Adjuvant Formulation-MF (SAF-MF) was weak and of short duration. The antibodies produced after immunization with ZZ-M2 coupled to influenza virus ISCOMs mainly recognized epitopes formed by two or more EENV subunits and were highly specific for Pf155/RESA. Furthermore, the antibodies efficiently inhibited merozoite reinvasion of erythrocytes in vitro, indicating that they recognized epitopes exposed on the native antigen. In addition, the ZZ-M2-conjugated ISCOMs also induced high titres of antibodies reacting with SpA or the influenza virus envelope protein. Taken together, these results suggest that the approach of coupling fusion proteins to preformed ISCOMs has the potential to be a suitable basis for the construction of anti-malarial subunit vaccines.

The peripheral (draining) lymph node, as the primary site of immune induction, determines the course of systemic responses to an injected antigen. Lymphatic duct cannulation procedures in sheep were used to investigate local immunoreactivity to human influenza virus antigen (Flu ag) admixed with the adjuvant ISCOMATRIX® (IMX). Compared to Flu ag or IMX alone, the co-administration of Flu ag and IMX (Flu ag+IMX) synergistically enhanced a number of immunological responses (lymphocyte and blast migration from the node, antigen-specific antibody levels and IL6 output in efferent lymph, and antigen-induced proliferation in cultured efferent lymph cells). Together, these results demonstrate that IMX is an immune modulator, and that lymphatic duct cannulation procedures may be used to evaluate antigen/adjuvant combinations for vaccine development.

ISCOMs® are typically 40 nm cage-like structures comprising antigen, saponin, cholesterol and phospholipid. ISCOMs® have been shown to induce antibody responses and activate T helper cells and cytolytic T lymphocytes in a number of animal species, including non-human primates. Recent clinical studies have demonstrated that ISCOMs® are also able to induce antibody and cellular immune responses in humans. This review describes the current understanding of the ability of ISCOMs® to induce immune responses and the mechanisms underlying this property. Recent progress in the characterisation and manufacture of ISCOMs® will also be discussed.

Lymphocyte recruitment from blood into the lymph node is thought to be initiated by the presence of antigen. In this study, we have used lymphatic cannulation in sheep to demonstrate that the adjuvant ISCOMATRIX can induce dramatic lymph node activation in the absence of antigen. Consistent patterns of node shutdown (decreased output) and cell recruitment (increased output) with minimal blast cell responses were observed indicating that an antigen-specific immune response is not required. Production of IL-6, IL-8 and IFN-gamma, and the transient presence of red blood cells and neutrophils in the efferent lymph were associated with changes in efferent lymph cell trafficking. These early events may facilitate the screening of low frequency antigen-specific cells for binding to antigen and the subsequent amplification of the immune response.