WO2015085242A1 - Skin innate response linked to protective intradermal vaccination against respiratory infection - Google Patents

Abstract

Intradermal (ID) vaccination aims at targeting the large network of skin antigen-presenting dendritic cells (DC) and constitutes a promising approach to improve anti-infectious immunity. Yet, its anti-viral protective efficacy for sub-unit vaccines and the underlying skin innate immune mechanisms remains to be explored for a wider usage in humans. Using a pig model of lethal respiratory infection with pseudorabies virus (PRV), we show that ID vaccination with adjuvant-free PRV glycoproteins triggers cellular and humoral immunity and confers protection from disease induction by nasal viral challenge, as efficiently as an oil- adjuvanted reference vaccine delivered intramuscularly.

Description

TITLE OF THE INVENTION

Skin innate response linked to protective intradermal vaccination against respiratory infection

This application claims priority to U.S. Provisional Patent Application number 61/912,350, filed December 5, 2013.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is Andreoni_PatentIn_sequences _ST25. The text file is 5KB; it was created on 13 November, 2014; and it is being submitted electronically via EFS-Web, concurrent with the filing of the specification.

FIELD OF THE INVENTION

The present disclosure relates generally to vaccines, methods of vaccine production and their administration. More specifically the present disclosure provides a composition, a method of manufacture and a method of administration of a non-adjuvanted porcine vaccine against pseudorabies virus (PRV).

BACKGROUND

Research in the vaccination against infectious disease has not typically focused on the intradermal (ID) route of administration. Most anti-infectious vaccines are currently delivered intramuscularly (IM), while the intradermal route is used for Bacillus Calmette-Guerin and rabies vaccines. There is limited knowledge of the ability of ID vaccination to induce protective anti-viral immunity at mucosal sites in humans. Although numerous studies documented the potential benefits of ID vaccination in mice, there is still a need to provide the proof of concept of ID vaccine protective efficacy and mode of action in animal models that could better translate to humans.

Renewed interest in intradermal vaccination is driven by the fact that epidermis and dermis of human skin are highly enriched in professional antigen-presenting dendritic cells (DC) including epidermal Langerhans cells (LC) and several subsets of dermal DC (dDC), with immune-stimulatory function in the priming and differentiation of effector T and B cells. Moreover, the unique anatomical characteristics of the dermis, with its dense network of lymphatic and blood vessels, strongly supports that direct delivery of a vaccine to this layer, rather than to muscle or sub-subcutaneous tissue, would favor efficient uptake and transport of antigen (Ag) by skin DC to draining lymph nodes (LN) for presentation to T cells.

Moreover, mouse studies indicated that the advantage of the cutaneous over the IM route is to induce cytotoxic CD8⁺ T cells in addition to CD4⁺ T cells and antibody responses, thus providing better protective immunity with smaller volume of vaccine. In this respect, recent progress in the design of new ID delivery devices now allows for accurate delivery of a small volume (e.g., about 100 μΐ) of vaccine to the superficial dermis without overt skin reactogenicity. Additionally, dose sparing and minimal training of personnel lend ID administration to widespread use for mass vaccination.

Indeed, the BD Soluvia® device is composed of a micron-sized needle, which is inserted 1.5 mm perpendicularly into the skin and targets the papillary dermis. Needle free technology is also known, such as that disclosed in EP 1 765 387 and US 7,582,302, both of which are incorporated by reference herein in their entirety. Clinical trials in humans vaccinated against seasonal flu with this device have documented the efficacy and superiority of the ID over the IM route for seroneutralisation, especially in elderly and in immune-compromised transplanted patients. Microneedle patch technology can also deliver vaccine intradermally. A microneedle patch contains an array of micron-scale, solid needles, which encapsulate vaccine in a water-soluble matrix (see, e.g., US 7,918,814, US 8,257,324, US 8,636,713 and US 2008/0213461, each to Prausnitz & the Georgia Tech Research Corporation, and incorporated into this disclosure, by reference, in their entirety). Within seconds of being applied, the microneedles puncture the skin, can separate from the patch, and can become embedded within the skin where they can dissolve. In the process, the microneedles can deliver the encapsulated vaccine without producing sharps waste. Yet, whether anti-infectious ID vaccination can be extended to sub-unit vaccines composed of recombinant or purified proteins or glycoproteins remains to be explored as well as the mechanisms behind its efficacy.

Along these lines, subunit vaccines often require coadministration of adjuvant to stimulate innate immunity through the induction of specific adaptive humoral and cell-mediated immune responses. Mouse studies have shown that the efficacy of adjuvants is linked to their ability to stimulate secretion of DC- and monocyte-attracting chemokines to recruit DC or their precursors at the immunization site. In addition, we demonstrated that the immunogenic property of several Ag delivered via pluri-stratified mucosae or skin, was directly driven by their intrinsic adjuvant properties and ability to stimulate local recruitment of immune- stimulatory inflammatory DC at the site of immunization. It is questionable, however, whether addition of adjuvants would be suitable for ID vaccination, as they are likely to provoke visible, sustained and potentially painful skin inflammation. Thus, it is of prime importance to evaluate whether the ID injection itself could provide the necessary mechanical stress to trigger appropriate skin innate immunity that could circumvent the need of adjuvant.

An important consideration for evaluation of vaccination strategies and mechanistic studies is to use appropriate models that are predictive of responses in humans. Besides non-human primates, who cannot be widely used for obvious ethical and financial reasons, domestic swine appears as one of the most relevant pre-clinical model in the field of cutaneous vaccination. Indeed, human and pig skin share numerous similarities in terms of anatomy, structure and thickness of the different skin layers and lipid composition and a similar microneedle device can be used in both species to perform ID injections. In addition, domestic pigs display a high degree of genetic and microbial diversity that is more relevant to humans compared to congenic animals hosted in specific pathogen-free conditions. Finally, the different DC subsets that populate pig epidermis and dermis are reminiscent to those described in human skin.

SUMMARY OF THE INVENTION

In the present study, we used a model of nasal infection with pseudorabies virus (PRV), the etiological agent of Aujeszky's disease, to study the cutaneous events conditioning efficient ID vaccination. We show in domestic swine that induction of cellular and humoral responses conferring anti-viral protection can be achieved by ID vaccination with an adjuvant- free sub- unit vaccine delivered with the micro-needle BD Soluvia® device and that vaccination efficacy is linked to a unique skin signature of early innate immunity integrating the adjuvant effect of ID mechanical stress and antigenic-stress, to mobilize inflammatory-type DC at the site of vaccine delivery.

In one aspect, the invention is a composition for inducing an immune response in swine to pseudorabies virus by intradermal administration of non-adjuvanted pseudorabies virus glycoprotein subunits.

In another aspect, the invention is a method for inducing an immune response in swine to pseudorabies virus by intradermal administration of non-adjuvanted pseudorabies virus glycoprotein subunits. In yet another aspect, the invention is a method of making a composition for inducing an immune response in swine to pseudorabies virus by intradermal administration of non- adjuvanted pseudorabies virus glycoprotein subunits.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure la: Cellular and humoral immune responses induced by ID immunization with PRV- IgM.

Figure lb: Cellular and humoral immune responses induced by ID immunization with PRV- IgG.

Figure lc: Cellular and humoral immune responses induced by ID immunization with PRV- IgA.

Figure Id: Virus neutralizing Ab titers in serum at day 21 after immunization.

Figure le: The frequency of PRV-specific IFNy producing cells in blood was analyzed at day 13 after vaccination by an ELISPOT assay after in vitro re-stimulation of PBMC with the NIA-3 PRV strain.

Figure If: The frequency of PRV-specific IL-2 producing cells in blood was analyzed at day 13 after vaccination by an ELISPOT assay after in vitro re-stimulation of PBMC with the NIA-3 PRV strain.

Figure 2a: ID immunization with PRV-gp confers protection from a lethal viral challenge with virulent NIA3 PRV strain.

Figure 2b: ID immunization with PRV-gp confers protection from a lethal viral challenge with virulent NIA3 PRV strain.

Figure 2c: PRV-specific IgA titers in nasal secretions at day 21, before challenge.

Figure 2d: PRV-specific IgA titers in nasal secretions at day 26, after challenge.

Figure 2e: Seric IgG titers at day 21 were compared between pigs that gained versus lost weight after viral challenge, irrespective of their experimental group.

Figure 2f: Frequency of IFNy-SFC in blood at day 13 were compared between pigs that gained versus lost weight after viral challenge, irrespective of their experimental group.

Figure 2g: Nasal IgA titers before (day 21) were compared between pigs that gained versus lost weight after viral challenge, irrespective of their experimental group.

Figure 2h: Nasal IgA titers after (day 26) were compared between pigs that gained versus lost weight after viral challenge, irrespective of their experimental group. Figure 3a: Ag distribution and fate after ID immunization. PBS-injected control.

Figure 3b: Higher magnification of Figure 3a.

Figure 3c: Ag distribution and fate after ID immunization. PRV-gB stain at 2 hours after ID injection of PRV-gp.

Figure 3d: Higher magnification of Figure 3c.

Figure 3e: Ag distribution and fate after ID immunization. PRV-gB stain at 24 hours after ID injection of PRV-gp.

Figure 3f: Higher magnification of Figure 3e.

Figure 3g: PRV-gB staining on frozen sections of draining lymph nodes.

Figure 3h: PRV-gB staining on frozen sections of draining lymph nodes at 2 hours after ID injection of PRV-gp.

Figure 3i: PRV-gB staining on frozen sections of draining lymph nodes at 24 hours after ID injection of PRV-gp.

