Scientific papers

Safety and immunogenicity, after nasal application of HIV-1 DNA gagp37 plasmid vaccine in young mice.

Jorma Hinkula, Marie Hagbom, Britta Wahren, Ulf Schröder.
Vaccine 26 (2008) 5101-5106

Background: There is a need for safe and potent adjuvants capable of delivering vaccine candidates over the mucosal barrier, with good capacity to stimulate both mucosal and systemic cell-mediated and
humoral immunity. An adjuvant aimed for intranasal delivery should preferably deliver the antigen and minimize the transfer into the close proximity of the central nervous system, thus avoiding damage on the olfactory tissues. Advantages with a mucosal delivery route would be to provide mucosal and systemic immunity, requiring lower vaccine doses then when given parentally. The aim of this study was to study if the N3 adjuvant intranasally administered with HIV-DNA plasmids would be transferred into the olfactory tissues and cause local inflammation and tissue damage.
Results: The N3 adjuvant alone or when combined with HIV-1 DNA gag plasmid and delivered intranasally did not cause detectable damage to the nasal epithelium or the olfactory epithelium or bulb over a period of 3 days after delivery. The intranasal administration of HIV-1 gagp37 DNA induced both a humoral and a cell-mediated immunity against the gag antigen. Significantly higher HIV-1-specific humoral, but not cell-mediated immune responses were seen in DNA/N3-immunized mice in comparison with HIV-1 DNA/saline-immunized animals.
Conclusions: A safe and convenient intranasal mode of HIV-1 DNA plasmid and adjuvant delivery was shown not to interfere with the tissues in close proximity to the central nervous system. The N3 adjuvant combined with HIV-1 plasmids enhances the HIV-1-specific immunogenicity and merits to be clinically tested.

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Andreas Bråve, David Hallengärd, Ulf Schöder, Pontus Blomberg, Britta Wahren and Jorma Hinkula.
Vaccine 26 (2008) 5075-5078

Abstract. One of the major challenges for the development of an HIV vaccine is to induce potent virus-specific immune responses at the mucosal surfaces where transmission of virus occurs. Intranasal delivery of classical vaccines has been shown to induce good mucosal antibody responses, but so far for genetic vaccines the success has been limited. This study shows that young individuals are sensitive to nasal immunization with a genetic vaccine delivered in a formulation of a lipid adjuvant, the Eurocine N3. Intranasal delivery of a multiclade/multigene HIV-1 genetic vaccine gave rise to vaginal and rectal IgA responses as well as systemic humoral and cellular responses. As electroporation might become the preferred means of delivering genetic vaccines for systemic HIV immunity, nasal delivery by droplet formulation in a lipid adjuvant might become a means of priming or boosting the mucosal immunity.

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DNA–VLP prime–boost intra-nasal immunization induces cellular and humoral anti-HIV-1 systemic and mucosal immunity with cross-clade neutralizing activity.

L. Buonaguro, C. Devito, M.L. Tornesello, U. Schröder, B. Wahren, J. Hinkula and F.M. Buonaguro
Vaccine 25 (2007) 5968-5977

Abstract. The immune response to HIV-1 virus-like particles (VLPs), presenting a clade A Ugandan gp120, has been evaluated in a mouse model by intra-nasal (i.n.) administration by a VLP + VLP homologous or a DNA+ VLP heterologous prime–boost immunization protocol, including a HIV-1 DNA gp160/rev plasmid. Furthermore, the effect of the Eurocine lipid-based mucosal L3 adjuvant on the VLP immunogenicity has been assessed as well.
The designed heterologous protocol is able to increase the env-specific humoral and cellular immune response, compared to the homologous protocol, which is to some extent increased by the administration of L3-adjuvanted VLP boosting dose. The anti-gag response is statistically increased in both homologous and heterologous protocols, particularly when the VLP boosting dose is adjuvanted. Immune sera from immunized animals exhibit >50% ex vivo neutralizing activity against heterologous A and B-clade viral isolates. An envelope B-cell epitope mapping shows an enhanced response against V3 epitopes all across the C2–V5 region in the heterologous prime–boost immunization strategy.
The induction of humoral immunity at mucosal sites, which represents the main port of entry for the HIV-1 infection, is extremely relevant. In this framework, theDNA–VLP heterologous prime–boost protocol appears a promising preventive vaccine approach which can significantly benefit from specific mucosal adjuvants, as the Eurocine L3.

