Fluzone® intra-dermal (Intanza®/Istivac® Intra-dermal): An updated overview

ABSTRACT

Influenza is a highly contagious respiratory acute viral disease which imposes a very heavy burden both in terms of epidemiology and costs, in the developed countries as well as in the developing ones. It represents a serious public health concern and vaccination constitutes an important tool to reduce or at least mitigate its burden. Despite the existence of a broad armamentarium against influenza and despite all the efforts and recommendations of international organisms to broaden immunization, influenza vaccination coverage is still far from being optimal. This, taken together with logistic and technical difficulties that can result into vaccine shortage, makes intra-dermal (ID) vaccines, such as Fluzone® ID and Intanza®, particularly attractive. ID vaccines are comparable and, in some cases, superior to intra-muscular/sub-cutaneous vaccines in terms of immunogenicity, safety, reactogenicity, tolerability and cross-protection profiles, as well as in terms of patient preference, acceptance and vaccine selection. Further advances, such as Fluzone® ID with alternative B strains and Quadrivalent Fluzone® ID or the possibility of self-administering the vaccines, make influenza ID vaccines even more valuable.

Introduction

Influenza

Influenza is a highly contagious respiratory acute viral disease characterized by a worldwide distribution, a short incubation period (1–3 days, generally 2 days), usually mild respiratory and systemic symptoms, such as high fever, cough, sore throat, headache, chills, anorexia and fatigue.^1,2 It can be asymptomatic in 30–50% of subjects.^2,3 On the other hand, it may result into severe complications, such as bacterial pneumonia and exacerbation of underlying chronic conditions (including heart or respiratory failure, chronic obstructive pulmonary diseases or COPDs, among others), hospitalization and deaths, especially in frail and high-risk subjects.²

The burden of seasonal influenza as well as of influenza pandemics is very heavy, both in terms of epidemiology and costs, in the developed countries⁴ as well as in the developing ones.⁵ Indeed, the World Health Organization (WHO) estimates that annual epidemics affect up to 15% of the total population worldwide, causing up to 4–5 million severe cases and up to 500,000 deaths.⁶

For these reasons, influenza represents a serious public health concern and vaccination constitutes an important tool to reduce and mitigate the tremendous socioeconomic burden generated by influenza.

Influenza is caused by a single-stranded, negative-sense RNA virus, Myxovirus influenzae, which belongs to the Orthomyxoviridae family together with Thogotovirus and Isavirus, and includes 3 serotypes or genera, A, B, and C. The genus A, first isolated in 1933,⁷ is the most clinically relevant and has the capacity to induce minor or major changes in its structure (antigenic drifts and antigenic shifts, respectively). In case of antigenic drifts, the virus causes interpandemic influenza (known also as annual epidemics or seasonal influenza). In case of antigenic shifts, it causes pandemic influenza. Pandemic influenza is caused by influenza virus genus A, while seasonal influenza by influenza genus A and genus B.²

Influenza virus has a quite complex replication cycle, achieved through various stages (Fig. 1).¹ First, the virus attaches to the (α-2,3)- or (α-2,6)-linked sialic acid receptors present on the free surface of the cells of the upper respiratory tract or erythrocytes. During this step (termed as virus adsorption), the role of hemagglutinin (HA), a cylinder-shaped, homotrimeric integral type 1 membrane glycoprotein found on the surface of influenza viruses, is crucial. The virus can then enter the cells (internalization) by exploiting different routes (clathrin-mediated endocytosis or CME, caveolae-dependent endocytosis or CDE, clathrin-caveolae-independent endocytosis, or macropinocytosis). CME is the most usual pathway; the virus is internalized into an endosomal compartment, from which it must emerge in order to release its nucleic acid into the cytosol (endosomal trafficking via endosomes/caveosomes/macropinosomes/lysosomes to the perinuclear compartment). During the phase of fusion of the viral envelope with the endosome membrane, after the pH has been reduced (pH-dependent fusion of viral and endosomal/organellar membranes and uncoating), HA plays again a major role. The ribonucleoprotein must then reach the nucleus (nuclear importation) in order to begin the process of translation of its genes and to transcribe and replicate its nucleic acid (transcription and replication). Subsequently, the RNA segments, surrounded by the nucleoproteins, must migrate to the cell membrane (nuclear exportation) in order to enable further molecular processing (namely, protein synthesis, post-translational processing and trafficking, and viral progeny assembly and packaging). Finally, the virus must be freed to invade other cells of the respiratory tract (budding and release). All this is achieved through a complex, finely tuned, highly dynamic, and synchronized action of a vast array of molecular complexes that perform multiple enzymatic and catalytic reactions, whose details are currently known only in part.¹

Figure 1.

The steps of the replication cycle of the influenza virus are the following: 1) virus adsorption; 2) internalization into cellular regions by means of clathrin-mediated endocytosis (CME), caveolae-dependent endocytosis (CDE), clathrin-caveolae-independent endocytosis, and macropinocytosis; 3) endosomal trafficking; 4) pH-dependent fusion of viral and endosomal / organellar membranes; 5) uncoating; 6) nuclear importation; 7) transcription and replication; 8) nuclear exportation; 9) protein synthesis; 10) post-translational processing and trafficking; 11) viral progeny assembly and packaging; 12) budding; and 13) release (modified from references¹ and ⁴ ).