Figure 4a: Cytokines and chemokines induced by ID immunization with viral Ag.

Figure 4b: Expression profile of ILl p, IL6, IL13, CCL2, CCL3, CCL8, CCL20 and CXCL8 in skin at 2, 24 and 72 hours after ID injection of PBS (white histograms) or PRV-gp (black histograms).

Figure 5a: Skin ILl gene signature of ID-induced mechanical stress at 0.5, 2, 4 and 24h after ID sham injection (e.g. prick without liquid injection, white circles), ID injection of 100 μΐ of PBS (black squares) or ID injection of 5μg PHA-L (i.e., phytohemagglutinin) in 100 μΐ of PBS (black triangles).

Figure 5b: Skin IL6 gene signature of ID-induced mechanical stress at 0.5, 2, 4 and 24h after ID sham injection (e.g. prick without liquid injection, white circles), ID injection of 100 μΐ of PBS (black squares) or ID injection of 5μg PHA-L (i.e., phytohemagglutinin) in 100 μΐ of PBS (black triangles).

Figure 5c: Skin CCL2 gene signature of ID-induced mechanical stress at 0.5, 2, 4 and 24h after ID sham injection (e.g. prick without liquid injection, white circles), ID injection of 100 μΐ of PBS (black squares) or ID injection of 5μg PHA-L (i.e., phytohemagglutinin) in 100 μΐ of PBS (black triangles).

Figure 5d: Skin CCL3 gene signature of ID-induced mechanical stress at 0.5, 2, 4 and 24h after ID sham injection (e.g. prick without liquid injection, white circles), ID injection of 100 μΐ of PBS (black squares) or ID injection of 5μg PHA-L (i.e., phytohemagglutinin) in 100 μΐ of PBS (black triangles).

Figure 5e: Skin CCL8 gene signature of ID-induced mechanical stress at 0.5, 2, 4 and 24h after ID sham injection (e.g. prick without liquid injection, white circles), ID injection of 100 μΐ of PBS (black squares) or ID injection of 5μg PHA-L (i.e., phytohemagglutinin) in 100 μΐ of PBS (black triangles).

Figure 5f: Skin CXCL8 gene signature of ID-induced mechanical stress at 0.5, 2, 4 and 24h after ID sham injection (e.g. prick without liquid injection, white circles), ID injection of 100 μΐ of PBS (black squares) or ID injection of 5μg PHA-L (i.e., phytohemagglutinin) in 100 μΐ of PBS (black triangles).

Figure 6a: HPS staining of paraffin-embedded skin sections at 24h after ID injection. Pigs were ID injected in the flank skin with PBS.

Figure 6b: HPS staining of paraffin-embedded skin sections at 24h after ID injection. Pigs were ID injected in the flank skin with 30 μg PRV-gp (100 μΐ).

Figure 6c: A higher magnification image of Figure 6a.

Figure 6d: A higher magnification image of Figure 6b.

Figure 6e: CD207 staining of epidermal sheets from un-injected skin (24h).

Figure 6f: CD207 staining of epidermal sheets from PBS-injected skin (24h).

Figure 6g: CD207 staining of epidermal sheets from PRV-gp-injected skin (24h).

Figure 6h: Quantitative analysis of LC showing the number of CD207⁺ cells per mm² of epidermis at 2 or 24h after ID injections with PBS or PRV-gp, in comparison to un-injected control skin. Each point represents an individual value and horizontal bars correspond to the mean (±SEM) counts of 4 fields from 2-3 epidermal sheets out of 3 pigs/group.

Figure 6i: IHS stain of skin frozen sections for CD207 at 24h after ID injection with PBS.

Figure 6j: IHS stain of skin frozen sections for CD207 at 24h after ID injection with PRV-gp.

Figure 6k: IHS stain of skin frozen sections for SLAII at 24h after ID injection with PBS.

Figure 61: IHS stain of skin frozen sections for SLAII at 24h after ID injection with PRV-gp.

Figure 6m: IHS stain of skin frozen sections for CD172a at 24h after ID injection with PBS.

Figure 6n: IHS stain of skin frozen sections for CD 172a at 24h after ID injection with PRV- g -

Figure 6o: IHS stain of skin frozen sections for CD1 at 24h after ID injection with PBS. Figure 6p: IHS stain of skin frozen sections for CD1 at 24h after ID injection with PRV-gp. Figure 7a: FACS dot plots depicting CDl 1R3 versus CD16 expression after ID injection of PBS, 24 hrs.

Figure 7b: FACS dot plots depicting CDl 1R3 versus CD16 expression after ID injection of 30 μg PRV-gp, 24 hrs.

Figure 7c: White histograms illustrate CD 14, CD 163, 2B2 and SWC8 expression by

CD l lR3⁺CD16^int cells and CDl lR3⁺CD16^high cells. Grey histograms correspond to isotype- matched controls. 24 hrs.

Figure 7d: Quantitative analysis of CDl lR3⁺CD16^mt granulocytes monocytes infiltrating the dermis at 24 and 72 hours after ID injection of PBS (hatched histograms) or PRV-gp (black histograms), comparatively to non-injected skin (white histograms).

Figure 7e: Quantitative analysis of CDl lR3 CD16^hlgh monocytes infiltrating the dermis at 24 and 72 hours after ID injection of PBS (hatched histograms) or PRV-gp (black histograms), comparatively to non-injected skin (white histograms).

Figure 7f: FACS dot plots depicting SLAII versus CD l expression after ID injection of PBS, 72 hrs.

Figure 7g: FACS dot plots depicting SLAII versus CDl expression after ID injection of 30 μg PRV-gp, 72 hrs.

Figure 7h: FACS dot plots depicting CD80/86 versus CD l expression after ID injection of PBS, 72 hrs.

Figure 7i: FACS dot plots depicting CD80/86 versus CDl expression after ID injection of 30 μg PRV-gp, 72 hrs.

Figure 7j: White histograms illustrate CD 14 and CD 163 expression on CDl^highSLAII⁺ and CDl^intSLAII⁺ cells. Grey histograms correspond to isotype-matched controls. 72 hrs.

Figure 7k: Quantitative analysis of CDl^highSLAII⁺ dendritic cells and CDl^intSLAII⁺ cells infiltrating the dermis at 24 and 72 hours after ID injection of PBS (hatched histograms) or PRV-gp (black histograms), comparatively to non-injected skin (white histograms).

Figure 71: Quantitative analysis of CDl^highSLAII⁺ dendritic cells and CDl^intSLAII⁺ cells infiltrating the dermis at 24 and 72 hours after ID injection of PBS (hatched histograms) or PRV-gp (black histograms), comparatively to non-injected skin (white histograms).

Figure 8a: The frequency of PRV-specific IFNy producing cells in the blood of pigs depicted in Figure 1 was analyzed at day 13 after immunization by an ELISPOT assay after in vitro re- stimulation of PBMC with 5-9 pools of overlapping peptides covering the entire PRV gB. Figure 8b: The frequency of IL-2 producing cells in the blood of pigs depicted in Figure 1 was analyzed at day 13 after immunization by an ELISPOT assay after in vitro re-stimulation of PBMC with 5-9 pools of overlapping peptides covering the entire PRV gC.

Figure 8c: The frequency of PRV-specific IFNy producing cells in the blood of pigs depicted in Figure 1 was analyzed at day 13 after immunization by an ELISPOT assay after in vitro re- stimulation of PBMC with 5-9 pools of overlapping peptides covering the entire PRV gB. Figure 8d: The frequency of PRV-specific IFNy producing cells in the blood of pigs depicted in Figure 1 was analyzed at day 13 after immunization by an ELISPOT assay after in vitro re- stimulation of PBMC with 5-9 pools of overlapping peptides covering the entire PRV gC.

Figure 9a: Correlates of protection of the experiment depicted in figure 2. Seric IgG titers at day 21 were compared between pigs that survived or died irrespective of group.

Figure 9b: Correlates of protection of the experiment depicted in figure 2. Frequency of IFNy-

SFC in blood at day 13 were compared between pigs that survived or died irrespective of group.

Figure 9c: Correlates of protection of the experiment depicted in figure 2. Nasal IgG titers after challenge (day 26) were compared between pigs that survived or died irrespective of group.

Figure 9d: Correlates of protection of the experiment depicted in figure 2. Nasal IgA titers after challenge (day 26) were compared between pigs that survived or died irrespective of group.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a", "an", and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicate otherwise.

It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of and "consists essentially of have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

The antigenic polypeptides of the invention are capable of protecting against pseudorabies virus, the etiological agent of Aujeszky's disease. That is, they are capable of stimulating an immune response in an animal. By "antigen" or "immunogen" means a substance that induces a specific immune response in a host animal. The antigen of the instant invention is a subunit or portion of an organism; a recombinant vector containing an insert with immunogenic properties; a piece or fragment of DNA capable of inducing an immune response upon presentation to a host animal; a polypeptide, an epitope, a hapten, or any combination thereof.

The term "immunogenic protein, polypeptide, or peptide" as used herein includes polypeptides that are immunologically active in the sense that once administered to the host, it is able to evoke an immune response of the humoral and/or cellular type directed against the protein. A protein fragment according to the invention has at least one epitope or antigenic determinant. An "immunogenic" protein or polypeptide, as used herein, includes the full- length sequence of the protein, analogs thereof, or immunogenic fragments thereof.

