WO2002053183A1

WO2002053183A1 - Hantavirus vaccin with adjuvant

Info

Publication number: WO2002053183A1
Application number: PCT/SE2001/002788
Authority: WO
Inventors: Ulf SCHRÖDER; Cristina Afua De Carvalho Nicacio; Åke LUNDKVIST
Original assignee: Eurocine AB
Current assignee: Eurocine AB
Priority date: 2001-01-03
Filing date: 2001-12-14
Publication date: 2002-07-11
Anticipated expiration: 2003-07-03
Also published as: SE0100011D0

Abstract

A vaccine formulation against a microorganism is disclosed. The formulation comprises as adjuvant, one or more substances selected from a) monoglyceride preparations having at least 80% monoglyceride content and b) fatty acids of the general formula CH3-(CH2)n-COOH where 'n' may be varied between 4 and 22, and where the acyl chain may contain one or more unsaturated bonds, and as immunizing component, an immunogenic product consisting of antigenically active N protein from a Hantavirus, the formulation may comprise, as adjuvant, a mixture of mono-oleic and oleic acid, and possibly soybean oil, and, as immunizing component, the N protein from a Hanta virus.

Description

HANTAVIRUS VACCIN WITH ADJUVANT

The present invention relates to a novel vaccine formulation against Hantavirus. The preferred route of administration of the vaccine formulation is via the mucosal membranes. BACKGROUND

The earliest described immunisation attempts were carried out in China over 900 years ago, where intranasal inoculation of dried and ground smallpox pustules was performed. In the classical immunology and in combination with vaccination against different types of infectious agents e.g. bacteria, virus or parasites the prevailing dogma has been to administer the vaccine subcutaneously or intramuscularly. However, research has during the last years shown that the body has a very effective immunological system that resides in the mucosa. It has also been shown that you can administer vaccines nasally, orally, rectally and vaginally. In the same way as for the classical immunisation, it has been shown that by mucosal vaccination there is also a need for enhancement of the immunological response by the addition of adjuvants.

The intranasal route has attracted increased attention because of the greater efficacy in inducing mucosal immune responses than the more conventional regimes of parenteral immunisation. Furthermore, the realisation that approximately 80% of the immune system reside in the mucosa combined with the fact that an equal percentage of the known pathogens enter our bodies via the mucosal membranes has pushed the interest towards the application of mucosal immunisation.

It has also been shown that parenteral vaccines do not induce immune response at mucosal sites. Thus, it is also clear that appropriate stimulation of a mucosal site such as the nose or the gut, can generate immune response at other mucosal sites. As an example, it is possible to apply a vaccine in the nose and obtain an immune response in the vagina. Furthermore, the mucosal immune response is very rapid with onset only hours after being subjected to stimulation by a pathogen, as compared to parenteral immunity having a response time of several days.

Hantaviruses belong to the Bunyaviridae family, which consists of more than 350 viruses, making them one of the largest groupings of animal viruses. Hantavirus has been implicated as aetiologic agents of two acute syndromes: haemorrhagic fever with renal syndrome (HFRS) and Hantavirus pulmonary syndrome (HPS). Both diseases are transmitted from rodents. HFRS viruses are carried by Old World rodents and HPS viruses by New World rodents.

HFRS is an acute febrile illness, first recognised in the western world after an outbreak among American troops stationed in Korea in the 1950ies. The disease, later referred to as Korean haemorrhagic fever (KHF), is characterised by fever and myalgia, followed by flushing and petechiae. The disease progresses to haemorrhage (gastrointestinal, subconjunctival), haemodynamic instability, and occasionally to shock. Thrombocytopenia, neutrophilia, atypical lymphocytes and haemoconcentration are common. There is acute renal failure, but the precise pathophysiologic lesion is uncertain. Mortality is due to shock or haemorrhage. Similar symptoms although generally less severe, are caused by the related SEO virus. The mortality rate in SEO virus infections appears to be lower (<1%) than in HTN virus infections (5-15%).

PUU is the causative agent of nephropathia epidemica (NE) a milder form of HFRS, which is endemic in Scandinavia, Finland, the European parts of Russia, and Central Europe. NE demonstrates strong clinical similarities to severe HFRS (caused by HTN or DOB hantaviruses) but is characterised by lower frequency of shock and haemorrhages.