Overview of the market

The pharmacological armamentarium against influenza includes different drugs and vaccines, which has been recently reviewed by Gasparini and collaborators.⁸ Current available drugs include NA inhibitors (NAIs), such as oseltamivir, zanamivir, and peramivir, and adamantane-based M2 protein blockers (amantadine and rimantadine). However, because of the biology of influenza virus and its frequent mutations, these drugs are plagued by the issue of resistance.^9,10

A variety of vaccines exists against influenza.⁸ They can be basically divided into 2 categories: pandemics and seasonal vaccines. Further, they can divided into inactivated influenza vaccines and live, attenuated influenza vaccines (called LAIVs). The first category includes: whole virus vaccine, subunit vaccine made up of purified HA and NA proteins, and split-virion vaccine.

Licensed prepandemic/pandemic vaccines (reviewed in ^11,12) are shown in Table 1. Other investigational vaccines against H7N9 or universal pandemic vaccines are still under clinical experimentation.¹³

Table 1.

Overview of the market with the main available influenza vaccines.

Commercial name	Marketing Authorisation Holder	Characteristics
Pandemic/prepandemic vaccines
AdimFlu-S®	Adimmune Corporation, Taichung, Taiwan	trivalent inactivated split-virion H1N1 pandemic vaccine
Aflunov®	Seqirus (bioCSL, formerly Novartis Vaccines and Diagnostics S.r.l. Siena, Italy)	monovalent inactivated surfacen antigen MF59-adjuvanted H5N1 prepandemic vaccine
Arepanrix®ˆ	GlaxoSmithKline, Rixensart, Belgium	monovalent inactivated split-virion AS03-adjuvanted H1N1 vaccine
Cantgrip®	INCDMI CANTACUZINO – Romania	monovalent inactivated H1N1 vaccine
Celtura®*	Seqirus (bioCSL, formerly Novartis)	monovalent inactivated MF59-adjuvanted H1N1 vaccine
Celvapan®*	Baxter International, Vienna, Austria	monovalent inactivated whole virus H1N1 vaccine
Daronrix®ˆ	GlaxSmithKline Biologicals, Rixensart, Belgium	monovalent whole virus inactivated H5N1 mock-up vaccine
Focetria®ˆ	Seqirus (bioCSL, formerly Novartis)	monovalent inactivated MF59-adjuvanted H1N1 vaccine
Foclivia®	Seqirus (bioCSL, formerly Novartis Vaccines and Diagnostics S.r.l., Siena, Italy)	monovalent inactivated surface antigen MF59-adjuvanted H5N1 mock-up vaccine
Green Flu-S®	Green Cross Corporation, Korea	monovalent inactivated split-virion H1N1 pandemic vaccine
Humenza®ˆ	Sanofi Pasteur, Lyon, France	monovalent inactivated split-virion AF03-adjuvanted H1N1 pandemic vaccine
Pandemrix® / D-Pan H1N1®ˆ	GlaxoSmithKline Biologicals, Rixensart, Belgium	monovalent inactivated split-virion AS03-adjuvanted H1N1 pandemic vaccine
Panenza®	Sanofi Pasteur, Lyon, France	monovalent inactivated split-virion H1N1 pandemic vaccine
Panvax® / Panvax® Junior	CSL Biotherapies, Parkville, Australia	monovalent inactivated split-virion H1N1 vaccine
Prepandrix® / D-Pan H5N1®	GlaxoSmithKline Biologicals, Rixensart, Belgium	monovalent inactivated split-virion AS03-adjuvanted H5N1 prepandemic vaccine
Pumarix®ˆ	GlaxoSmithKline Biologicals, Rixensart, Belgium	monovalent inactivated split-virion AS03-adjuvanted H5N1 pandemic vaccine
Q-Pan H5N1®	GlaxoSmithKline Biologicals, Rixensart, Belgium	monovalent inactivated split-virion AS03-adjuvanted H5N1 vaccine
Vepacel®*	Baxter International, Vienna, Austria	monovalent inactivated whole virus H5N1 vaccine
Seasonal vaccines
Addigrip® / Adiugrip®	Sanofi Pasteur (bioCSL, formerly Novartis Vaccines and Diagnostics S.r.l., Siena, Italy)	inactivated MF59-adjuvanted vaccine
Adjuvanted Influpozzi®	Seqirus (bioCSL, formerly Novartis)	inactivated MF59-adjuvanted split-virion vaccine
Alorbat®	Asta Pharma, Ho Chi Minh City, Vietnam	inactivated whole virus vaccine
AnFlu®	Sinovac Kexing Biological Product Co., Ltd	inactivated vaccine
Agriflu®	Seqirus (bioCSL, formerly Novartis Vaccines and Diagnostics Limited)	inactivated subunit vaccine
Agrippal®	Seqirus (bioCSL, formerly Novartis)	inactivated subunit vaccine
Batrevac®	Abbott srl	inactivated split-virion vaccine
Begrivac®	Chiron, France	inactivated split-virion vaccine
Biaflu ®/ Biaflu zonale®	KEDRION SpA, Italy (formerly Farmabiagini, Italy)	inactivated whole virus vaccine
CAIV-T®	Medimmune LLC, Maryland, USA / AstraZeneca (formerly Aviron, Mountain View, CA, USA)	LAIV, needle-free
Chiroflu®	Seqirus (bioCSL, formerly Novartis)	inactivated subunit vaccine
Dotaricin®	Alentia Biotech S.L., Granada, Spain	inactivated subunit MF59-adjuvanted vaccine
Elvarix®	VEB Sachsesches Serumwerk Dresden	inactivated split-virion vaccine
Evagrip®	Seqirus (bioCSL, formerly Novartis)	inactivated vaccine