As discussed the invention encompasses active fragments and variants of the antigenic polypeptide. Thus, the term "immunogenic protein, polypeptide, or peptide" further contemplates deletions, additions and substitutions to the sequence, so long as the polypeptide functions to produce an immunological response as defined herein. The term "conservative variation" denotes the replacement of an amino acid residue by another biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue does not change or is another biologically similar residue. In this regard, particularly preferred substitutions will generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids. For example, amino acids are generally divided into four families: (1) acidic-aspartate and glutamate; (2) basic-lysine, arginine, histidine; (3) non-polar— alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar— glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another hydrophobic residue, or the substitution of one polar residue for another polar residue, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like; or a similar conservative replacement of an amino acid with a structurally related amino acid that will not have a major effect on the biological activity. Proteins having substantially the same amino acid sequence as the reference molecule but possessing minor amino acid substitutions that do not substantially affect the immunogenicity of the protein are, therefore, within the definition of the reference polypeptide. All of the polypeptides produced by these modifications are included herein. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.

The term "epitope" refers to the site on an antigen or hapten to which specific B cells and/or T cells respond. The term is also used interchangeably with "antigenic determinant" or

"antigenic determinant site". Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.

As used herein, the term "multivalent" means a vaccine containing more than one antigen whether from the same species, an antigen from a different species, or a vaccine containing a combination of antigens from different genera (for example, a vaccine comprising antigens from pseudorabies virus, Pasteurella multocida, Salmonella, Escherichia coli, Haemophilus somnus and Clostridium).

An "immunological response" to a composition or vaccine is the development in the host of a cellular and/or antibody-mediated immune response to a composition or vaccine of interest. Usually, an "immunological response" includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, and/or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered viral titer in the infected host.

Synthetic antigens are also included within the definition, for example, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens. See, e.g., Bergmann et al, 1993; Bergmann et al, 1996; Suhrbier, 1997; Gardner et al, 1998. Immunogenic fragments, for purposes of the present invention, will usually include at least about 3 amino acids, at least about 5 amino acids, at least about 10-15 amino acids, or about 15-25 amino acids or more amino acids, of the molecule. There is no critical upper limit to the length of the fragment, which could comprise the full-length of the protein sequence, or even a fusion protein comprising at least one epitope of the protein.

Accordingly, a minimum structure of a polynucleotide expressing an epitope is that it has nucleotides encoding an epitope or antigenic determinant of a pseudorabies virus polypeptide. A polynucleotide encoding a fragment of a pseudorabies polypeptide may have a minimum of 15 nucleotides, about 30-45 nucleotides, about 45-75, or at least 57, 87 or 150 consecutive or contiguous nucleotides of the sequence encoding the polypeptide. Epitope determination procedures, such as, generating overlapping peptide libraries (Hemmer et al., 1998), Pepscan (Geysen et al, 1984; Geysen et al, 1985; Van der Zee R. et al, 1989; Geysen, 1990;

Multipin. RTM. Peptide Synthesis Kits de Chiron) and algorithms (De Groot et al, 1999; PCT/US2004/022605) can be used in the practice of the invention.

The term "nucleic acid" or "polynucleotide" refers to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant

polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches. The sequence of nucleotides may be further modified after polymerization, such as by conjugation, with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides or solid support. The polynucleotides can be obtained by chemical synthesis or derived from a microorganism.

The term "gene" is used broadly to refer to any segment of polynucleotide associated with a biological function. Thus, genes include introns and exons as in genomic sequence, or just the coding sequences as in cDNAs and/or the regulatory sequences required for their expression. For example, gene also refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences.

The invention further comprises a complementary strand to a polynucleotide encoding a pseudorabies virus antigen, epitope or immunogen. The complementary strand can be polymeric and of any length, and can contain deoxyribonucleotides, ribonucleotides, and analogs in any combination. The terms "protein", "peptide", "polypeptide" and "polypeptide fragment" are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymer can be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component. An "isolated" biological component (such as a nucleic acid or protein or organelle) refers to a component that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant technology as well as chemical synthesis.

The term "purified" as used herein does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified polypeptide preparation is one in which the polypeptide is more enriched than the polypeptide is in its natural environment. That is the polypeptide is separated from cellular components. By "substantially purified" it is intended that such that the polypeptide represents several embodiments at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%, or more of the cellular components or materials have been removed. Likewise, the polypeptide may be partially purified. By "partially purified" is intended that less than 60% of the cellular components or material is removed. The same applies to polynucleotides. The polypeptides disclosed herein can be purified by any of the means known in the art.

The antigenic polypeptides or fragments or variants thereof are partially purified pseudorabies virus glycoproteins (i.e., pseudorabies virus antigenic polypeptides; PRV-gp). Fragments and variants of the disclosed polynucleotides and polypeptides encoded thereby are also encompassed by the present invention. By "fragment" is intended a portion of the

polynucleotide or a portion of the antigenic amino acid sequence encoded thereby. Fragments of a polynucleotide may encode protein fragments that retain the biological activity of the native protein and hence have immunogenic activity as noted elsewhere herein. Fragments of the polypeptide sequence retain the ability to induce a protective immune response in an animal. "Variants" is intended to mean substantially similar sequences. "Variant" protein is intended to mean a protein derived from the native protein by deletion or addition of one or more amino acids at one or more sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is they have the ability to elicit an immune response. As used herein, the term "derivative" or "variant" refers to a polypeptide, or a nucleic acid encoding a polypeptide, that has one or more conservative amino acid variations or other minor modifications such that (1) the corresponding polypeptide has substantially equivalent function when compared to the wild type polypeptide or (2) an antibody raised against the polypeptide is immunoreactive with the wild-type polypeptide. These variants or derivatives include polypeptides having minor modifications of the pseudorabies virus polypeptide primary amino acid sequences that may result in peptides which have substantially equivalent activity as compared to the unmodified counterpart polypeptide. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. The term "variant" further contemplates deletions, additions and substitutions to the sequence, so long as the polypeptide functions to produce an immunological response as defined herein.

The term "conservative variation" denotes the replacement of an amino acid residue by another biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue does not change or is another biologically similar residue. In this regard, particularly preferred substitutions will generally be conservative in nature.

The term "recombinant" means a polynucleotide semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in an arrangement not found in nature.

"Heterologous" means derived from a genetically distinct entity from the rest of the entity to which it is being compared. For example, a polynucleotide may be placed by genetic engineering techniques into a plasmid or vector derived from a different source, and is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter.

The present invention relates to adjuvant free vaccines or pharmaceutical or immunological compositions which may comprise an effective amount of pseudorabies virus antigens and a pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle. As used herein, the terms "pharmaceutically acceptable carrier" and "pharmaceutically acceptable vehicle" are interchangeable and refer to a fluid vehicle for containing vaccine antigens that can be injected into a host without adverse effects. Suitable pharmaceutically acceptable carriers known in the art include, but are not limited to, sterile water, saline, glucose, dextrose, or buffered solutions. Carriers may include auxiliary agents including, but not limited to, diluents, stabilizers (i.e., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, viscosity enhancing additives, colors and the like.

METHODS

Animals and vaccine antigens

Large White/Landrace conventional hybrid pigs (9-10 weeks females, 20-28 kg) were maintained in conventional housing conditions on a complete composite food diet. All experiments were performed according to institutional guidelines and were previously approved by an ethical committee (Comite Regional d'Ethique pour Γ Experimentation Animale). For all experiments, pigs were randomized according to their weight.

Immunogenicity and protection studies were performed at Merial (Saint Vulbas, France), while experiments dedicated to the analysis of cellular and molecular skin events were performed at NASMA Biomatech (Chasse sur Rhone, France) and VetAgro Sup (Marcy I'Etoile, France). Partially purified glycoproteins from pseudorabies virus 1 (PRV-gp) and the oil-adjuvanted Geskypur® vaccine containing the same PRV-gp were provided by Merial. Immunizations and viral challenge

Pigs were tranquilized by intramuscular (IM) injection of Stresnil® (Janssen-Cilag, Issy les Moulineaux, France) and immunized ID at day 0 (dO) in the posterior flank with 10, 30 or 100 μg of PRV-gp in 200 of PBS using the BD Soluvia™ microinjection device, allowing accurate and reproducible ID vaccine delivery as in humans (Laurent et al). A group of pigs was immunized IM in the upper part of the neck with the Geskypur® reference vaccine. At d21, all pigs were inoculated intranasally in each nostril with 1 ml of the virulent PRV strain Northern Ireland Aujeszky-3 [NIA-3, 8.54 loglO tissue culture infective dose per ml (TCID50/ml)] diluted at 1 :5 in MEM pyruvate. Clinical signs were recorded daily until sacrifice at d28 and body weight (BW) was measured just before challenge and at the day sacrifice or death. The mean relative weight gain (MRWG) following the viral challenge was calculated as follows: [(BW d28 - BW d21) - (BW d21 x 7)]xl00. Nasal swabs were collected just before the viral challenge and at d26 to measure viral titers using PK15 cells as described elsewhere (Vandeputte et al). Anti PRV mAb titration

PRV-neutralizing mAbs were measured in serum at dl3 as described elsewhere (Vandeputte et al). Anti-PRV IgG, IgA and IgM Abs were quantified in serum at d-1, 13 and 21 by indirect ELISA, using PRV-gp for coating, and peroxidase-conjugated anti-pig IgG or IgM mAb (AbD Serotec, Oxford, UK), or anti-pig IgA Ab (Nordic, Tilburg, NL) and peroxidase labeled anti-rabbit IgG (Rockland, Gilbertsville, PA). The optical density (OD) was measured at 450nm-650nm and Ig titers were calculated using the Codunit Software using a titration curve of a reference serum included in each plate. The IgG, IgA and IgM titers expressed in arbitrary ELISA units (EU), correspond to the log 10 of the reciprocal serum dilution giving an OD of 1.0. The threshold of antibody detection in serum was 1.0 log.