The mortality rates for NE (0.1-1%) are considerably lower than for HTN DOB/SEO hantavirus infections, and only a few cases with a fatal outcome are known. The most common clinical findings in NE patients are acute onset of symptoms; fever, headache, nausea, backpain, vomiting, myalgia, abdominal pain and visual disturbances. One third of the patients have haemorrhagic manifestations and the majority have signs of renal failure, i. e. increased levels of serum creatinin, proteinuria and haematuria. There are currently at least nine serologically different hantaviruses indigenous to Old World rodents, including the four that cause HFRS; PUU, HTN, DOB and SEO viruses, and at least eight among indigenous American rodents, including four that cause HPS; SN, BCC, BAY and NY-1 viruses.

The Hanta viral particles are spherical/pleomorphic with varying diameters of 75-115 nm. The nucleocapsids have a helical, filamentous structure, 200-3000 nm. long (depending on arrangement) with a diameter of 2-2.5 nm. Hantaviruses are enveloped and have a three-segment negative stranded RNA genome packed in helical nucleo capsids. The genome encodes four structural proteins: an RNA polymerase, two envelope glycoproteins (Gl and G2) and a nucleocapsid (N) protein. Distinct spikes of about 10 nm. consisting of the glycoproteins extend from the envelope. The virus has no internal matrix protein; therefore, the virion structure is stabilised by direct interaction of the internal nucleocapsids with the cytoplasmic domain of the inserted glycoproteins. Each virion contains approximately 25 copies of the L protein associated with 2100 molecules of the N protein, and 270-1400 widely distributed spike proteins extending from the surface. The virions are composed of 1-2 % nucleic acid, over 50 % protein, 20-30 % lipid, and 2-7% carbohydrates, by weight.

The route by which the virus is transmitted to humans is via aerosols of infected excreta of rodents. In Scandinavia, the rodent reservoir has been identified as being the bank vole. Infected rodents appear to be persistently infected.

In Scandinavia and Finland, the bank vole populations vary in size in a cyclic pattern, which correlates to the number of NE cases. The population peaks occur every 3 to 4 years, and an incidence of 30 cases per 100,000 inhabitants has been reported during peaks.

Primaiy replication of PUU virus in humans appears to occur in the lungs, and involvement of the respiratory tract has been reported to occur in one third of the patients. Some patients have a prolonged renal dysfunction after disease.

In the Balkans, both a severe and a milder form of HFRS have been reported and attributed to the presence of at least two different hantaviruses in the region. Serological data from patients shows that both DOB (fatality rate 5-12%) and PUU viruses are causing HFRS in Bosnia.

HPS was first recognised in 1993, when a cluster of three unexplained adult respiratory distress syndrome cases occurring in the Four Corners region of the United States. HPS is characterised by an initial fever followed by the abrupt onset of acute pulmonary oedema and shock. The most common prodromal symptoms are fever, myalgia, dyspnea, gastrointestinal symptoms, and headache. Physical findings are tachypnea, tachycardia and hypotension.

The mortality of HPS exceeds 50%. In Asia, KHF appears to occur throughout the year, with a biphasic peak incidence in May to July and in October to December.

The basic response after immunising a mammal may be described as being humural or cellular: A humural response is characterised by the formation of antibodies against the invading pathogen and the cellular is characterised by the activation of the T cells in the immune system.

Presently, there is no vaccine available against any hantavirus in the western world. There are a few vaccines, based on inactivated mice-brain cultured virus preparations, available in Asia, but the efficiency as well as the safety of these vaccines are not known. A number of investigations have been performed in order to establish the basic criteria for an efficient vaccine. As with other virus vaccines there is, among the persons skilled in the art, a basic understanding in that, in order to achieve protective vaccination, there is a need for a cellular immune response. A number of attempts has been tried during the last years to evoke protective immunity using the envelope glycoproteins of hantaviruses, however, so far without great success, partly because of the known difficulty in producing cellular immunity against carbohydrates, which are essential components of the Gl and G2 envelope proteins. Surprisingly, it has now been found that the N protein is capable of generating protective immunity based on a cellular response, not only against virus challenge from the homologous strain of the virus, but also from distant hantaviruses (e.g. DOB and AND) causing the more severe form of HFRS and HPS.