Fluad® / Fluad pediatric / Chiromas / Gripguard	Seqirus (bioCSL, formerly Novartis, formerly Chiron Vaccines, Siena, Italy)	inactivated subunit MF59-adjuvanted vaccine
Fluarix®	GlaxoSmithKline Biologicals, Rixensart, Belgium	inactivated split-virion vaccine
Fluarix Tetra® / Influsplit Tetra / α-RIX-Tetra	GlaxoSmithKline Biologicals, Rixensart, Belgium	inactivated split-virion vaccine
Flublok®§	Protein Sciences Corporation, Meriden, CT and Pearl River, NY, USA	inactivated split-virion vaccine, trivalent, recombinant
Flucelvax® / Optaflu®*	Seqirus (bioCSL, formerly Novartis)	inactivated split-virion vaccine
FluINsure	ID Biomedical Canada (formerly Intellivax International)	LAIV (inactivated subunit ProteosomeTM-adjuvanted vaccine)
FluLaval®	GlaxoSmithKline Biologicals, Rixensart, Belgium	inactivated split-virion vaccine
Flumist® / Fluenz®	Medimmune LLC, Maryland, USA / AstraZeneca (formerly Aviron)	LAIV
Flumist quadrivalent® / Fluenz Tetra®	Medimmune LLC, Maryland, USA / AstraZeneca (formerly Aviron, Mountain View, CA, USA)	LAIV
FluShield®	Wyeth Lederle Vaccines, USA	inactivated vaccine
FluVaccinol®	BGP GmbH, Vienna, Austria / STADA Medical GmbH, Germany	inactivated vaccine
Fluval AB®	Omninvest Vaccine Manufacturing, Researching and Trading Ltd., Hungary	inactivated aluminum phosphate gel-adjuvanted vaccine
Fluvax® / Afluria® / Enzira® / Nilgrip® / X-Flu™, Influenza Vaccine Ph. Eur.	CSL Biotherapies, Parkville, Australia / bioCSL GmbH, Marburg, Germany / Pfizer Vaccines, UK / Instituto Biológico Argentino SAIC, Argentina	inactivated split-virion vaccine
Fluviral®	GlaxoSmithKline Biologicals, Rixensart, Belgium	inactivated split-virion vaccine
Fluvirin®	Seqirus (bioCSL, formerly Novartis Vaccines and Diagnostics Limited)	inactivated subunit vaccine
Focusvax®	Crucell, Leiden, the Netherlands (formerly Berna Biotech Italia S.r.l., formerly Istituto sieroterapico Berna S.r.l.)	inactivated subunit virosome-adjuvanted vaccine
Gripax®	Hebrew University	inactivated whole virus vaccine
Gripguard®	Socopharm / Chiron, France	inactivated vaccine
Gripovax®	GlaxoSmithKline, Belgium	inactivated whole virus vaccine
Grippol® / Grippol Plus® / Grippol Neo®*	Petrovax, Russia	inactivated subunit Polyoxidonium-adjuvanted vaccine
Immugrip®	Pierre Fabre Médicament
Immuvac®	Nezel / Solvay Pharma	inactivated subunit vaccine
Imovax gripe®	Aventis Pasteur	inactivated vaccine
Inflexal® V / Infectovac Flu® / Isiflu® / Viroflu®	Crucell, Leiden, The Netherlands / Infectopharm / Janssen-Cilag GmbH, Neuss, Germany	inactivated virosome-adjuvanted
Influ-kovax®	Korea Vaccine, Korea	inactivated vaccine
Influmix®	Schiapparelli, Italy	inactivated whole virus vaccine
Influpozzi®	Seqirus (bioCSL, formerly Novartis)	inactivated subunit vaccine
Influsome-Vac	Hebrew University, Israel	inactivated liposome-based IL-2/GM-CSF-adjuvanted vaccine
Influvac® / Influvac sub-unit / Influvac S	Abbott Biologicals	inactivated subunit vaccine
Influvac Plus®	Abbott Biologicals (formerly Solvay Pharmaceuticals)	inactivated virosome-adjuvanted vaccine
Influvac® TC ˆ	AbbVie (formerly Abbott Biologicals, formerly Solvay Pharmaceuticals)	inactivated subunit vaccine
Influvirus®	Nuovo Istituto Sieroterapico Milanese S.r.l., Milan, Italy	inactivated split-virion vaccine
Invivac®	Solvay Pharmaceuticals, Vienna, Austria	virosome-adjuvanted vaccine
Isigrip®	KEDRION SpA, Italy	inactivated split-virion vaccine
Levrison®	Laboratorios Farmacéuticos ROVI, S.A., Spain	inactivated split-virion vaccine
Mastaflu®	MASTA Ltd., London, UK	inactivated subunit vaccine
MFV®	Servier, UK	Inactivated whole virus vaccine
MFV-ject®	Sanofi	inactivated whole virus vaccine
Monogrippol® / Monogrippol Neo® / Monogrippol Plus®	Petrovax Pharm, Russia	Inactivated
Munevan®ˆ	CellTech (formerly Medeva)	inactivated whole virus vaccine
Nasovac®	Serum Institute of India	LAIV
Nivgrip®	Nicolau Institute of Virology, Romania	inactivated whole virus vaccine
Preflucel®*	Baxter International, Vienna, Austria	inactivated split-virion vaccine
Previgrip®ˆ	Socopharm / Chiron, France	inactivated split-virion vaccine
Prodigrip®	Aventis Pasteur MSD	inactivated MF59-adjuvanted vaccine
Sandovac®	Sanofi Pasteur, Lyon, France	inactivated subunit vaccine
Subinvira®	Imuna, Czech Republic	inactivated split-virion vaccine
Tetagrip®	Sanofi Pasteur, Lyon, France	inactivated vaccine
Vacuna antigripal Pasteur®	Sanofi Pasteur MSD	inactivated split-virion vaccine
Vacuna antigripal polivalente Leti®	Leti, Spain	inactivated split-virion vaccine
Vaxigrip® / Vaxigrip® Junior / Istivac® / Mutagrip®/ Gripavac® / Inactivated Influenza Vaccine (Split Virion)®	Sanofi Pasteur, Lyon, France	inactivated split-virion vaccine
Xanaflu®	Abbott Biologicals B.V., the Netherlands	inactivated subunit vaccine