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A novel DNA adjuvant, N3, enhances mucosal and systemic immune responses induced by HIV-1 DNA and peptide immunizations.

Jorma Hinkula, Claudia Devito, Bartek Zuber, Reinhold Benthin, Denise Ferreira, Britta Wahren, Ulf Schröder.
VACCINE 24 (2006) 4494-4497

AIMS: The study was designed to evaluate a novel cationic lipid DNA adjuvant (N3) and its function for HIV-1gp160/rev DNA plasmid delivered intranasally. The primary N3/HIV-DNA plasmid immunizations were boosted intranasally with a gp41 peptide in a anionic L3 adjuvant. This novel prime-boost strategy of mucosal immunization provided a broad HIV-1 envelope specific immunity, and recognition of viruses of subtypes A, B and C. CONCLUSIONS: Intranasal N3-adjuvanted gp160/rev DNA prime followed by one L3-peptide boosting immunization, induced broadly neutralizing antibodies against HIV-1 in the mucosa and systemically. The needle-free intranasal prime-boost strategy using two different adjuvant formulations reduced significantly the dose of DNA needed.

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Nasal boost with adjuvanted heat killed BCG or arabinomannan-protein conjugate improves primary BCG-induced protection in C57BL/6 mice

M. Haile, B. Hamasur, T. Jaxmar, D. Gavier-Widen, M.A. Chambers, B. Sanchez, U. Schröder, Källenius, S.B. Svenson, A. Pawlowski.
Tuberculosis (2005) 85, 107-114

Abstract: Today it is generally accepted that the Bacillus Calmette-Guerin (BCG) vaccine protects against childhood tuberculosis (TB) but this immunity wanes with age, resulting in insufficient protection against adult pulmonary TB. Hence, one possible strategy to improve the protective efficacy of the BCG vaccine would be to boost in adulthood. In this study, using the mouse model, we evaluated the ability of two new TB vaccine candidates, heat-killed BCG (H-kBCG) and arabinomannan-tetanus toxoid conjugate (AM-TT), given intransally in a novel Eurocine adjuvant, to boost a primary BCG-induced immune response and to improve protection. Young C57BL/6 mice were vaccinated with conventional BCG and, 6 months later, boosted intranasally with adjuvanted H-kBCG or AM-TT, or subcutaneously with BCG. Ten weeks after the booster, mice were challenged intravenously with M. tuberculosis (Mtb) strain H37Rv. In spleens, there was a significant reduction of cfu counts in mice boosted with either H-kBCG or AM-TT vaccines compared to the non-boosted BCG-vaccinated mice. None of the boosting regimens significantly reduced bacterial loads in lungs, compared to non-boosted BCG vaccination. However, the extent of granulomatous inflammation was significantly reduced in the lungs of mice that received two of the booster vaccines (AM-TT and conventional BCG), as compared with sham-vaccinated mice. All boosted groups, except for mice boosted with the AM-TT vaccine, responded with a proliferation of spleen T cells and gamma interferon production comparable to that induced by a single BCG vaccination.