The amino acid sequence of the N-protein from PUU-KAZ has been determined (Lunkvist, A. et al, J. Virology (1997) 71:9515-9523) and the 433 amino acids has the following sequence:

MSDLTDIQEEITRHEQQLVVARQKLKDAERAVEVDPDDVNKNTLQARQQTVSA LEDKLADYKRRMADAVSRKKMDTKPTDPTGIEPDDHLKERSSLRYGNVLDVN AIDIEEPSGQTADWYTIGNYVIGFTIPπLKALYMLSTRGRQTVKENKGTRIRFKD DTSFEDT GIRRPKHLYVSMPTAQSTMKAEELTPGRFRTINCGLFPTQIQVRNIMS PVMGVIGFSFF\^KDWPERIRDFMEKESPFIKPEVKPGTPAQEIEFLKRNRNYFMT RQD\O.DKΝHVADIDKLIDYAASGDPTSPDDIESPΝAPWVFACAPDRCPPTCIYV AGMAELGAFFSILQDJVffiNTIMASKTVGTAEEKLKKKSSFYQSYLRRTQSMGIQL DQRIILLYMLEWGKEMVDHFHLGDDMDPELRGLAQSLIDQKVKEISNQEPLKI. Thus, the present invention is directed to virus vaccines, which comprise, as an immunising component, at least one member of the group consisting of a) a recombinant N protein b) fragments of said protein which comprise B-cell and/or T-cell epitopes and, c) amino-acid sequences which are at least 80% homologous to the sequences of a) or b) and which comprise B-cell and/or T-cell epitopes.

It is believed that some amino-acid substitutions, deletions, and extensions may occur in the above defined protein a) and fragments b), while such homologous amino- acid sequences defined in c) will still provoke the same type of protective immunity. The closer the homologous sequence is to the parent sequence the more likely the properties are the same. Thus, homologies of 95%, 90% and 85% are preferred.

Thus, as can be seen in Example 3, the voles were partially protected after challenge with DOB and AND viruses, both of which are known as "killer virus" and furthermore, coming from distant parts of the world, as compared to the PUU virus from which the rN protein was cloned.

Adjuvants are a heterogeneous group of substances that enhance the immunological response against an antigen that is administered simultaneously. Almost all adjuvants used today for enhancement of the immune response against antigens are particles or are forming particles together with the antigen. In the book "Vaccine Design - the subunit and adjuvant approach" (Ed: Powell & Newman, Plenum Press, 1995) almost all known adjuvants are described both regarding their immunological activity and regarding their chemical characteristics. As described in the book more than 80% of the adjuvants tested today are particles or polymers that together with the antigens (in most cases proteins) are forming particles. The type of adjuvants that are not forming particles are a group of substances that are acting as immunological signal substances and that under normal conditions consist of the substances that are formed by the immune system as a consequence of the immunological activation after administration of particulate adjuvant systems.

Using particulate systems as adjuvants, the antigens are associated or mixed with or to a matrix, which has the characteristics of being slowly biodegradable. Of great importance using such matrix systems are that the matrices do not form toxic metabolites. Choosing from this point of view, the kinds of matrices that can be used are mainly substances originating from a body. With this background there are only a few systems available that fulfil these demands: lactic acid polymers, poly-amino acids (proteins), carbohydrates, lipids and biocompatible polymers with low toxicity. Combinations of these groups of substances originating from a body or combinations of substances originating from a body and biocompatible polymers can also be used. Lipids are the preferred substances since they display structures that make them biodegradable as well as the fact that they are the most important part in all biological membranes. Lipids are characterised as polar or non-polar. The lipids that are of most importance in the present invention are the polar lipids since they have the capacity to interact and form particulate systems in water. Another way of defining these lipids are as amphiphilic due to their chemical structure with one hydrophobic and one hydrophilic part in the molecule thereby being useable as surface active substances. Examples of main groups of polar lipids are mono-glycerides, fatty acids, phospholipids and glycosphingolipids. These main groups can be further characterised depending on the length of the acyl chain and the degree of saturation of the acyl chain. Since the number of carbon atoms in the acyl chain can be in the range of 6 to 24, and the number of unsaturated bonds can be varied, there is an almost infinite number of combinations regarding the chemical composition of the lipid. Particulate lipid systems can be further divided into the different groups as discussed in the scientific literature such as liposomes, emulsions, cubosomes, cochleates, micelles and the like.

In a number of systems the lipids may spontaneously form, or can be forced to form, stabile systems. However, under certain circumstances other surface-active substances have to be introduced in order to achieve stability. Such surface-active systems can be of non-lipid character but possess the characteristics of the polar lipids having hydrophobic and hydrophilic parts in their molecular structure.

Another factor that has been shown to be of importance is that lipids exhibit different physical chemical phases, these phases have in different test systems been shown to enhance uptake of biological substances after administration to mucous membranes. Examples of such physical chemical phases described are L2, lamellar, hexagonal, cubic and L3.