Quantification of PRV-specific cytokine-producing T cells by ELISPOT

MultiScreen HA 96-well plates (Millipore, Bedford, MA) were coated overnight at 4 °C with 5 μg/ml anti-IFNy mAb (P2G10) or anti-IL2 mAb (6.6.1.1) (BD Biosciences, San Diego, USA) in carbonate/bicarbonate buffer (0.05 M, pH 9.6). PBMCs were incubated in RPMI (Invitrogen, Cergy Pontoise, France) culture medium for 20-24 h at 37 °C with either the NIA-3 ST4 PRV strain (MOI 1) or pools of 22-26 overlapping 15aa peptides (^g/ml for each peptide, Sigma-Aldrich, St-Quentin Fallavier, France) covering the entire PRV-gB (203 peptides) and gC (1 11 peptides) proteins. Wells were then incubated for 2h with biotinylated anti-IFNy (P2C1 1) or anti-IL2 (B33-2) mAb (both from BD Biosciences) and spot forming cells (SFC) were then revealed by sequential incubation with streptavidin-peroxidase (Southern Biotechnology) and AEC (Sigma-Aldrich). The number of SFC was then counted using an ELISPOT plate reader (Microvision Instruments, Evry, France).

Skin biopsy of ID injection sites

Pigs were anesthetized with tiletamine-zolazepam (VIRBAC, Carros, France) followed by inhalation of isoflurane. Each posterior flank was clipped free of fur and received 5 ID injections of either PBS or 30 μg PRV-gp (100 μΐ) using the BD Soluvia® device (10 injections in total, distant from each other by >3 cm in all directions). The quality of each ID injection was checked using 20MHz 2D ultrasonic echography (Dermascan, Cortex,

Denmark) to confirm Ag/PBS delivery at the dermo-epidermal junction (data not shown) (Laurent et al.). At 2, 24 or 72 hours after injections, pigs were anesthetized and were injected with butorphanol (Fort Dodge Animal Health, UK). Skin punch biopsy (8 mm) of each injected sites (3 pigs per stimulus and time point) were processed for FACS (4 pooled biopsies/pig), immuno-histochemistry (IHC, 2 biopsies/pig), Hemalun-Phloxine- Saffron (HES) staining (2 biopsies/pig) and real time RT-PCR (2 biopsies/pig).

Flow cytometry analysis of dermal cells

Epidermal sheets were separated from the dermis after overnight incubation of skin biopsies at 4°C with HBSS medium (Invitrogen) containing 0,4 mg/ml dispase (Roche Diagnostics, Meylan, France). The dermis was cut into small pieces, digested lh at 37°C in RPMI containing 2% FBS, 1 mg/ml type-IA collagenase (Sigma-Aldrich) under magnetic agitation, and a cell suspension was obtained after filtration over a 100 μιη cell strainer. Multicolor FACS stainings were performed using anti-CD 172a (74-22-15), -CD1 (76-7-4) (Southern biotechnology, Birmingham, CA), anti-SLA-II (2E9/13), -CD 14 (MIL2), -CD 16 (G7), - CD 11R3 (2F4/1 1), -CD163 (2A100/11), -Swc8 (MIL3), -granulocytes (2B2) Abs (all from AbD Serotec, Oxford, UK). Anti-SLA-II was coupled to PE using a Zenon® labeling kit (Invitrogen), while anti-Swc8 was revealed by anti-IgM-APC Abs (1B4B1, Southern biotechnology). Anti-CD 14, -CD 163, -granulocytes Abs were labeled in house using an APC labeling kit (AbD Serotec). CD80 and CD86 expression was detected using a human CTLA4- mouse-immunoglobulin fusion protein (ANC152.2/8H5, Ancell, Bayport, MN). Biotinylated mAbs were detected using streptavidin-PEcy7 (BD Biosciences). Data were acquired on a LSRII flow cytometer (BD Biosciences) and analyzed with the FlowJo software (Tree Star, Ashland, Oregon).

Histological and immune-histochemical analysis

Skin biopsies were snap-frozen in Cryomount (Histolab, G5teborg, Sweden) for IHC staining or paraffin-embedded for histological staining with HPS. Cryostat sections were fixed in cold acetone and processed for staining with the Discovery autostainer (Ventana, Illkirch, France). After inhibition of endogenous peroxidase, primary Abs including, anti-CD 1 (76-7-4, Southern biotechnology, Birmingham, CA), -SLAII DQ (K274.3G8, AbD Serotec), anti- CD207 (929F3, Dendritics, Lyon, France) and -CD 172a Abs (74-22- 15 A, BD Biosciences) were manually applied and incubated for lh. PRV-gp were detected using anti-gB

(AJY632F2), -gC (AJY631E10) and -gD (AJY22M24) mAbs kindly provided by Merial. Tissue sections were then subsequently incubated for 30 min with biotinylated rat anti-mouse IgGl (A81-1), IgG2a (R19-15) or IgG2b mAb (R12-3) (BD Biosciences), 16 min with SA- HRP and 8 min DAB-H202. Tissues were finally counterstained with Haematoxylin and bluing reagent, mounted using Pertex (Microm, Francheville, France) and observed using an Eclipse E400 microscope (Olympus, Ville Rungis, France) coupled to an image analysis workstation.

Real Time quantitative RT-PCR

Total R A was prepared from skin biopsies using a Trizol extraction kit (Invitrogen, Cergy Pontoise, France). Genomic DNA was removed by DNA-free treatment (Applied Biosystems, Courtaboeuf, France) and mRNAs were reverse transcribed into cDNA using polydT oligonucleotides, random primers and murine leukemia virus reverse transcriptase

(Invitrogen). Real-Time PCR was performed using a Stratagene MX 3000 (Stratagene, La Jolla, CA, USA) and SYBR Green Master Mix (Invitrogen). The oligonucleotides used in this study are presented in Table 1. HPRT and TUBB4 were used as endogenous control genes to normalize for variations in the starting amount of RNA. Relative expression was calculated using the 2^" method (Livak & Schmittgen, 2001) and results were expressed as fold increase relative to non- injected skin.

EXAMPLES

Virus-specific antibodies and T cell responses are induced after a single ID

immunization with PRV glycoproteins.

We investigated the immune response induced by a single ID immunization with 10, 30 or 100 μg adjuvant- free glycoproteins from PRV (PRV-gp), compared to IM vaccination with the oil-adjuvanted Geskypur® reference vaccine containing the same PRV-gp, as positive control. Pre-vaccination levels of PRV-specific serum IgG and IgA were negligible (Fig. 1B- C), as expected, and low levels of specific IgM were detected (Fig. 1A), possibly reflecting cross-reactive low affinity natural IgM. IM vaccination with Geskypur® induced by day 13 robust IgM and IgG responses and a moderate IgA response, which all persisted at d21, with a slight increase of specific IgG. Pigs immunized ID with the adjuvant-free PRV-gp vaccine developed specific IgG and IgA responses that were optimal with the dose of 30 μg PVR-gp and reached maximal levels by day 21 (Fig. 1B-C). Importantly, PRV-neutralizing serum Abs were present at day 21 in most pigs immunized ID with 30 or 100 μg of PRV-gp, and in pigs vaccinated IM with Geskypur®, but not in pigs injected with either PBS or 10 μg of PRV-gp (Fig. ID).

The PRV-gp-specific T cell response was determined at day 13 by analyzing the frequency of IL-2 and IFNy-producing T cells in blood by an ELISPOT assay after in vitro stimulation of PBMC with either the intact virus or pools of overlapping peptides covering the gB and gC sequences. No PRV-specific IL-2 or IFNy-spot-forming cells (SFC) were detected in either PBS-injected or lC^g ID immunized pigs (Fig. 1 E-F and Fig. 8 A-B). In contrast, ID immunization with 30 or 100 μg PRV-gp and IM immunization with the Geskypur® vaccine induced in most, but not all, pigs the differentiation of virus-specific IL2 and IFNy-producing T cells (Fig. 1 E-F) directed against both gB and gC glycoproteins (Fig. 8 A-B). Detailed analysis of the PRV-specific IFNy⁺ T cell response in the 3 best responders of the 30 μg PRV- gp ID group and of the Geskypur® IM group revealed that vaccination through ID or IM routes induced T cells directed against similar immuno-dominant epitopes profiles for gB (e.g., peptides pools B, C, D, E and G) and gC (e.g., peptide pools B, D and E) (Fig. 8C-D).

Together, these data demonstrated that ID vaccination with 30 μg adjuvant free-PRV-gp induced potent anti-viral humoral and T cell-mediated immunity, as efficiently as IM vaccination with the oil-adjuvanted IM Geskypur® vaccine containing PRV-gp.

ID vaccination with PRV-gp protects against a lethal nasal challenge with PRV.