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Intranasal HIV-1-gp160-DNA/gp41 peptide prime-boost immunization regimen of mice results in longterm HIV-1 neutralizing humoral, mucosal and systemic immunity

C. Devito, B. Zuber, U. Schröder, R. Benthin, K. Okuda, K. Broliden, B. Wahren and J. Hinkula.
J.Immunology (2004) 173: 7078-7089

Abstract: An intranasal DNA vaccine prime followed by a gp41 peptide booster immunization was compared with gp41 peptide and control immunizations. Serum HIV-1-specific IgG and IgA as well as IgA in feces and vaginal and lung secretions were detected after immunizations. Long-term humoral immunity was studied for up to 12 mo after the booster immunization by testing the presence of HIV-1 gp41- and CCR5-specific Abs and IgG/IgA-secreting B lymphocytes in spleen and regional lymph nodes in immunized mice. A long-term IgA-specific response in the intestines, vagina, and lungs was obtained in addition to a systemic immune response. Mice immunized only with gp41 peptides and L3 adjuvant developed a long-term gp41-specific serum IgG response systemically, although over a shorter period (1-9 mo), and long-term mucosal gp41-specific IgA immunity. HIV-1-neutralizing serum Abs were induced that were still present 12 mo after booster immunization. HIV-1 SF2-neutralizing fecal and lung IgA was detectable only in the DNA-primed mouse groups. Intranasal DNA prime followed by one peptide/L3 adjuvant booster immunization, but not a peptide prime followed by a DNA booster, was able to induce B cell memory and HIV-1-neutralizing Abs for at least half of a mouse's life span.

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Immunization with heat-killed Mycobacterium bovis bacille Calmette–Guerin (BCG) in EurocineTM L3 adjuvant protects against tuberculosis

M. Haile, U. Schröder, B. Hamasur, A. Pawlowski, T. Jaxmar, G. Källenius, S.B. Svenson.
VACCINE 22 (2004) 1498-1508

Abstract: The current live attenuated vaccine against tuberculosis, BCG, poses a risk of disseminated infections in immunocompromised subjects. Therefore, in this study we compared the protective effect of a heat-killed bacille Calmette-Guerin (H-kBCG) vaccine given in a new adjuvant (Eurocine L3) with the protection provided by the conventional live attenuated BCG vaccine in mice (C57BL/6 and BALB/c) challenged with virulent Mycobacterium tuberculosis (strain Harlingen). The H-kBCG vaccine alone, in accordance with earlier studies, did not give any or only gave slight protection compared to sham-vaccinated controls. However, the same vaccine given with Eurocine L3 adjuvant, either formulated as a suspension or as an emulsion, afforded significant levels of protection. This protection was at least as good as that of the control live attenuated BCG vaccine. The Eurocine L3 adjuvant is approved for human use as a nasal vaccine adjuvant and a successful phase I trial with nasal immunization with diphtheria vaccine has recently been performed in Sweden. Here we show that, in mice, intranasal priming with H-kBCG in Eurocine L3 adjuvant followed by intranasal booster resulted in the same level of protection as subcutaneous priming followed by intranasal booster. All H-kBCG formulations in the Eurocine L3 adjuvant elicited mycobacterial antigen-specific serum IgG and IFN gamma responses. In general, among the different vaccine formulation(s) in the Eurocine L3 adjuvant those that produced a relatively high Th2 response, as measured by IgG1/IgG2a ratio and IFN gamma production in vitro, were the most protective. In conclusion, H-kBCG in Eurocine L3 adjuvant could represent a safe and a more stable alternative to the conventional live BCG vaccine.

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Mycobacterium tuberculosis arabinomannan–protein conjugates protect against tuberculosis

Beston Hamasur, Melles Haile, Andrzej Pawlowski, Ulf Schröder, Ann Williams, Graham Hatch, Graham Hall, Philip Marsh, Gunilla Källenius, Stefan B. Svenson.
VACCINE 21 (2003) 4081–4093

