Vaccine 21 (2003) 776–780
`
`Immunity under the skin: potential application for
`topical delivery of vaccines
`, A.-S. Beignon a, F. Mawas b, G. Belliard a, J.-P. Briand a, S. Muller a
`a UPR 9021, CNRS, Immunologie et Chimie Thérapeutiques, Institut de Biologie Moléculaire et Cellulaire,
`15 rue René Descartes, Strasbourg F-67084, France
`b Bacteriology Division, National Institute for Biological, Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, UK
`
`C.D. Partidos a,∗
`
`Abstract
`
`With the technological advances in biomedical sciences and the better understanding of how the immune system works, new immunisation
`strategies and vaccine delivery options, such sprays, patches, and edible formulations have been developed. This has opened up the possibility
`of administering vaccines without the use of needles and syringes. Already topical immunisation is a reality and it has the potential to make
`vaccine delivery more equitable, safer, and efficient. Furthermore, it would increase the rate of vaccine compliance and greatly facilitate
`the successful implementation of worldwide mass vaccination campaigns against infectious diseases. This review gives a brief account of
`the latest developments of application of candidate vaccine antigens onto bare skin and describes some of our recent observations using
`peptide and glycoconjugate vaccines as immunogens.
`© 2002 Elsevier Science Ltd. All rights reserved.
`
`Keywords Skin immunisation; CTL responses; Haemophilus influenzae type b
`
`1. Advantages of skin delivery of vaccines
`
`2. The immune barrier of the skin
`
`Application of antigens onto bare skin is a simple immu-
`nisation procedure that promises to revolutionise the way
`vaccines would be administered in the future. There are sev-
`eral advantages that make this approach of immunisation at-
`tractive: (1) it increases compliance due to the elimination
`of multiple dosing schedules and usage of needles and sy-
`ringes. The practical importance of compliance is apparent.
`If people do not comply, the prophylactic goal of vaccina-
`tion would not be achieved and the cost for emergency care
`required would become enormous; (2) the fact that no nee-
`dles and syringes are needed would make the immunisation
`practice painless. This is particularly relevant for children
`that normally associate the site of a needle with pain; (3) vac-
`cine delivery is safe, since reuse of needles and syringes that
`is a common practice in developing countries has the risk
`of transmission of bloodborn infections [1]; (4) vaccine ad-
`ministration would become practical and simple, since they
`can be self-administered with a patch without requiring any
`medical personnel and finally; and (5) topical immunisation
`elicits both systemic and mucosal immunity. The latter is of
`great importance, since the majority of pathogens enter the
`host via the mucosal surfaces.
`
`∗
`
`Corresponding author. Tel.: +33-3-88-417028; fax: +33-3-88-610680.
`E-mail address h.partidos@ibmc.u-strasbg.fr (C.D. Partidos).
`
`The skin is the principal interface with the external envi-
`ronment, keeping water and nutrients in, and unwanted toxic
`substances and pathogens out. It also acts as an immune
`barrier, protecting the host from invading pathogens. For
`this purpose, the skin is equipped with immunocompetent
`cells, such as keratinocytes, Langerhans cells (LCs), sub-
`sets of T lymphocytes and strategically located lymph nodes
`that constitute the skin-associated lymphoid tissue (SALT)
`[2]. Keratinocytes, apart of being responsible for establish-
`ing the physical barrier of the skin and guaranteeing the
`structural integrity of the epidermis, produce a wide range
`of cytokines upon activation by various stimuli [3]. These
`cytokines shape the local microenvironment to help main-
`tain the appropriate balance of skin immune responses, and
`stimulate the maturation and migration of LCs.
`The LCs are powerful antigen-presenting cells that cover
`nearly 20% of the surface area through their horizontal ori-
`entation and long protrusions. They are located at the basal
`layer of the epidermis as immature cells playing a sentinel
`role in the epidermis. LCs capture and process antigens and
`during their migration via the efferent lymphatics to the
`paracortical T cell areas of the draining lymph nodes, they
`mature and present antigenic peptides to na¨ıve T cells [4].
`At this stage, they express co-stimulatory molecules of the
`B7 family, they upregulate the surface expression of MHC
`
`0264-410X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
`PII: S 0 2 6 4 - 4 1 0 X ( 0 2 ) 0 0 5 9 7 - 2
`
`Page 1 of 5
`
`YEDA EXHIBIT NO. 2041
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`C.D. Partidos et al. / Vaccine 21 (2003) 776–780
`
`777
`
`class I and class II molecules bound to peptides, and secrete
`high levels of proinflammatory cytokines, such as IL-12 and
`IL-1.