We next investigated whether ID vaccination with PRV-gp conferred protection from a lethal nasal challenge with PRV and prevented Aujeszky's disease. This was assessed by following survival, body weight gain and virus excretion in nasal fluids. As expected, lethal nasal challenge of PBS-injected control pigs with the virulent PRV NIA-3 strain at day 21 induced wasting disease manifested by body weight loss (Fig. 2A) and high levels of virus excretion in nasal swabs at d26 (Fig. 2B), causing death of 4/6 pigs within 5-7 days (black symbols in Fig. 2A-B). IM vaccination with Geskypur® fully protected against Aujeszky's disease as revealed by 100% survival, prevention of weight loss and decreased virus titers in nasal secretions in 5/6 pigs. Importantly, ID vaccination with 30 μg of PRV-gp also conferred good protection since only 1/6 pigs died and displayed high virus excretion, while 4/6 pigs were fully protected and one pig only showed mild weight loss and virus production in nasal fluids (Fig. 2A-B). Partial protection with 50% survival was achieved by ID vaccination with 100 μg (but not 10 μg) of PRV-gp. Interestingly, viral challenge raised the titer of PRV-specific IgA in nasal fluids of pigs vaccinated ID with 30 μg of PRV-gp or IM with Geskypur® (Fig. 2 C-D). Interestingly, plotting levels of PRV-specific nasal IgA, serum IgG or blood IFNy-T cells (of all vaccinated or un-vaccinated pigs) with clinical protection revealed that the humoral, rather than the cellular, response best correlated with protection against wasting disease (Fig. 2E) and survival (Fig. 9). These data demonstrate that protection against Aujeszky's disease is achieved equally well by ID vaccination with 30 μg of adjuvant free PRV-gp and IM injection of the adjuvanted Geskypur® vaccine and highlights the ability of both routes to induce nasal IgA responses.

PRV-gp transiently accumulate in skin and diffuse to draining lymph nodes after ID immunization.

To track the distribution and fate of PRV-gp after ID injection, cryostat sections of the injected skin sites and draining LN were stained with mAbs specific for PRV-gB at 2 and 24h post ID immunization. Immunohistochemical (IHC) analysis revealed that PRV-gB transiently (2h) accumulated in basal keratinocytes, at the dermo-epidermal junction and in the papillary dermis, while it was hardly detected at 24h (Fig. 3A). PRV-gB was detected in the deep dLN at 2h and was mainly localized in, and in the close vicinity of, subcapsular and trabecular sinuses, especially in the central LN area where B cell follicles are mostly located in pigs (Binns & Pabst). By 24h, only low level of native Ag remained in discrete areas surrounding trabecular sinuses. Similar results were obtained using anti-gC or anti-gD mAbs (data not shown). Thus, ID-injected PRV-gp distributed in native form in various layers of the skin and rapidly diffused to dLN B cell areas, where it remained detected for up to 24h post immunization.

Skin innate gene signature in response to antigenic versus mechanical stress induced by ID.

We next evaluate by real time RT-PCR the local skin innate immune gene signature in response to antigenic stress (ID PRV-gp) versus mechanical stress (ID puncture with or without PBS). RNA levels of a set of inflammatory cytokines and chemokines (Table 2) were quantitated from skin biopsies at 2, 24 and 72h after PBS- or PRV-gp-injection and from intact naive skin (Fig. 4A-B). At 2h post injection, PBS and PRV-gp similarly induced the transcription of a set of genes coding for inflammatory cytokines involved in DC

differentiation, activation or migration (ILl , IL6, IL13, TNFa and GM-CSF) and chemokines attracting neutrophils (CXCL8) or monocytes and immature DC (CCL2, CCL3, CCL8 and CCL20) (Fig. 4A). Only IL6 and CXCL8 were significantly better induced by the viral Ag as compared to PBS alone at 2h (Fig. 4B). Importantly, whereas RNA levels of the set of genes had returned to steady state level of transcription by 24h after PBS injection (Fig. 4A), sustained transcription of several genes up to 24h (ILl , CXCL8) and even 72h (CCL2, 3 and 8) was achieved by Ag injection (Fig. 4A-B). To decipher the respective contribution of the skin puncture and liquid injection into the dermis to the mechanical stress caused by ID vaccination, transcripts of 6 genes were analyzed in sham-ID injected skin (e.g. a simple prick without liquid injection) versus PBS ID-injected skin. Skin ID-injected with Phytohemagglutinin-L (PHA-L), a plant lectin widely used to induce skin inflammation, were included as positive controls. The mere skin puncture without liquid injection induced the rapid transcription as soon as 30' of ILl , CXCL8 (Fig. 5A & 5F), and to a lesser extent of IL6, CCL2 and CCL8 (Fig. 5B-C & 5E), which all peaked at 4h and then decreased at 24h to reach steady state levels (except for CXCL8). Injection of PBS further increased the transient transcription of all these genes but CXCL8, and induced CCL3 transcripts (Fig. 5D). As expected, PHA-L induced far higher and sustained levels of transcription of all tested cytokines and chemokines. The level and kinetics of gene transcription was not merely modified by increasing the volume of injected PBS or PHA-L from 50 μΐ to 200 μΐ (data not shown).

Altogether, these data demonstrate that micro-needle insertion into the skin and liquid injection into the dermis create a mechanical stress and induce the rapid but transient transcription of numerous genes coding for inflammatory cytokines and chemokines, which is further amplified and maintained over time by co-administration of viral Ag.

ID immunization with PRV-gp induces emigration of epidermal LC and recruitment of monocytes and inflammatory DC into dermis.

We next determined the impact of ID vaccination on skin histology and dynamics of cell subsets. At 24h, PBS-injected skin had a similar histology to naive skin (data not shown), while PRV-gp injected skin displayed a moderate perivascular dermal infiltration with the presence of neutrophils and few eosinophils and dermal micro-inflammation with

extravasated red blood cells (Fig. 6A).

Staining of epidermal sheets with an anti-CD207 mAb revealed a significant decrease of LC at 24h after injection of viral Ag compared to naive or control PBS-injected animals (Fig. 6B), suggesting LC emigration into to draining LN after reaching dermal lymphatics. Accordingly, IHC staining of skin cryosections at 24h revealed an increased number of CD207⁺ cells in the papillary dermis at the Ag injection site but not in PBS-injected control site (Fig. 6C). This was associated with an enhanced density of CD1⁺, SLA-II⁺ and CD172a⁺ cells in the dermis of Ag-injected skin compared to PBS-injected skin, indicating that DC accumulate at the site of Ag delivery. To further identify the cellular infiltrate at the injection site, flow cytometry analysis of dermal cell suspensions was carried out at 24 and 72h after ID injection of PRV-gp or PBS. There was low to undetectable T or B cell infiltration at either time point or condition (data not shown). Using Abs to CD1 1R3 (a likely pig equivalent of CD 1 lb (Bullido et al.)) and CD 16/FcyRIII, we identified two cell populations among CD172a/SIRPa⁺ dermal myelo- monocytic cells (Ezquerra et al), which were increased by ID PRV-gp injection (Fig 7B): CD HR3⁺CD16^mt granulocytes, which express the granulocyte markers 2B2 and SWC8 but no or low levels of monocyte/macrophage markers CD 14 and CD 163, and CD l lR3⁺CD 16^hlgh monocytes, which harbor the reverse phenotype (Fig. 7C). CD HR3⁺CD16^int granulocyte infiltration manifested at 24h reaching up to 15% of dermal myeloid cells, and decreased to nearly steady state levels by 72h (Fig. 7D), consistent with the detection of neutrophils made by HPS (Fig. 6A and data not shown). CD1 lR3 CD16^hlgh monocyte infiltration was also prominent by dl after ID injection of PRV-gp but persisted at 72h accounting for up to 18% of dermal myeloid cells (Fig. 7E).

Importantly, two distinct DC subsets were identified among dermal DC, by co-expression of MHC class II (SLAII), CD80/86 co-stimulatory molecules and various levels of CD1 (Fig. 7F-I): the CDl^hlgh subset, corresponding to resident dDC (Bautista et al, Marquet et al), and CDl^int DC, most likely representing newly recruited inflammatory DC derived from blood monocytes, as evidenced by co-expression of the monocyte-associated markers CD 14 and CD 163, and lower levels of SLA-II compared to resident dermal DC. Whereas the proportion of CDl^hlgh resident dDC (2-3% of dermal myeloid cells) was not affected by ID injection of either PBS (Fig. 7F,H) or PRV-gp (Fig. 7G,I), CD l^int DC gradually increased from 24h to reach 8% of dermal myeloid cells at 72h after PRV-gp ID vaccination (Fig. 7K).

Together, these data demonstrate that ID injection of PRV-gp induces an early but transient recruitment of neutrophils and rapid and sustained recruitment of monocyte, followed by monocyte-derived inflammatory DC, reminiscent to the CD163⁺ DC recently identified in pig skin {Marquet, 201 1 #9024} .

ID vaccination is believed to improve anti-infectious immunity by directly targeting skin antigen-presenting DC. Yet, whether ID delivery of adjuvant-free sub-unit vaccines can confer anti-viral protection and whether this is linked to a unique skin immune signature in animal species that translates to human, remains to be explored. Here we show, in domestic swine, whose skin structure and function closely relates to human skin, that a single ID vaccination with PRV glycoproteins without adjuvant is sufficient to trigger specific humoral and cellular immunity and confer protection against Aujeszky's disease after nasal challenge with a virulent PRV strain, as efficiently as the IM oil-adjuvanted Geskypur® vaccine.

Analysis of the quality and magnitude of the PRV-specific humoral and cellular immune response showed appearance of PRV-specific IgG and IgA Abs in serum by 13 days peaking at 21 days after ID or IM vaccination. The dose of 3C^g of PRV-gp was optimal to induce specific serum IgG and IgA, sero-neutralization titers as well as specific IFN-γ or IL-2- producing T cells, to levels comparable to those in pigs vaccinated IM with oil-adjuvanted Geskypur®, albeit doses of 10 or 100 μg were less immunogenic. It could be noted that, although low frequencies of viral-specific cytokine-producing T cells in PBMC were found after either ID or IM vaccination, similar T cell repertoires of PRV-gB or gC specific T cells were generated, as demonstrated by the reactivity to pools of overlapping peptides. Together these data indicated that both routes of vaccination stimulated quantitatively and qualitatively comparable adaptive responses.