Abstract: Lipoarabinomannan (LAM) is a major structural surface component of mycobacteria. Arabinomannan (AM) oligosaccharides derived from LAM of Mycobacterium tuberculosis H37Rv were isolated and covalently conjugated to tetanus toxoid (TT) or to short-term culture filtrate proteins (antigen 85B (Ag85B) or a 75kDa protein) from M. tuberculosis strain Harlingen. The different AM oligosaccharide (AMOs)-protein conjugate vaccine candidates proved to be highly immunogenic, inducing boosterable IgG responses against the AMOs portion of the conjugates in rabbits and guinea-pigs. Proliferation of T-cells from C57BL/6 mice immunized with the conjugates was seen upon in vitro stimulation with PPD. In C57BL/6 mice subcutaneous immunization with the AMOs-antigen 85B conjugate in alum provided significant protection compared to sham (alum only) immunized mice (P < 0.021) as estimated by long term survival against intravenous challenge with 10(5) M. tuberculosis H37Rv. Subcutaneous immunization followed by nasal boost with an AMOs-TT conjugate in Eurocine L3 adjuvant provided high (P < 0.025) protection as determined by long term survival after intranasal challenge with 10(5) virulent M. tuberculosis strain Harlingen. This level of protection was comparable to that obtained with the conventional live attenuated BCG vaccine. In guinea-pigs, immunization with AMOs-Ag85B in Eurocine L3 adjuvant followed by aerogenic challenge with M. tuberculosis H37Rv resulted in increased survival and reduced pathology in lungs and spleens relative to non-immunized animals.

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Immunogenicity and protective efficacy of a formalin-inactivated rotavirus vaccine combined with lipid adjuvants

Kari Johansen, Ulf Schröder and Lennart Svensson.
VACCINE 21 (2003) 368-375

Abstract: The immunogenicity and protective efficacy of inactivated rotavirus vaccine administered intramuscularly with lipid adjuvants; MPL (monophosphoryl lipid A from Salmonella minnesota) or L3 (monooleate/lauric acid) was evaluated in an infant mouse model. Purified and formalin-inactivated rhesus rotavirus (I-RRV) combined with one of the adjuvants were administered to female balb/c mice at 0, 4 and 8 weeks. High serum IgG antibody titers developed in all vaccinated groups; I-RRV (GMT 45524+/-9819), I-RRV-MPL (GMT 190637+/-64250) and I-RRV-L3 (GMT 126266+/-27553). The formalin-inactivation procedure preserved neutralizing epitopes and elicited high neutralizing antibody titers; I-RRV (GMT 43053 S.E.M.+/-4189), I-RRV-MPL (GMT 66398 S.E.M.+/-20202) and I-RRV-L3 (GMT 60887 S.E.M.+/-10750). All offsprings to immunized dams were protected against clinical diarrhea upon oral challenge with RRV. The IgG1/IgG2a ratio was in all immunized groups approximately 1 suggesting development of a balanced Th1/Th2 response.

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Nasal and parenteral immunizations with diphtheria toxoid using monoglyceride/fatty acid lipid suspensions as adjuvants

Ulf Schröder & Stefan B. Svenson.
VACCINE 17 (1999) 2096-2103.

Abstract: A novel suspension system was developed where monoglycerides were formulated together with fatty acids and subsequently admixed with antigens. In the present study, diphtheria toxoid was used as a model antigen primarily due to its weak immunological properties as well as to its importance as a future human vaccine for mucosal, particularly nasal immunization. The formulations were administered parenterally and/or nasally to mice whereafter the immune response was determined. In the present study, we have shown that mono-olein/oleic acid vesicles enhance the immunogenicity of admixed diphtheria toxoid in mice to the same level as Alum adsorbed (or Freund's complete adjuvant) when administered parenterally or nasally. It was also shown that the immunogenicity was linked to the length of the acyl chain of the lipids, where shorter acyl chains resulted in reduced titers. Furthermore, shorter acyl chains also gave rise to more pronounced toxic reactions at the injections sites, such as necrosis and alopeci, both of which were lacking when the optimal formulation consisting of mono-olein and oleic acid was used. Thus, this lipid matrix has in our view a great potential as an immunological adjuvant with an exceptionally simple and efficient preparation procedure without organic solvents and with low cost endogenous lipid based raw materials.

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