Abbreviations: IM (intramuscular), IN (intranasal), LAIV (Live Actenuated Influenza Vaccine), SC (subcutaneous).

^*Cell-culture derived

^§Insect-cell derived

^ˆCurrently discontinued

Seasonal vaccines can be divided into inactivated split-virion, inactivated subunit and LAIV vaccines (revised in ^14-23). Recently, also seasonal adjuvanted vaccines such as those adjuvanted by MF59 or virosomes have become available (reviewed in²⁴). Inactivated vaccines can be administered via the IM route in subjects aged 6 mo and older,²⁵ while LAIVs may be given intra-nasally to healthy, non-pregnant people aged 2–49 y. They may safely be administered at the same time as other vaccines.²⁶ For further details, the reader is referred to Table 1.

Generally speaking, the overall efficacy of influenza vaccines is 59% against laboratory-confirmed cases of influenza according to the recent meta-analysis by Osterholm and collaborators.²⁷ According to another meta-analysis it varies from 17% against influenza like illness (ILI) to 73% against confirmed influenza.²⁸ There is therefore the need to develop more effective vaccines.

Influenza vaccination coverage

Despite the fact that annual vaccination represents an important strategy for curbing influenza-related complications, the vaccination coverage is still far from being optimal. For example, in the USA, according to the National Immunization Survey-Flu and the National Internet Flu Survey, only 39.0% of all individuals aged ≥6 mo, that is to say 2 subjects out of 5, were vaccinated against influenza, leaving therefore most population unprotected (CDC, 2015). Also among health-care personnel, the coverage was unacceptably low: according to the most recently available data released by National Health Interview Survey, in the 2007–08 flu season, vaccination coverage among healthcare workers was 48%.²⁹

Further, due to the broadening of vaccination recommendations that extended the suggestion of being vaccinated to all subjects aged 6 mo and older, vaccine shortage can be experienced, together with manufacturing difficulties.

Intradermal (ID) vaccines, such as Fluzone® ID and Intanza®, are therefore an attractive option to properly overcome these critical issues.

Fluzone® ID and intanza®

An ID trivalent split-virion influenza vaccine (Fluzone® ID, Sanofi Pasteur, Swiftwater, PA) has been approved by the Food and Drug Administration (FDA) on 10^th May 2011 and been available in the US since the 2011/2012 influenza season for adults aged 18–64 y. Fluzone® ID is available as single-dose, preservative-free pre-filled syringe that contains 9μg hemagglutinin (HA) per strain and exploits the innovative BD's Soluvia™ microinjection device with a glass barrel, a stainless steel barrel with a 1.5-mm, 30-gauge needle and an elastomeric plunger stopper, produced by Becton-Dickinson (BD, Franklin Lakes, NJ).^30-32

Intanza 9μg vaccine (also known in some countries as IDflu™ 9μg, Sanofi Pasteur, administered with a micro-injection system), identical to Fluzone® ID for antigen content, way of administration and injection system, received marketing authorization in the EU in February 2009, licensed by the European Medicines Agency (EMA) for adults 18–59 y of age since the 2010/11 season in Europe, and in Canada in September 2010.

Istivac® ID is identifical to Fluzone ID and is commercialized in Argentina for adults 18–59 y of age, used successfully since 2010.

For subjects older than 64 y in the USA and 60 y in Europe and Canada, Fluzone® ID high Dose and Intanza® 15μg (also known in some countries as IDflu™ 15μg, Sanofi Pasteur; administered with a micro-injection system) are available.

High-quality reviews on Intanza® and Fluzone® are already available in the extant literature^30,33-36 and the reader is recommended to refer to them for information concerning trials and studies published until 2012. The thrust of our current manuscript intends to update the current evidences on ID vaccines and to provide new insights and prospects on future developments in the field.