It is believed from studies using classical IM or SC vaccination routes, that only live or live- attenuated vaccines can induce robust immune response able to confer protection against infection. Alternatively, sub-unit vaccines, such as purified or recombinant viral proteins, often require additional components like adjuvants to become immunogenic (Coffman et al), although evidence for their protective efficacy beyond the mouse species is still scarce. In our study, a single ID vaccination with PRV-gp alone protected pigs against respiratory virus challenge. The optimal dose of 30 μg appeared as efficient as the oil-adjuvanted Geskypur® vaccine given IM, as evidenced by survival, body weight gain and decreased virus titers in nasal swabs, indicating correlation with adaptive immunity. Interestingly, despite the low levels of PRV-specific nasal IgA titers before challenge, high levels of specific IgA in nasal secretions were induced one week after challenge, supporting that boosting memory IgA B cells occurred upon mucosal exposure to virulent PRV. Interestingly, comparative analysis in all groups of both vaccinated and non-vaccinated pigs, of IgG and IgA antibody titers and frequency of IFNy⁺-producing specific T cells in blood (Sup. Fig 2), showed that pig surviving viral challenge exhibited the highest titers of PRV-specific serum IgG and nasal IgA titers, but not of IFNy⁺ T cells, indicating that humoral rather than cellular immunity best correlated with protection against wasting disease. Although mucosal immunity is best afforded by mucosal immunization due to the compartmentalization of the mucosal immune system (Brandtzaeg & Johansen), our data illustrate that parenteral immunization using the ID route can induce protection in the respiratory mucosa, although the relative contribution of IgA and IgG in viral exclusion from the respiratory mucosa, limitation of systemic virus spreading and protection from the wasting disease remains to be explored.

The finding that protection against pseudorabies infection can be conferred after a single ID vaccination with PRV-gp in the absence of added adjuvants, suggests that Ag delivery by puncture induced the appropriate tissue stress to trigger innate skin immunity. Because protective immunity relies on the quality and strength of local innate immune events, we thoroughly analyzed the skin site of immunization within 24h after immunization to decipher: i) Ag bio-distribution, ii) up-regulation of cytokines and chemokines genes and iii) dynamics of various myeloid cells subsets in epidermis and dermis. Although the rationale of ID immunization is to target vaccines to dermal APC (i.e., antigen presenting cells), there is so far limited information regarding the in vivo bio-distribution of sub-unit vaccine Ag in the skin and dLN. Mouse studies documented earlier that fluorescently-labeled inactivated influenza virus were partially retained for several days at the ID injection site (del Pilar Martin et al), while a fluorescent soluble protein Ag rapidly reached draining lymph nodes after SC (Roozendaal et al.) or ID (Pape et al, Itano & Jenkins 2003, Carrasco & Batista 2007) delivery. Because Ag coupling to a fluorescent tracer may modify Ag conformation and impact on its bio-distribution, we used PRV-gB and gC specific mAb to directly track the native Ag (e.g., with conformational B cell epitopes) in pig tissues. PRV-gp were detected within 2 hours after ID injection in several skin layers including basal keratinocytes and papillary dermis, supporting its accessibility to both epidermal LC and dDC. Concomitantly, Ag appeared in skin dLN and localized in sub-capsular sinus and surrounding trabecular sinuses in the central region where B cell follicles mostly localize in pigs (Binns & Pabst 1994). PRV-gp were virtually undetectable in the skin within 24h after ID injection, indicating that most of the Ag has been either drained out of the injection site or processed by local APC. These data are reminiscent to the first wave of soluble Ag reaching the LN via the lymph reported in mice (Itano & Jenkins 2003) and support that native PRV-gp Ag, rapidly diffusing from the dermis via lymph, reach B cell follicles for initiation of the humoral response.

Remarkably, thorough analysis of the early innate immune events at the skin injection site revealed that Ag delivery by micro-puncture provides the adequate mechanical and antigenic tissue stress promoting sustained up-regulation of key cytokine and chemokines genes and recruitment of monocytes, neutrophils and inflammatory DC, recapitulating immuno- stimulatory conditions. Indeed, the infra-clinical trauma caused by needle puncture and saline injection into the dermis initiates the early transcription of genes encoding inflammatory cytokines involved in DC differentiation, activation or migration (ILl , IL6, IL13, TNFa and GM-CSF) and chemokines, attracting monocytes, neutrophils and immature DC (CCL2, CCL3, CCL8, CCL20 and CXCL8). A similar set of chemokines and cytokines is induced in humans skin after tape stripping (Nickoloff & Naidu 1994, Dickel et al.) and in mice after skin insertion of uncoated microneedle patches (del Pilar Martin et al.) or ID injection of saline buffer (Liu et al). It may thus be postulated that the mechanical tissue stress induced by ID injection creates a local microenvironment prone to initiation of efficient adaptive immune responses. In this respect, liquid injection in mouse dermis was found to induce TLR9 mRNA, promoting subsequent responsiveness to a CpG-containing vaccine preparation (Liu et al). Importantly, in our study, while up-regulation of gene transcription by liquid injection was transient for most cytokines and returned to steady state level by 24h (suggesting that production of biologically relevant amounts of proteins may require additional stimuli), ID injection of viral Ag caused sustained transcription of the pro-inflammatory cytokine ILl and of several chemokines (CCL2, CCL3, CCL8, CXCL8) responsible for monocyte and polymorphonuclear cell attraction.

Accordingly, IHC analysis revealed several dynamic changes in skin DC subsets induced by ID vaccination with PRV-gp. Firstly, LC rapidly emigrated from the epidermis, as evidenced at 24h by the decrease density of Langerin⁺ cells in epidermal sheets and occasional detection on cryostat sections of Langerin⁺ foci in the papillary dermis, just beneath the dermo- epidermal junction. This is reminiscent to observations in mice after ID injection of a modified vaccinia Ankara vaccine (Liard et al.) and in human skin explants after ID injection of Influenza VLP using hypodermic needles (Pearton et al, PloS one) or microneedles (Pearton et al, Vaccine). Thus, ID immunization with different types of vaccines (VLP, attenuated virus, subunit) triggers epidermal LC migration to LN. Importantly, the trauma caused by the ID injection itself was not sufficient to trigger migration of LC, consistent with the fact that ILl and TNFa, which are essential for the migration process (Wang et al), were only transcribed at high levels after PRV-gp delivery. We and others have shown in humans that, at odds to monocyte-derived DC and related dermal CD14⁺ DC, LC are poorly efficient in stimulating B cell differentiation (Klechevsky et al, Dubois et al), suggesting that LC are not responsible for initiating humoral immunity after ID vaccination, consistent with recent results in mice (Liard et al). Alternatively, emigrated LC might be involved in the induction of the cellular immunity (Klechevsky et al.) that we observed after ID vaccination.

The overall frequency of dermal DC was not significantly changed after ID vaccination, suggesting either very limited emigration of dDC to LN or, more likely, migration of a discrete fraction of dDC to LN compensated by newly recruited DC. In this respect, analysis of the dynamics of skin myeloid cell subsets after ID PRV-gp vaccination revealed a massive recruitment of myeloid cells expressing CD11R3 (the pig equivalent of CD1 lb), suggesting that the relevant chemokines that were detected at the gene level were produced locally at functionally relevant quantities. Based on CD 16 expression levels analyzed by flow cytometry and compatible with histological detection of skin infiltrates, we identified that this myeloid cell infiltrate comprised both neutrophils (CD16^int) and monocytes (CD16^hlgh), which were maximal at 24h after ID injection of PRV-gp. We and others have shown earlier in mice that local recruitment of these cells occurs in response to skin or mucosal immunization with potent immunogens (Le Borgne et al, Abadie et al, Duffy et al.) or infection (Leon et al). The present data is to our knowledge the first demonstration in a model close to humans that monocytes and neutrophils are rapidly recruited to the ID immunization site and may contribute to the efficiency of ID vaccination. Through their capacity to produce various effector molecules (Mantovani et al), neutrophils may induce local tissue inflammation and attract and/or activate monocytes and DC (Mantovani et al, Soehnlein et al), which both play a critical role for in vivo priming of cellular and humoral immunity (Klechevsky et al., Le Borgne et al, Kool et al). They may also capture and transport the Ag from skin to draining LN (Abadie et al.) and bone-marrow (Duffy et al), where they may directly present the Ag to T cells (Beauvillain et al.) or modulate Ag-bearing DC (Yang et al).

Remarkably, we demonstrate recruitment of monocytes followed by monocyte-derived inflammatory DC at the site of ID delivery of the vaccine. The majority of recruited monocytes were CD163⁺ and CD14^low and differ from less mature CD 163^" monocytes by expression of some MHC class-II molecules, production of higher levels of TNFa (Chamorro et al, 2005) and more efficient differentiation into immune-stimulatory DC (Chamorro et al, 2004), reminiscent of the human CD16⁺ subset (Randolph et al), that contribute to local innate surveillance of tissues (Geissman et al, Cros et al). That these recruited monocytes may give rise to DC is supported by appearance of a cell population typified by expressing SLAII, CD80/CD86 costimulatory molecules and intermediate level of CD1, which later accumulated in the dermis. This subset co-expresses CD163, a marker of inflammatory DC, barely represented in normal dermis (Marquet et al). These inflammatory DC are reminiscent to human CD14⁺ dermal DC, specialized in induction of T helper cells necessary for plasma cell differentiation (Klechevsky et al), suggesting that they may be instrumental for inducing IgG and IgA protective anti-viral responses after ID vaccination.