`
`3. Immunogenicity of antigens applied onto
`bare skin
`
`The skin represents a readily accessible surface area for
`absorption (2 m2 in adult humans). This offers a distinct
`advantage of exploiting its immune system for delivering
`vaccines. Antigens applied onto bare skin penetrate across
`the continuous stratum corneum mainly via the intracellular
`or intercellular routes [5]. However, appendages including
`hair follicles, sebaceous or sweat glands can also serve as
`portal of antigen entry.
`For a long time it was thought that the skin barrier was
`impermeable to large molecules, but studies by Glenn et al.
`[6] have demonstrated that topical application of cholera
`toxin (CT) (secreted by Vibrio cholerae) onto hydrated skin
`can induce strong systemic and mucosal immune responses
`and confer protection against lethal mucosal toxin chal-
`lenge [7]. A similar effect was demonstrated with another
`ADP-ribosylating exotoxin, the heat-labile enterotoxin (LT)
`of Escherichia coli [8] or its mutants LTK63 and LTR72 [9].
`Also preliminary results from a phase I trial conducted in hu-
`man volunteers [10] have shown that topical application of
`LT with a patch: (i) induces only minimal local or systemic
`
`Table 1
`Type of antigens that can be applied onto bare skin
`
`Viruses: HSV, adenovirus, inactivated rabies virus, recombinant
`Mengo virus
`Parasites: Dirofilaria immitis
`Plasmid DNA: HBsAg, influenza Ags
`Bacterial toxins (CT, LT), toxoids (TTx, DTx)
`Proteins: BSA, ␤-galactosidase
`Peptides: Th, B, CTL epitopes
`
`adverse reactions; (ii) elicits anti-LT IgG responses that were
`boosted after the second and third topical application of LT;
`and (iii) induce long lasting immunity and detectable anti-LT
`IgG or IgA antibodies in urine and stools.
`Parallel to their immunogenic potential, CT and LT act
`as adjuvants, enhancing immune responses to topically
`co-applied antigens, including toxoids, proteins, peptides
`and viruses [7–14]. It is the combination of their binding
`activity and built-in adjuvanticity that make CT and LT
`powerful immunogens and adjuvants. Several other adju-
`vants,
`including CpG motifs,
`lipopolysaccharide (LPS),
`muramyl dipeptide (MDP), alum, IL-2, and IL-12 have been
`shown to enhance the antibody titres to topically co-applied
`antigens [13]. However, the responses were short lived and
`weaker to those induced in the presence of CT or LT [13].
`Following these initial observations, the potential of the
`skin as a non-invasive route for vaccine delivery has been
`demonstrated with several types of antigens (Table 1).
`
`Fig. 1. Peptide- and virus-specific IFN-␥ secreting T cells. Groups of BALB/c mice (two mice per group) were immunised onto bare skin with 25 ␮g
`empty flagella (Fla) or 25 ␮g flagella expressing the influenza nucleoprotein CTL epitope (Fla/CTL) with 25 ␮g of either LT or its mutants LTK63 or
`LTR72. A total volume of 50 ␮l was applied onto bare skin. Mice were boosted 2 weeks later by the same route and dose of antigen. Four weeks after
`the priming, splenocytes were collected and IFN-␥ secreting T cells were measured by a standard ELISPOT assay. Results represent the mean of triplicate
`cultures. Cells were restimulated in vitro with 10 ␮g peptide or 3 × 103 pfu of heat-inactivated influenza virus (strain A/NT 60/68; H3N2)/culture. M:
`medium, P: test peptide, C: control peptide, V: virus.
`
`Page 2 of 5
`
`YEDA EXHIBIT NO. 2041
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`778
`
`C.D. Partidos et al. / Vaccine 21 (2003) 776–780
`
`4. Induction of CTL responses following
`immunisation onto bare skin
`+
`CD8
`cytotoxic T lymphocytes (CTL) play a critical role
`in eliminating virus-infected cells. CTL responses can be
`elicited after systemic or mucosal immunisation with pep-
`tide epitopes administered with various delivery systems.
`Flagella is a bacterial component that has been extensively
`tested as a carrier protein to cloned epitope sequences that
`are expressed at the surface of flagellin, the flagellar ma-
`jor subunit [15]. This has prompted us to study the effi-
`cacy of a flagella fusion protein expressing a conserved cy-
`totoxic T cell epitope from influenza virus nucleoprotein
`representing residues 147–158 (TYQRTRALVRTG) [15] to
`elicit peptide- and virus-specific CTL responses after appli-
`cation onto bare skin. To potentiate immune responses, LT
`or its LTK63 and LTR72 mutants were used as adjuvants.
`Two weeks following the booster application, splenocytes
`were tested for peptide- and virus-specific IFN-␥ secreting T
`cells. As shown in Fig. 1, the flagella fusion in the presence
`of mutant LTR72 elicited higher numbers of peptide- and
`virus-specific IFN-␥ secreting T cells. However, when the
`flagella fusion was applied topically in the presence of LT
`or LTK63, the number of peptide- and virus-specific IFN-␥
`secreting T cells was very low and not higher to the number
`
`produced by the flagella fusion given alone. These findings
`are in a good agreement with a recent report demonstrating
`the preferential stimulation of IFN-␥ by LTR72 mutant after
`immunisation onto bare skin [9].