Mechanisms of action

ID vaccination is not a novelty, being already known and performed since 1908.³³

Although the intradermal route of vaccine delivery was extensively studied for several vaccination including typhoid fever, measles, cholera, rabies, hepatitis B and poliomyelitis,³³ only influenza vaccines have been broadly administered by this route: possible issues that hampered the intradermal delivery for other vaccines include the high costs of technical development and the unacceptable local reactogenicity due to adjuvants contained in traditional formulations.³⁷

Skin represents an optimal site for vaccination and elicits both innate and adaptive immune responses. Skin, covering an impressive surface area of 1.6–1.85 m² and being situated at the interface between human body and environment,³⁸ is an important barrier, both passive and active, against chemical, physical and microbial insults. From an anatomic point of view, skin is composed of an upper laying (the epidermis), a basement membrane and a lower laying (the dermis). The epidermis is 150–200 μm thick and comprises the stratum corneum, the stratum germinativum, the stratum lucidum (present only in some specific parts of the human body), the stratum granulosum, the stratum of Malpighi or stratum spinosum, and the stratum basale.³⁷ The stratum corneum is particularly thick, consisting of dead cells (corneocytes) surrounded by lipid drafts and regions in the lamellar phases,^39-40 and has a great importance, in that optimal strategies for drug/vaccine delivery have to overcome the presence of this ‘formidable’ physical barrier.³⁸ As summarized in an excellent way by Gill and collaborators, there are different ways for an optimal drug/vaccine skin delivery: the first strategy is based on stratum corneum disruption (use of chemical enhancers, ultrasounds, and electroporation), the second is based on stratum corneum removal (tape stripping, abrasion, thermal ablation, and microdermabrasion), and the third is based on stratum corneum penetration (use of jet injectors, gene gun, and micro-needles).⁴¹

The dermis is 1.5–3 mm thick and, in its turn, can be divided into a papillary compartment (stratum papillare) and a reticular compartment (stratum reticulare), containing thin and thick collagen fibers, respectively.³⁸ Hypodermis or subcutaneous tissue is 3–100 mm thick and is a layer of loose connective tissue and elastin.³⁸

Further, skin harbours immune cellular components (epidermal dendritic cells or epidermal DCs, known also as Langerhans cells or LCs, dermal DCs or DDCs known also as interstitial or migratory DCs, αβ T cells, γδ T cells, natural killer or NK cells, B cells, mast cells, and macrophages), thus constituting a immunocompetent, multi-tasking organ^38-44 or a complex system (skin immune system or SIS).⁴⁵ Skin includes, indeed, skin-associated lymphoid tissues (SALTs),⁴⁶ which are responsible of a continuous cross-talk between skin (and its cellular components) and lymph nodes.³⁸ SALTs are constituted by lymphoid follicles, vessels, antigen-presenting cells (APCs), and lymphocytes. Some scholars, referring to the unique, natural immune enhancer effect of the skin, have proposed the existence of a skin-mediated ‘adjuvant’ mechanism.^38-44 However, the exact nature of this mechanism is complex and still poorly known. It can be hypothesized that the effect of ID vaccination may be the result of 3 concurring, complementary pathways (summarized in Table 2 and Fig. 2). Antigens are captured at skin level by resident, highly efficient APCs, like epidermal keratinocytes and specialized DCs, expressing high levels of class II major histocompatibility complex.³⁸ Skin DCs belong to the non-lymphoid tissue (NLT) DC group, while the LT DC group comprises 3 subsets (namely, the conventional DCs or cDCs type 1, the cDCs type 2, and, finally, the plasmacytoid DCs or pDCs).⁴⁷ Skin NLT DCs can be subdivided into 4 subsets: LCs within the suprabasal layers of the epidermis, expressing langerin; DDCs lacking langerin, which in their turn include DDCs expressing CD1a and DDCs expressing CD14; and finally, recruited macrophages or other innate immune cells, activated by the expression of Toll-like receptors (TLRs) and nucleotide-binding domain leucine-rich repeat receptors.³⁸ CD141, known also as BDCA-3, THBD or thrombomodulin, is an important marker in differentiating the different DC subsets. LCs can mature interacting with e-cadherin expressed by keratinocytes and can crosstalk with T_h1, T_h2, T_h17, T_h22 (cells producing IL-22, but not IL-17), T_reg, and can prime naïve CD8⁺ T cells into effector cytotoxic T lymphocytes (CTLs), while CD1a⁺ DDCs crosstalk with T_h2 and CTLs, CD1a⁺ CD141⁺ DDCs with T_h17, and CD14⁺ CD141⁺ DDCs with T_h1, T_fh, T_reg, and TC2, as well as induce the differentiation of naïve B cells into IgM-secreting plasma cells.⁴⁸

Table 2.

Overview of the 3 mechanisms at the basis of the action of intra-dermal vaccines.

PATHWAY	DESCRIPTION	REFERENCES
Dermal dendritic cells-dependent pathway	Involvement of skin-resident antigen-presenting cells	33,51,52,107,108
Dermal dendritic cells-independent pathway	Passive drainage of small antigenic components (<400 nm) to lymph nodes Role of micro-vascular structures and lymphatic vesselsInvolvement of lymph nodes-resident antigen presenting cells	51,53,107,108
Mechanical insults due to micro-needles	Formation of apoptotic and necrotic cells Activation of different signaling pathways	54

Figure 2.