Altogether, our data, obtained in a pig model highly relevant to the human situation, demonstrate that protection against viral respiratory infection can be achieved by ID immunization with adjuvant-free viral glycoproteins and is tightly linked to an early skin innate response manifested by up-regulation of key cytokines and chemokines and recruitment of neutrophils, monocytes and inflammatory DC at the vaccination site. This molecular and cellular skin signature might serve for the design of efficient subunit ID vaccines.

FIGURE LEGENDS

Figure 1 : Cellular and humoral immune responses induced by ID immunization with PRV-gp.

Groups of 6 pigs were injected ID at day 0 with 10, 30 or 100μg of PRV-gp in the flank. Pigs injected ID with PBS or injected IM in the neck with the oil-adjuvanted inactivated

Geskypur^® vaccine, as negative and positive controls, respectively. (A-C) PRV-specific IgM (A), IgG (B) and IgA (C) serum Ab titers were determined before (day-1) and after immunization (day 13 and 21). The data are presented as mean ¹⁰log titers ± SEM (n=6). (D) Virus neutralizing Ab titers in serum at day 21 after immunization. Each point represents an individual pig and horizontal bars indicate the arithmetic mean. (E, F) The frequency of PRV- specific IFNy producing cells (E) and IL-2 producing cells (F) in blood was analyzed at day 13 after vaccination by an ELISPOT assay after in vitro re-stimulation of PBMC with the NIA-3 PRV strain. Results are expressed as the number of SFC/10⁶ PBMC, each point corresponding to an individual pig and horizontal bars representing the arithmetic mean. *p<0.05, **p<0.01 by Mann Whitney test.

Figure 2: ID immunization with PRV-gp confers protection from a lethal viral challenge.

The pigs (n=6 per group) depicted in Figure 1 were challenged at day 21 after immunization by intra-nasal instillation of a lethal dose of the virulent NIA3 PRV strain, and protection was assessed by survival, body weight daily gain and viral excretion. (A) Mean relative daily weight gain during the week after challenge. (B) Viral titers in nasal secretions at day 26. *p<0.05, **p<0.01 and ***p<0.001 versus the PBS control group by one-way ANOVA with Dunnett's multiple comparison test. (C, D) PRV-specific IgA titers in nasal secretions at day 21, e.g. before challenge (C), and at day 26 (D). (E) Correlates of protection. Seric IgG titers at day 21, frequency of IFNy-SFC in blood at day 13, and nasal IgA titers before (day 21) and after (day 26) were compared between pigs that gained versus lost weight after viral challenge, irrespective of their experimental group. Each symbol corresponds to an individual pig, horizontal bars correspond to the arithmetic mean ± SEM, and white and black circles (A, B) indicate whether pigs survived or not to the viral challenge, respectively. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by Mann Whitney test.

Figure 3 : Ag distribution and fate after ID immunization.

PRV-gB staining (red) on frozen sections of skin injection sites (A) and draining lymph nodes (B) at 2 and 24h after ID injection of PRV-gp. Tissues were counterstained with hematoxylin (blue). For each skin section, a higher magnification of the area depicted by a square is presented in the right panel. PBS-injected skin sections, presented as controls revealed some nonspecific background staining of the dermo-epidermal junction and of several structures in the hypodermis like the membrane of adipocytes, sweet glands and pilo-erector muscles.

Figure 4: Cytokines and chemokines induced by ID immunization with viral Ag.

Pig flank skin was ID injected with either PBS or 3C^g PRV-gp and skin biopsies were harvested at various times for analysis of mRNA levels of a set of genes by real time RT- PCR. (A) Cluster analysis of the expression profile of a selected set of genes encoding inflammatory cytokines and chemokines at 2 and 24 hours after injection of PBS or PRV-gp. Each line corresponds to one skin biopsy (4 per group) and mRNA expression levels are represented as fold change compared with non- injected skin. (B) Expression profile of ILl , IL6, IL13, CCL2, CCL3, CCL8, CCL20 and CXCL8 in skin at 2, 24 and 72 hours after ID injection of PBS (white histograms) or PRV-gp (black histograms). Results are expressed as the mean mRNA fold change ± SEM of 6 skin biopsies compared with non- injected skin. *p<0.05, **p<0.01 between PBS-and PRV-gp-injected skin by the Mann Whitney test.

Figure 5: Skin gene signature of ID-induced mechanical stress.

Skin biopsies were harvested at 0.5, 2, 4 and 24h after ID sham injection (e.g. prick without liquid injection, white circles), ID injection of 100 μΐ of PBS (black squares) or ID injection of 5μg PHA-L in 100 μΐ of PBS (black triangles), and the level of mRNA coding for ILl (A), IL6 (B), CCL2 (C), CCL3 (D), CCL8 (E) and CXCL8 (F) was determined by real time RT-PCR. Results are expressed as mRNA fold change compared with non-injected skin and correspond to the mean ± SEM of 5 skin biopsies collected from different pigs.

Figure 6: Immunohistochemical analysis of skin dendritic cells dynamics after ID

immunization with PRV-gp.

Pigs were ID injected in the flank skin with either PBS or 30 μg PRV-gp (100 μΐ) and skin biopsies of injected sites were collected 2 and 24h later to prepare tissue sections. (A) HPS staining of paraffin-embedded skin sections at 24h after ID injection. A representative low magnification image is shown for each group (left panels), as a well as a high power field (right panels) of a perivascular area showing infiltrating cells and extravasated red blood cells after PRV-gp injection. (B) Representative CD207 staining of epidermal sheets from un- injected, PBS- or PRV-gp-injected skin (24h), and quantitative analysis of LC showing the number of CD207⁺ cells per mm² of epidermis at 2 or 24h after ID injections with PBS or PRV-gp, in comparison to un-injected control skin. Each point represents an individual value and horizontal bars correspond to the mean (±SEM) counts of 4 fields from 2-3 epidermal sheets out of 3 pigs/group. Statistical significance was determined using an unpaired t test. (C) IHS stain of skin frozen sections for CD207, CD l, SLAII and CD 172a (red) at 24h after ID injections. Tissues were counterstained with hematoxylin (blue).

Figure 7: ID immunization with PRV-gp induce recruitment of neutrophils and

monocytes/DC in the dermis.

Pigs were ID injected in the flank skin with either PBS or 30 μg PRV-gp (100 μΐ) and skin biopsies of injected sites were collected 24 and 72h later to prepare dermal cell suspensions for flow cytometry analysis. (A, C) Representative FACS dot plots depicting CDl 1R3 versus CD 16 expression (A) and SLAII or CD80/86 versus CDl expression (C) on gated CD172a⁺ dermal myeloid cells at 24 hours (A) or 72 hours (C) after ID injection of either PBS or PRV- gp. White histograms illustrate CD 14, CD 163, 2B2 and SWC8 expression by

CD l lRS^Die"¹' cells and CDl lR3⁺CD16^high cells (A), and CD14 and CD163 expression on CDl^highSLAII⁺ and CD l^intSLAII⁺ cells (C) from PRV-gp injected skin. Grey histograms correspond to isotype-matched controls. (B, D) Quantitative analysis of CDl lR3⁺CD16^int granulocytes and CDl lR3⁺CD16^high monocytes (B) and of CDl^highSLAII⁺ dendritic cells and CDl^intSLAII⁺ cells (D) infiltrating the dermis at 24 and 72 hours after ID injection of PBS (hatched histograms) or PRV-gp (black histograms), comparatively to non-injected skin (white histograms). Data are mean % ± SEM among CD 172a dermal cells of 4 pigs per group. *p<0.05 by Mann Whitney test.

Figure 8: gB- and gC-specific cellular immune responses induced by ID immunization with PRV-gp. The frequency of PRV-specific IFNy producing cells (A, C-D) and IL-2 producing cells (B) in the blood of pigs depicted in Figure 1 was analyzed at day 13 after immunization by an ELISPOT assay after in vitro re-stimulation of PBMC with 5-9 pools of overlapping peptides covering the entire PRV gB (A, C) and gC glycoproteins (B, D). (A, B) Total number of IFNy SFC specific of gB or gC obtained by adding the number of SFC obtained for each individual pool of peptides. Each point corresponds to an individual pig and horizontal bars represent the arithmetic mean. (C, D) Analysis of the diversity of epitopes recognized by IFNy producing T cells in 3 pigs responding to ID immunization with 30 μg PRV-gp (white histograms) versus IM immunization with Geskypur® (black histograms). Results are expressed for each pig as the proportion of T cells specific for each pool of peptides. *p<0.05, **p<0.01 by Mann Whitney test.

Figure 9: Correlates of protection of the experiment depicted in figure 2. Seric IgG titers at day 21, frequency of IFNy-SFC in blood at day 13, and nasal IgA titers before (day 21) and after (day 26) were compared between pigs that survived or died after the viral nasal challenge, irrespective of their experimental group. Each symbol represents an individual pig and horizontal bars correspond to the arithmetic mean ± SEM. **p<0.01 by Mann Whitney test.