In the same way as within the classical immunology where vaccines (antigens) are administered parenterally, there is within mucosal immunization a great interest in directing the immunological response towards development of humoral and/or cellular response. If you obtain a humoral response, it would be important to direct the response in a way that a certain class of antibodies would be obtained. In order to obtain such a goal, specific immune stimulating agents can be added to the formulation of antigens and adjuvants.

A formulation, which fulfils these goals, is described inPCT/SE97/01003, the content of which is incorporated herein by reference. The disclosed formulation comprises monoglycerides and fatty acids. The monoglycerides comprise one or more substances selected from monoglycerides wherein the acyl group contains from 6 to 24 carbon atoms, preferably 8 to 20 carbon atoms, even more preferably 14 - 20 carbon atoms and where the acyl chain may contain unsaturated bonds.

The acyl chain of the fatty acid may be varied between 4 and 22, preferably 8 to 18 and where the acyl chain may contain one or more unsaturated bonds. A combination of the monoglyceride mono-olein and oleic acid has shown to be an L3 phase, which can be described as sponge-like structure, in contrast to liposomes that form onion-like lamellar structures.

Said combination of monoglycerides and fatty acids may be further formulated by the addition of a biocompatible and biodegradable oil thus forming an oil in water (o/w) or w/o/w emulsion. Such emulsions have been shown in the literature to be very effective in enhancing the cellular response against an antigen after administration to an animal (Singh, M., et al 1997, Vaccine 15, 1773-78). It is generally accepted that in order to have an acceptable vaccine against most viruses there is a need for a cellular immune response.

Thus, there is a need for a simple way of administering a vaccine combined with an antigen that is easily documented and formulated. One way of producing such a system would be to use antigenic components from virus which would have the capacity to provoke an immune response in a body, preferably producing a protective immunity against the pathogen which was the origin of the antigen.

Description of the invention

The present invention is directed to a vaccine formulation against a microorganism comprising, as adjuvant, one or more substances selected from a) monoglyceride preparations having at least 80 % monoglyceride content and having the general formula CH — CH CH₂

I ^z I o o O

I I I

R-, R₂ R3

wherein R\ and R₂ is H and R₃ is one acyl group containing from 6 to 24 carbon atoms, and where the acyl chains may contain one or more unsaturated bonds and b) fatty acids of the general formula CH₃ - (CH₂)_n - COOH where "n" may be varied between 4 and 22, and where the acyl chain may contain one or more unsaturated bonds, and the recombinantly produced N protein from a Hantavirus.

The adjuvant of the vaccine formulation of the invention preferably has a monoglyceride preparation content of at least 90 %, preferably at least 95 %, and the acyl chains of the monoglyceride preparation contains 8 to 20 carbon atoms, preferably 14 to 20 carbon atoms, and the acyl chains optionally contains one or more unsaturated bonds, and the recombinantly produced N protein from a Hantavirus. The vaccine formulation according to the invention may further comprise pharmaceutical excipients selected from the group consisting of biocompatible oils, such as such as rape seed oil, sunflower oil, peanut oil, cotton seed oil, jojoba oil, squalan or squalene, physiological saline solution, preservatives and osmotic pressure controlling agents, carrier gases, pH-controlling agents, organic solvents, hydrophobic agents, enzyme inhibitors, water absorbing polymers, surfactants, absorption promoters, and anti-oxidative agents.

A most preferred embodiment of the invention is a vaccine formulation which comprises, as adjuvant, a mixture of mono-olein and oleic acid, and possibly soybean oil, and, as immunizing component, the recombinantly produced N protein from a Hantavirus.

In another preferred embodiment of the vaccine formulation according to the invention, the formulation is formulated into a preparation for mucosal administration, such as nasal, pulmonary, oral, rectal or vaginal administration. Another aspect of the invention is directed to an aerosol or spray package comprising a vaccine formulation according to the invention.

Yet another aspect of the invention is directed to a nose-drop package comprising a vaccine formulation according to the invention. A further aspect of the invention is directed to a method of vaccinating a mammal against a virus having the recombinantly produced N protein from a Hantavirus, which comprises mucosal administration to the mammal of an protection- inducing amount of a Hanta vaccine formulation according to the invention.

The present invention describes a formulation that may be prefabricated, and therefore no need for skilled personnel is needed upon nasal administration, thereby eliminating injection systems such as needles and syringes which in developing world often are contaminated and thus is spreading diseases between patients. Furthermore, a device for multidose aerosol delivery of a nasal vaccine can easily be constructed in way that no person-to-person infection can occur. The invention will now be illustrated by way of the following examples, which, however, are not to be interpreted as limitation to the scope of protection according to the appended claims.