A pictorial scheme of the different mechanism at the basis of the action of intra-dermal vaccines. Abbreviations: DCs: dendritic cells; DDCs: dermal dendritic cells.

This pathway plays undoubtedly a major role. The maturation, the differentiation, the acquisition of adequate immune abilities, and the migration to the paracortical area of the regional draining lymph nodes as afferent lymph veiled cells (ALVCs) through high endothelial venules (HEVs), are modulated by different signaling pathways, including increased expression of MHC antigens, co-stimulatory molecules and pro-inflammatory cytokines such as IL-1β, IL-6, IL-12 and TNF-α. These event lead to the subsequent activation of lymphocyte T CD8+, releasing interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), granzyme B, among other molecules, and high cytotoxic activity and memory B-cells.

This schematic representation, even though more complex, basically corresponds to the classical Ralph Steinman's theory, which postulates the existence of at least 2 distinct states of DCs: a) immature DCs committed to antigen capture and b) mature DCs working as APCs.^49,50

There is also another complementary pathway, independent on the first: antigens and especially small vaccine components (<400 nm) are passively drained by the unique micro-vascular and lymphatic structures of the skin and transported directly to the lymph nodes, where they are captured by lymph node resident DCs, medullary macrophages and subcapsular sinus macrophages.^51-53 This second path, even though less known and studied, is not nevertheless less fundamental.⁵³

Moreover, there are some physical aspects due to the mechanic action of the adopted injection system. The microneedle device generates localized transient stresses invoking cell necrosis and apoptosis around each projection,⁵⁴ favoring the release of damage associated molecular patterns (DAMPs). This is the well-known Matzinger's ‘danger hypothesis.’^55,56 Further, the device generates high immunoglobulin G (IgG) responses. Also immunoglobulin E (IgE) in immunity against influenza virus could play a role, as recently demonstrated by Smith-Norowitz and colleagues.⁵⁷

On the contrary muscle quite inefficient to capture antigens²⁶ and the IM route is therefore inferior to ID route in terms of biological mechanism and action.The molecular mechanisms of Fluzone® ID have been recently uncovered also using innovative cutting-edge biotechnologies, such as transcriptomic assays, which have shown a strong signature of adaptive immunity activation following vaccination.⁵⁸ In particular, Fluzone® ID seems to elicit temporary transcriptional changes in the circulating myeloid compartment. Vaccination upregulates modules linked to NF-κB-driven inflammation, IFN-γ response, TNF and CD40 signaling. These pathways, involved in T-cell activation and development of adaptive immunity, were detected in symptomatic individuals, but not asymptomatic ones.

As regard as the cross-protection potential of influenza ID vaccines, that is the ability of an influenza vaccine to elicit an effective antibody response against circulating viruses presenting antigenic patterns different from those of the vaccine strains, the exact involved mechanism is still not understood and under debate, even though a number of studies provided evidences of cross-protection elicited by ID vaccines (see Cross-protection section). In particular, Ansaldi et al. attributed the enhanced and broaden antibody response elicited by ID vaccine to the re-stimulation of B cells specific for selected antigenic sites of hemagglutinin surface protein, previously primed either by vaccination or infection. However, new and specific evaluations are needed in order to deeply investigate this aspect.

Cutaneous drug and vaccine delivery

Current ID drug and vaccine delivery relies mainly upon 6 technologies: namely, jet injection, ballistic injection, tattooing devices, transcutaneous patches, micro-injection and microneedles. First generation approaches relied upon Mantoux and bifurcated needles.²⁶

Jet injectors consist in a needle-free syringe and in a high-pressured propulsion system made of reusable, disposable pre-filled cartridges. While jet injectors are used to deliver liquids, ballistic injectors are able to transfer solid particles, such as gold nanoparticles. Tattooing devices consist in a short injection needle (or multiple needles) penetrating the skin through vibrations at a high frequency, while transcutaneous patches contain a dry formulation of the vaccine antigen, the adjuvant or of the vaccine and adjuvant mix.

Microneedle device represents an important achievement within the experimental technologies for a proper skin drug delivery. Solid-silicon microneedles and solid metal microneedle arrays, hollow-silicon microneedles, polymers- or carbohydrates-based microneedles, macroneedles, patches are other possible devices, which are being currently investigated.^58-60

ID devices are used in other fields, such as diabetology (such as SQ-PEN technology, InsuJet^TM, Injex23^TM, Injex30^TM, Vision® MediJector II^TM, or ClickSoft^TM for the delivery of insulin),^61,62 immunology/allergology,⁶³ anaesthesiology (J-Tip Needle-Free Injector, Medajet-XL),^64-65 endocrinology (for the delivery of growth hormones, including for example Bioject® Cool Click™, SeroJet, Antares' Medi-Jector Vision technology),⁶⁶ dermatology and cosmetology (for example, MTS ROLLER™, ScalpRoller^TM, DERMAROLLER®, MICRO HYALA®, or LITE CLEAR®, for the treatment of anti-aging, scars, hair-loss, and acne).⁶⁷

The Soluvia BD device represents the first FDA-approved delivery system in the field of vaccinology. Subsequently, the USA FDA has approved on August 14, 2014 the use of the PharmaJet Stratis 0.5 ml Needle-free Jet Injector for delivery of one particular flu vaccine (AFLURIA® by bioCSL Inc.) in people 18 through 64 y of age.