Table 1 : Seq ID numbers of primers used for real time RT-PCR

8 DNA IL13 reverse primer

9 DNA TNFa forward primer

10 DNA TNFa reverse primer

11 DNA GM-CSF forward primer

12 DNA GM-CSF reverse primer

13 DNA CCL2 forward primer

14 DNA CCL2 reverse primer

15 DNA CCL3 forward primer

16 DNA CCL3 reverse primer

17 DNA CCL8 forward primer

18 DNA CCL8 reverse primer

19 DNA CCL20 forward primer

20 DNA CCL20 reverse primer

21 DNA CXCL8 forward primer

22 DNA CXCL8 reverse primer

23 DNA CX3CL1 forward primer

24 DNA CX3CL1 reverse primer

25 DNA CXCL12 forward primer

26 DNA CXCL12 reverse primer

27 DNA Actin b forward primer

28 DNA Actin b reverse primer

29 DNA HPRT forward primer

30 DNA HPRT reverse primer

31 DNA Tubb4 forward primer

32 DNA Tubb4 reverse primer

Table 2. Primer sequences for RT-PCR.

Gene symbol Forward primer Reverse primer

ILla 5' -AACCCGACTGTTTGTGAGTGCTC-3 ' 5'-ACTTTGGATGGGCGGCTGATTTG-3'

ILlb 5' -TGGTGTTCTGC ATGAGCTTTGTG-3 ' 5'-AGGGTGGGCGTGTCATCTTTC-3'

IL6 5'-CAGCAAGGAGGTACTGGCAGAAAAC-3' 5'-TTGAACCCAGATTGGAAGCATCCG-3'

IL13 5' -GTCATTGCTCTC ACCTGCTTTGG-3 ' 5'-CACACCATGCTGCCGTTGC-3' TNFa 5'-TGAGCACTGAGAGCATGATCCG-3' 5'-GGGGCCGATAACCTCGAAGTG-3'

GM-CSF 5' -AAGCCCTGAGCCTTCTAAACAACAG-3 ' 5'-GGCGAGTCTGCACGCATGTC-3'

CCL2 5' -AGTCACCTGCTGCTATACACTTACC-3 ' 5'-ATCACTGCTTCTTTAGGACACTTGC-3'

CCL3 5'-GTAGCCGACTATTTTGAGACCAGCAG-3' 5'-AGGCATCCTCGGGGTTGGC-3'

CCL8 5' -GAGACCCACTTAGAAATCACC AACG-3 ' 5'-GCAGCAGGTGATCGGGATGG-3'

CCL20 5' -ACAGACCATATTCTTCACCCCAGATTTATC-3 ' 5'-TCCAATTCCATAGCAGGGAGCATC-3'

CXCL8 5'-AAATACGCATTCCACACCTTTCCAC-3' 5'-GCAGACCTCTTTTCCATTGACAAGC-3'

CX3CL1 5'-GCCTTCCTTGGTCTCCTTTTCTTCC-3' 5'-CCCTTCTGCCATGTCCCCTGAC-3'

CXCL12 5'-GCCGCCTACTCCTGCCATG-3' 5'-ATCGGCAAGGGCATCTGTAGC-3'

Actinb 5'-CTGGCACCACACCTTCTACAACG-3' 5'-CGGTTGGCTTTGGGGTTCAGG-3'

HPRT 5'-AAGGACCCCTCGAAGTGTTGGC-3' 5'-GTCAAGGGCATAGCCTACCACAAAC-3'

Tubb4 5' -TGAGGAAGGAGGCGGAGAGC-3 ' 5'-GAGGGCACCACGCTGAAGG-3'

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Annex to the Disclosure

SEQUENCE LISTING

<110> erial

Andreoni, Christine

<120> Skin Innate Response Linked Intradermal Vaccination Against

Respiratory Infection

<130> MER 13-224

<160> 32

<170> Patentln version 3.5

<210> 1

<211> 23

<212> DNA

<213> ILla forward primer

<400> 1

aacccgactg tttgtgagtg etc 23

<210> 2

<211> 23

<212> DNA

<213> ILla reverse primer

<400> 2

actttggatg ggcggctgat ttg 23

<210> 3

<211> 23

<212> DNA

<213> ILlb forward primer

<400> 3

tggtgttctg catgagcttt gtg 23

<210> 4

<211> 21

<212> DNA

<213> ILlb reverse primer

<400> 4

agggtgggcg tgtcatcttt c 21

<210> 5

<211> 25

<212> DNA

<213> IL6 forward primer

<400> 5

cagcaaggag gtactggcag aaaac <210> 6

<211> 24

<212> DNA

<213> IL6 reverse primer

<400> 6

ttgaaoccag attggaagca tccg

<210> 7

<211> 23

<212> DNA

<213> IL13 forward primer

<400> 7

gtcattgctc tcacctgctt tgg

<210> 8

<211> 19

<212> DNA

<213> IL13 reverse primer

<400> 3

cacaccatgc tgccgttgc.

<210> 9

<211> 22

<212> DNA

<213> TNFa forward primer

<400> 9

tgagcactga gagcatgatc eg 22

<210> 10

<211> 21

<212> DNA

<213> TNFa reverse primer

<400> 10

ggggecgata acctcgaagt g 21

<210> 11

<211> 25

<212> DNA

<213> GM-CSF forward primer

<400> 11

aagecctgag ccttctaaac aacag 25

<210> 12

<211> 20

<212> DNA <213> G -CSF reverse primer

<400> 12

ggcgagtctg cacgcatgtc 20

<210> 13

<211> 25

<212> DNA

<213> CCL2 forward primer

<400> 13

agtcacctgc tgctatacac ttacc

<210> 14

<211> 25

<212> DNA

<213> CCL2 reverse primer

<400> 14

atcactgctt ctttaggaca cttgc

<210> 15

<211> 26

<212> DNA

<213> CCL3 forward primer

<400> 15

gtagccgact attttgagac cagcag 26

<210> 16

<211> 19

<212> DNA

<213> CCL3 reverse primer

<400> 16

aggcatcctc ggggttggc 19

<210> 17

<211> 25

<212> DNA

<213> CCL8 forward primer

<400> 17

gagacccact tagaaatcac caacg 25

<210> 18

<211> 20

<212> DNA

<213> CCL8 reverse primer

<400> 18

gcagcaggtg atogggatgg 20 <210> 19

<211> 30

<212> DNA

<213> CCL20 forward primer <400> 19

acagaccata ttcttcaccc cagatttatc

<210> 20

<211> 24

<212> DNA

<213> CCL20 reverse primer <400> 20

tccaattcca tagcagggag catc

<210> 21

<211> 25

<212> DNA

<213> CXCL8 forward primer <400> 21

aaatacgcat tccacac.c.tt tccac

<210> 22

<211> 25

<212> DNA

<213> CXCL8 reverse primer <400> 22

gcagacctct tttccattga caagc

<210> 23

<211> 25

<212> DNA

<213> CX3CL1 forward primer <400> 23

gccttccttg gtctcctttt c.ttcc

<210> 24

<211> 22

<212> DNA

<213> CX3CL1 reverse primer <400> 24

cccttctgcc atgtcccctg ac

<210> 25

<211> 19

<212> DNA <213> CXCL12 forward primer

<400> 25

googcctact cctgccatg 19

<210> 26

<211> 21

<212> PNA

<213> CXCL12 reverse primer

<400> 26

atcggcaagg gcatctgtag c 21

<210> 27

<211> 23

<212> DNA

<213> Actin b forward primer

<400> 27

ctggcaccac accttctaca acg 23

<210> 28

<211> 21

<212> DNA

<213> Actin b reverse primer

<400> 23

cggttggctt tggggttcag g 21

<210> 29

<211> 22

<212> PNA

<213> HPRT forward primer

<400> 29

aaggacccct cgaagtgttg gc 22

<210> 30

<211> 25

<212> PNA

<213> HPRT reverse primer

<400> 30

gtcaagggca tagcctacca caaac 25

<210> 31

<211> 20

<212> PNA

<213> Tubb4 forward primer

<400> 31

tgaggaagga ggcggagagc 20 <210> 32

<211> 19

<212> DNA

<213> Tubb4 reverse primer

<400> 32

gagggcacca cgctgaagg 19

Claims

1. A method of inducing an immune response to pseudorabies virus in swine comprising the step of administering non-adjuvanted pseudorabies virus glycoprotein subunits by intradermal administration.

2. The method of claim 1, wherein the step of administering non-adjuvanted pseudorabies virus glycoprotein subunits comprises administration with a single microneedle injector.

3. The method of claim 1, wherein the step of administering non-adjuvanted pseudorabies virus glycoprotein subunits comprises administration with a microneedle patch having two or more needles.

4. The method of claim 1, wherein the step of administering non-adjuvanted pseudorabies virus glycoprotein subunits comprises administration with a needle free injection system.

5. The method of claim 1, wherein the step of administering non-adjuvanted pseudorabies virus glycoprotein subunits comprises administration of pseudorabies virus glycoprotein subunit IgM.

6. The method of claim 1, wherein the step of administering non-adjuvanted pseudorabies virus glycoprotein subunits comprises administration of pseudorabies virus glycoprotein subunit IgG.

7. The method of claim 1, wherein the step of administering non-adjuvanted pseudorabies virus glycoprotein subunits comprises administration of pseudorabies virus glycoprotein subunit IgA.

8. The method of claim 1, wherein the step of administering non-adjuvanted pseudorabies virus glycoprotein subunits by intradermal administration comprises glycoprotein subunits of pseudorabies virus strain NIA-3.

9. An adjuvant free immunogenic composition against pseudorabies virus, the etiological agent of Aujeszky's disease, comprising pseudorabies virus IgM glycoprotein.

10. An adjuvant free immunogenic composition against pseudorabies virus, the etiological agent of Aujeszky's disease, comprising pseudorabies virus IgG glycoprotein.

11. An adjuvant free immunogenic composition against pseudorabies virus, the etiological agent of Aujeszky's disease, comprising pseudorabies virus IgA glycoprotein.