EXAMPLE 1 Production of recombinant nucleocapsid protein. The open reading frame

(ORF) of the PUUV (strain Kazan-E6) N protein gene was cloned and sequenced. The N ORF, encoding amino acids (aa) 1-433, was amplified from cDNA with primers 5' TTG CAT GCT TAT GAG TGA CTT GAC AGA CAT CCA. A 3' and 5' TTG TCG ACT TAA TCA TAT CTT TAA GGG CTC CTG 3', containing a Sphl and a Sail restriction site, respectively. The N ORF was cloned into the pQE-32 vector (Qiagen), containing a T5 promotor and encoding a polyhistidine tag to facilitate purification, according to the manufacturer's instructions. Competent Ml 5 [pREP4_/ E. coli cells (Qiagen) were transformed and spread on Luria-Bertani agar containing 100 μg/ml ampicillin and 25 μg/ml kanamyan. Selected colonies were grown in super broth medium containing antibiotics, as above, and induced with IPTG (1 mM). Induced mini cultures were screened for expression of N protein by immunoblotting with a pool of PUUV N- specific monoclonal antibodies (MAbs). Colonies containing a fusion protein of the expected size were selected for further amplification and purification. The rN protein was extracted and purified, using the polyhistidine tag, on a column containing nickel- agarose (Qiagen). An irrelevant protein, mouse dihydrofolate reductase (DHFR), provided by the manufacturer and expressed in the same system was used as a control. The purified protein was dialysed against PBS (Slide-A-Lyser 10,000 MWCO; Pierce), and the protein concentration was determined by measuring the absorbance at 280 nm. The purity and size was determined by Coomassie Blue staining of standard SDS-PAGE gels and immunoblotting as above or with a MAb specific for the polyhistidine tag (Tetra His MAb; Qiagen).

The sequence of the cloned S gene was confirmed by nucleotide sequence analysis using sequencing primers provided by the vector supplier (Qiagen).

The expressed rN protein gave a band of the expected size (about 54 kDa) by immunoblot with a pool of PUUV-specifϊc MAbs or a polyhistidine-specifϊc Mab. The rN protein was also characterized with a panel of 13 N-specific MAbs which recognized all the epitopes earlier seen in E. cotϊ-expressed recombinant proteins.

EXAMPLE 2

Localization of T-helper cell recognition sites. Immunizations were carried out on groups of five to six mice (each group of a different haplotype) intraperitoneally with 20 μg of rN protein emulsified in Freund's complete adjuvant (FCA). The mice were boosted 4 weeks later subcutaneously with 50 μg of rN protein emulsified in Freund's incomplete adjuvant and sera were collected by retroorbital bleedings at 2, 4 and 6 weeks.

For proliferation assays and cytokine detection, groups of mice were immunized subcutaneously in the base of the tail with 50 μg of rN protein emulsified in FCA. For peptide immunizations, mice were injected with 100 μg of peptide emulsified in FCA.

The rN protein raised PUUV-specific antibodies in inbred mice and all IgG subclasses were detected. Epitope mapping using peptides spanning the N protein reveal that the B-cell recognition sites were mainly located at the aminoterminal part of the protein. Proliferative T-helper (Th) lymphocyte responses were detected in all haplolypes after a single immunization with rN. Several Th-recognition sites, spanning amino acids 6-27, 96-117, 211-232 and 256-277, were identified using overlapping peptides. Peptides representing the identified sites could also prime Th-lymphocytes to proliferate in response to recall with rN protein, thereby confirming the authenticity of the identified sites. The rN-primed Th lymphocytes produced predominantly interleukin (IL)-2 and gamma interferon, together with lower levels of IL-4 and IL-6, indicating a mixed Thl/Th2 response.

EXAMPLE 3

The crossprotection after immunization with recombinant N proteins from other hantaviruses (DOB and AND) was measured after challenge with PUU. Regarding amino acid homology and nucleotide homology, there is only a 55-70% similarity between DOB/AND and PUU viruses. Thus, voles were immunized with rN from the different virus strains, whereafter the voles were challenged nasally with the PUU virus strain. The cross-protection against PUUV infection in bank voles after rN immumzation is seen below.