The need of a more effective activation of the immune system than that achieved by traditional IM vaccine delivery, along with the concerns of influenza vaccine shortages and the possibility of using ID route to spare vaccine dose, connected the research for new ID devices with the clinical development of ID influenza vaccines, in particular Fluzone®. A number of clinical and pre-clinical trials, evaluating the ID flu vaccine efficiency, have been performed by using the Soluvia BD device or with precursors of this micro-injection system.^68,69

Microneedle allows precise tissue localization of delivery and overcome the limitations of other chemical, biochemical and physical methods of transdermal drug delivery.^70-72

Immunogenicity

The immunogenicity profile of Fluzone® ID has been extensively investigated.^73-76

Immunogenicity has been explored also in immunocompromised patients: in transplanted patients,^77,78 in HIV-infected patients,⁷⁹ and in cancer patients.⁸⁰

Immunogenicity profiles of ID vaccines are not inferior to IM vaccines, as proven by some recently published meta-analyses: those of Marra and colleagues carried out among immunocompetent subjects⁸¹ and of Pileggi and collaborators performed among immunocompromised individuals.⁸² Marra and collaborators found an IM/ID GMTR ratio of 0.92 (95% CI = 0.77-1.09), of 0.97 (95% CI = 0.80–1.18) and of 0.93 (95% CI = 0.80–1.08), a seroconversion ratio of 0.94 (95% 0.86–1.02), of 0.89 (95% CI 0.80–0.99) and of 0.91 (95% CI = 0.80–1.04), and a seroprotection ratio of 0.97 (95% CI = 0.94–1.00), of 0.98 (95% CI = 0.96–0.99) and of 0.97 (95% CI = 0.91–1.03) for A/H1N1, A/H3N2 and B strains, respectively.⁸¹ Pileggi and co-workers found an overall risk ratio (RR) of seroprotection was 1.00 (95 % CI = 0.91–1.10) for A/H1N1 strain, 1.00 (95 % CI = 0.90–1.12) for A/H3N2 and 0.99 (95 % CI = 0.84–1.16) for B strain.⁸²

In conclusion ID vaccines proved to be statistically non inferior to other influenza vaccines, such as Fluzone® IM, Fluad®, Vaxigrip®, Inflexal V® and Flumist®.^73,83-87

Cross-protection

Ansaldi and collaborators performed a randomized trial to investigate the ability of Intanza® 15μg versus Vaxigrip® of conferring cross-protection against heterologous circulating H3N2 strains in 50 adults aged 60 y and older during the 2006–2007 influenza season. This study demonstrated the broader immune response elicited by an ID influenza vaccine vs. a standard IM influenza vaccine against heterologous viruses.^87,88

Also Camilloni and coworkers found that ID vaccine confers cross-protection against drifted H3N2 strains.⁷³

Safety and reactogenicity

Both pre-licensure and post-licensure studies have confirmed the safety profile of Fluzone® ID. According to the most recently available data, based on the Vaccine Adverse Effects Reporting System (VAERS), 9 out of 466 received reports (1.9%) were serious, including one reported fatality in an 88-year-old vaccinee. The most common adverse effects (AEs) included injection site reactions, which accounted for approximately half of all the reported AEs.⁸⁹

Tsang and collaborators performed a randomized, controlled, multicenter, phase II study in older adults (≥65 years of age) who were randomly assigned to ID vaccine with 15μg (HA)/strain (n = 636), ID vaccine with 21μg HA/strain (n = 634), standard IM vaccine with 15μg HA/strain (n = 319) and high-dose IM vaccine with 60μg HA/strain (n = 320), respectively. Subjects immunized with ID vaccine were more likely to report more injection-site reactions.⁸⁵

An interesting study carried out by Gorse and co-workers assessed the safety and reactogenicity in case of revaccination with ID preparation, performing a phase II, active-controlled, multi-center, open-label trial in 1,250 adults aged 18–64 y randomly assigned to ID or IM vaccine. Vaccines with ID vaccine reported erythema, induration, swelling, pruritus and ecchymosis more often than those receiving IM vaccine, especially if they had been immunized with ID vaccine in the previous year.^90,91

Patient acceptance and preference

Besides the unique properties of ID vaccination, due to the immunological and micro-vascular properties of the skin and its extreme richness in specific resident and recruited antigen-presenting cells (APCs), capable of eliciting stronger immune responses, ID vaccination offers further advantages in terms of patient acceptance and preference. ID vaccination is, indeed, painless and the microneedle device exorcises the fear of needle-sticks.⁴¹

Frenck and coworkers, indeed, found that adults rated the experience of receiving Fluzone ID vaccine as less (50%) or the same (24%) painful as their previous experience of receiving the vaccine via IM route.⁷⁵