`
`5. Topical application of a H aemophilus inf luenzae
`type b conjugate vaccine with CT elicits protective
`immunity
`
`Haemophilus influenzae type b (Hib) conjugate vac-
`cines have successfully reduced the burden of invasive
`Hib disease in developed countries and there is clearly
`a case for implementation of these vaccines worldwide.
`Since topical delivery of vaccines simplify vaccine admin-
`istration and has the potential to promote compliance and
`cost-effectiveness, we were interested in evaluating the
`immunogenicity and efficacy of a Hib conjugate vaccine
`following skin immunisation. Two applications of 50 ␮g of
`poly-ribosyl-ribitol phosphate (PRP) oligosaccharide con-
`jugated to the non-toxic, cross-reacting mutant protein of
`diphtheria toxin (CRM197) (PRP-CRM197) onto bare skin of
`rats with or without CT (50 ␮g/rat) as an adjuvant elicited
`substantial levels of both IgG anti-PRP and anti-CRM197
`antibodies as measured by ELISA [16] (Fig. 2). However,
`
`Fig. 2. Immunogenicity of Haemophilus influenzae type b conjugate vaccine following skin immunisation. Rats (Sprague Dawely, three per group) were
`immunised with 50 ␮g of PRP-CRM with or without CT (50 ␮g/rat) on days 0 and 21 and bled 3 weeks after the boost. Each bar represents antibody
`responses from individual rats.
`
`Page 3 of 5
`
`YEDA EXHIBIT NO. 2041
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`C.D. Partidos et al. / Vaccine 21 (2003) 776–780
`
`779
`
`Table 2
`Passive protection of infant rats from Haemophilus influenzae type b
`bacteraemia by anti-PRP antibodies induced after immunisation onto bare
`skin
`
`Immunogen
`
`PRP-CRM
`PRP-CRM + CT
`Normal rat serum
`
`Number of rats bacteraemic at 24 h/number
`of rats challenged
`1/4
`0/3
`4/4
`
`Immune sera from rats immunised with two doses of PRP-CRM or
`PRP-CRM + CT were injected i.p. (50 ␮l/rat) into 5-day-old infant rats
`(Sprague Dawely) 24 h before challenge (i.p.) with 104 Haemophilus
`influenzae type b. Rats were bled 24 h after challenge and blood was
`cultured on chocolate agar for the presence of bacteraemia.
`
`responses against both the polysaccharide (PRP) and the
`carrier protein (CRM197) were higher in the group of rats
`receiving CT as an adjuvant. Moreover, adoptive transfer of
`immune sera from both groups protected infant rats from
`Hib-induced bacteraemia (Table 2).
`
`6. Perspectives for skin delivery of vaccines
`
`The realisation that the skin is easily accessible has an ef-
`fective immune system, and its physical barrier is not so im-
`permeable as previously thought makes it an attractive route
`for non-invasive delivery of vaccines. Studies in several an-
`imal species and clinical trials in humans have established
`the proof of principle. However, for effective vaccine deliv-
`ery several variables related to the nature of the antigen and
`characteristics of the skin barrier must be first overcome.
`For example, the diffusion of an antigen through the stra-
`tum corneum is dependent on its physicochemical properties
`and its molecular interactions with skin constituents. This
`could explain the differences in immunogenicity of several
`antigens after their application onto bare skin. For example,
`in a recent study where the safety and immunogenicity of
`a prototype enterotoxigenic E. coli vaccine was assessed in
`adult volunteers after topical application, only 68 and 53%
`were found to have serum anti-colonising factor CS6 IgG
`and IgA antibodies, respectively, while all responded to LT
`[17].
`Furthermore, the skin of humans and animals poses a
`unique barrier due to differences in anatomy and phys-
`iology between species [18]. Therefore,
`these variables
`make the task of extrapolating the findings of studies per-
`formed in different animal species to the target species
`difficult.
`Current research efforts are focused: (i) in the selection
`of suitable delivery systems that will allow efficient pen-
`etration across the stratum corneum and selective uptake
`of antigens by LCs; (ii) in understanding the mechanisms
`involved in the induction of systemic and mucosal im-
`mune responses after topical immunisation; and (iii) to
`identify approaches to enhance and modulate immune re-
`sponses.
`
`Acknowledgements
`
`The authors would like to thank Prof. Ruth Arnon (Weiz-
`mann Institute of Science, Rehovot Israel) and Dr. Rino Rap-
`puoli (IRIS Research Center, Chiron S.P.A, Siena, Italy) for
`kindly providing the recombinant flagella construct and the
`LT mutants, respectively.