No. of positives/challenged animals

Vaccine Ag-ELISA RT-PCR

Puumala-rN 0/9 0/9

Dobrava-rN 1/10 2/10

Andes-rN 0/8 3/8

DHFR (control) 8/8 8/8

Unvaccinated 8/8 8/8

These results indicate that rN protects 100% of the voles which were vaccinated with the homologous N protein, determined as detectable antigen (Ag-ELISA) or RNA (RT-PCR) in the lung tissue of the challenged animal. Furthermore, 65 to 100% of the voles challenged with non-homologous strain were also protected.

EXAMPLE 4

Protection of bank voles after intranasal/s.c. immunization and (s.c.) challenge with Hantavirus (strain PUU) by immunization with the rN protein in the L3 lipid adjuvant formulations.

The emulsion was produced by mixing the rN protein with 100 μl of soybean oil and 100 μl of a mixture of mono-olein and oleic acid (1:1). The amount of rN protein was adjusted so that a dose of 10 μg was given to the mice in 100 μl (parenteral) or in 10 μl (nasal). This mixture was sonicated briefly for a few seconds whereafter 1.0 ml of 0.1M TRISS buffer and 20 μl of 4 M NaOH was added. Sonication was performed for 2 minutes whereafter the vaccine formulation was used for immunization.

Immunization 1; 0 weeks (nasal).

Immunization 2; 2 weeks (nasal)

Immunization 3; 4 weeks (parenteral)

Challenge; 6 weeks

Three weeks after challenge, the voles were killed and the amount of virus was determined in the lung tissue by two different methods, either using a method which detects proteins from the virus (known as an antigen-ELISA) or using a PCR method for detection of virus RNA in the tissue.

No. of positives/challenged animals

As can bee seen, these results indicate that the voles are protected from infection of the virus after being vaccinated with the L3/rN-protein formulation according to the present invention.

Claims

1. Vaccine formulation against a virus comprising, as adjuvant, one or more substances selected from a) monoglyceride preparations having at least 80 % monoglyceride content and having the general formula

CHo — CH — CHo o o o

I I I

Rl F*2 ^R3

wherein Ri and R₂ is H and R₃ is one acyl group containing from 6 to 24 carbon atoms, and where the acyl chains may contain one or more unsaturated bonds and b) fatty acids of the general formula

CH₃ - (CH₂)„ - COOH where "n" may be varied between 4 and 22, and where the acyl chain may contain one or more unsaturated bonds, and as immunizing component, at least one member of an immunogenic product consisting of antigenically active a) recombinant N protein originating from a Hantavirus b) fragments of said protein which comprise B-cell and or T-cell epitopes, and c) amino-acid sequences which are at least 80% homologous to the sequences of a) or b) and which comprise B-cell and/or T-cell epitopes.

2. Vaccine formulation according to claim 1, wherein the immunologically active component contain a recombinantly produced N protein originating from a Hantavirus.

3. Vaccine formulation according to claim 1 or 2, wherein the adjuvant has a monoglyceride preparation content of at least 90 %, preferably at least 95 %, and the acyl chains of the monoglyceride preparation contains 8 to 20 carbon atoms, preferably 14 to 20 carbon atoms, and the acyl chains optionally contains one or more unsaturated bonds, and the immunologically active components are derived from polypeptides and are selected from the N protein originating from a Hantavirus.

4. Vaccine formulation according to any one of claims 1 - 3, which further comprises pharmaceutical excipients selected from the group consisting of biocompatible oils, physiological saline solution, preservatives and osmotic pressure controlling agents, carrier gases, pH-controlling agents, organic solvents, hydrophobic agents, enzyme inhibitors, water absorbing polymers, surfactants, absorption promoters, and anti-oxidative agents.

5. Vaccine formulation according to claim 3 or 4, wherein the adjuvant is a mixture of mono-olein and oleic acid, and possibly soybean oil, and the immunizing component is the N protein originating from a Hantavirus.

6. Vaccine formulation according to any one of claims 1-5, wherein the formulation is formulated into a preparation for mucosal administration.

7. Vaccine formulation according to claim 6, wherein the mucosal administration is selected from nasal, pulmonary, oral, rectal and vaginal administration.

8. Aerosol or spray package comprising a vaccine formulation according to any one of the claims 1 - 7.

9. Nose-drop package comprising a vaccine formulation according to any one of the claims 1 - 6.

10. A method of vaccinating a mammal against a virus having antigenically active N protein originating from a Hantavirus, which comprises mucosal administration to the mammal of a protection-inducing amount of a vaccine formulation according to any one of claims 1 - 7.