`
`References
`
`[1] Kane A, Lloyd J, Zaffran M, Simonsen L, Kane M. Transmission of
`hepatitis B, hepatitis C and human immunodeficiency viruses through
`unsafe injections in the developing world: model-based regional
`estimates. Bull World Health Organ 1999;77:801–7.
`[2] Streilein JW. Skin-associated lymphoid tissues (SALT): origins and
`functions. J Invest Dermatol 1983;80:S12–6.
`[3] Uchi H, Terao H, Koga T, Furue M. Cytokines and chemokines in
`the epidermis. J Dermatol Sci 2000;24:S29–38.
`[4] Debenedictis C, Joubeh S, Zhang G, Barria M, Ghohestani RF.
`Immune functions of the skin. Clin Dermatol 2001;19:573–85.
`[5] Barry BW. Novel mechanisms and devices to enable successful trans-
`dermal drug delivery. Eur J Pharm Sci 2001;14:101–14.
`[6] Glenn GM, Rao M, Matyas GR, Alving CR. Skin immunization
`made possible by cholera toxin. Nature 1998;391:851.
`[7] Glenn GM, Scharton-Kersten T, Vassell R, Mallett CP, Hale
`TL, Alving CR. Transcutaneous immunization with cholera toxin
`protects mice against lethal mucosal toxin challenge. J Immunol
`1998;161:3211–4.
`[8] Beignon A-S, Briand J-P, Muller S, Partidos CD. Immunization onto
`bare skin with heat-labile enterotoxin of Escherichia coli enhances
`immune responses to co-administered protein and peptide antigens
`and protects mice against
`lethal
`toxin challenge.
`Immunology
`2001;102:344–51.
`[9] Beignon A-S, Briand J-P, Rappuoli R, Muller S, Partidos CD.
`The LTR72 mutant of heat-labile enterotoxin of Escherichia coli
`+
`enhances the ability of peptide antigens to elicit CD4
`T cells and
`secrete IFN-␥ after co-application onto bare skin. Infect Immun
`2002;70:3012–9.
`[10] Glenn GM, Taylor DN, Li X, Frankel S, Montemarano A, Alving
`CR. Transcutaneous immunization: a human vaccine delivery strategy
`using a patch. Nat Med 2000;6:1403–6.
`[11] El-Ghorr AA, Williams RM, Heap C, Norval M. Transcutaneous
`immunisation with herpes simplex virus stimulates immunity in mice.
`FEMS Immunol Med Microbiol 2000;29:255–61.
`[12] Hammond SA, Tsonis C, Sellins K, Rushlow K, Scharton-Kersten T,
`Colditz I, et al. Transcutaneous immunization of domestic animals:
`opportunities and challenges. Adv Drug Deliv Rev 2000;43:45–55.
`[13] Scharton-Kersten T, Yu J, Vassell R, O’Hagan D, Alving CR,
`Glenn GM. Transcutaneous immunization with bacterial ADP-ribo-
`sylating exotoxins, subunits, and unrelated adjuvants. Infect Immun
`2000;68:5306–613.
`[14] Beignon A-S, Briand J-P, Muller S, Partidos CD. Immunization
`onto bare skin with synthetic peptides:
`immunomodulation with
`a CpG-containing oligodeoxynucleotide and effective priming of
`influenza virus-specific CD4+ T cells. Immunology 2002;105: 204–
`12.
`[15] Levi R, Arnon R. Synthetic recombinant influenza vaccine induces
`efficient
`long-term immunity and cross-strain protection. Vaccine
`1996;14:85–92.
`[16] Bolgiano B, Mawas F, Yost SE, Crane DT, Lemercinier X, Corbel MJ.
`Effect of physico-chemical modification on the immunogenicity of
`Haemophilus influenzae type b oligosaccharide-CRM(197) conjugate
`vaccines. Vaccine 2001;19:3189–200.
`
`Page 4 of 5
`
`YEDA EXHIBIT NO. 2041
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`780
`
`C.D. Partidos et al. / Vaccine 21 (2003) 776–780
`
`[17] Guerena-Burgueno F, Hall ER, Taylor DN, Cassels FJ, Scott
`DA, Wolf MK, et al. Safety and immunogenicity of a prototype
`enterotoxigenic Escherichia coli vaccine administered transcutane-
`ously. Infect Immun 2002;70:1874–80.
`
`[18] Wester RC, Maibach HI. Animal models for transdermal delivery.
`In: Kydonieus AF, Berner B, editors. Transdermal delivery of drugs,
`vol. I. Bocca Raton, FL: CRC Press; 1987. p. 61–70.
`
`Page 5 of 5
`
`YEDA EXHIBIT NO. 2041
`MYLAN PHARM. v YEDA
`IPR2015-00644