Eizenberg and collaborators carried out a study among 1,666 vaccinees with Intanza 9μg and 46 prescribers in Australia and Argentina during the 2010 influenza. 98% of vaccinees were satisfied or very satisfied. 95% of vaccinees reported that they would prefer to receive the same vaccination next year. Furthermore, 85% of prescribers were satisfied or very satisfied with the vaccine.⁹²

Durando and collaborators performed an observational multicenter study in Italy among 1,600 subjects aged 60 y and older, using a validated, self-administered questionnaire, namely the 21-items Vaccinees' Perception of Injection (VAPI®), which assessed 4 dimensions (bother, arm movements, sleep, and acceptability). 75.5% and 94.9% of the interviewees were favorable and very favorable to Intanza® 15 μg, respectively. Also the compliance by healthcare professionals (n = 130) with the novel ID vaccine was favorable. No serious adverse event occurred during the 6-month follow-up period, while solicited local reactions were significantly higher in the ID-vaccine group than in the IM-vaccine group.⁹³

Arnou and collaborators performed an online survey in France and Germany among 483 physicians and 2,778 members of the general public aged 50 y and older. 61–78% of practitioners would strongly recommend Intanza® over the corresponding IM vaccine. More than two-thirds of the unvaccinated general public would prefer Intanza®. More than 82% of the physicians agreed that Intanza may help increase vaccination coverage rates.⁹⁴

Further, from some studies it is emerging that subjects would prefer ID versus IM vaccine in terms of pre-injection anxiety and post-injection pain, and in particular self-administration vs. nurse-led administration, if given the possibility of freely choosing.

Self-administration has been previously investigated and used with live attenuated influenza vaccines, and the performed studies reported high percentage of compliance with this practice and a few or no problems with administration.^95,96

Self-administration has the potential to reduce the time of influenza vaccination, in comparison with nurse-administration, and to increase influenza vaccine uptake, especially among healthcare workers. This approach would be particularly useful for the management of possible pandemics or urgent mass vaccination settings, such as in response to a severe influenza epidemic, both at country and global level.

In order to assure a safe and effective self-administration, a proper education about the correct techniques and an appropriate responses to any immediate adverse reactions, such as hypersensitivity reactions, should be guaranteed. For these reason, a supervision by trained staff must be available to manage possible anaphylactic reactions following administration of the vaccine

Coleman and colleagues recruited 228 adults aged 18–59 y who were randomized to either self-administered (n = 115) or nurse-administered (n = 113) ID vaccine. Self-administering participants rated pain less than nurse-led vaccines, even though they reported larger areas of redness post-vaccination. 70% of all participants said they would prefer ID vaccinations in the future.⁹⁷

Foy and colleagues compared Fluzone® ID versus Fluzone® IM and Flumist (Medimmune) intranasal vaccine. Of the 367 participants vaccinated, 249 (67.8%) chose the ID vaccine and 99.6% of these subjects reported being satisfied with the route and method of administration.³²

Dhont and colleagues investigated the acceptability of Intanza® 15 g among 837 vaccinees and 105 general practitioners (GPs) during the 2010–2011 influenza season. The majority of vaccinees was very satisfied (70.0%) or satisfied (27.9%) with the ID vaccine. Most vaccinees (91.1%) who had previously received IM influenza vaccination preferred the ID vaccine, and 98.5% of vaccinees reported they would consider receiving the ID vaccine the following year. The majority of GPs was very satisfied (78.6%) or satisfied (18.4%) with the ID vaccine, and most GPs (87.6%) expressed a preference for the ID vaccine over IM influenza vaccine.⁹⁸

Coleman and co-workers performed an interesting trial among 810 healthcare workers and found that ID self-administration could be a valuable asset for increasing the coverage of influenza vaccination among people working in health-care settings. 401 subjects were randomized to self-administered ID influenza vaccine (Intanza®, n = 401), while 409 were assigned to receiving nurse-administered IM vaccine (Vaxigrip®). Acceptability was high: 96% were very or somewhat certain that they administered the vaccine correctly, 83% would choose ID influenza vaccine again and of those, 75% would choose self-administration again.⁹⁹

Future prospects

Future prospects concern the increase in influenza strain coverage, adding for example a second B-lineage strain or using an alternative B-lineage strain. In the 1970s, the influenza B virus split into 2 main antigenically different lineages, named B/Yamagata and B/Victoria after named after their first representatives, B/Victoria/2/87 and B/Yamagata/16/88, respectively. Further, since 2001, the 2 B strain lineages have co-circulated with varying prevalence, making it difficult to predict the next season's dominant lineage.^100,101 The result has been frequent mismatches between the B strain lineage in influenza vaccines and the B strain lineage circulating in the community. Between 1999–2000 and 2012–2013, the B strain lineage in trivalent influenza vaccines has not matched the dominant circulating strain in half of the influenza seasons. For this reason, quadrivalent vaccines containing both B strain lineages have been developed.¹⁰² Quadrivalent Fluzone® IM has already shown non inferiority to trivalent Fluzone® IM for all matched strains and superior immunogenicity for the additional B-lineage strain in a study carried out in the USA during the 2013/2014 influenza season for persons ≥6 mo of age.¹⁰³

Gorse and collaborators investigated the immunogenicity and safety of an ID quadrivalent split-virion influenza vaccine administered to 1,672 adults 18–64 y of age in the US during the 2012–2013 influenza season vs. 837 participants vaccinated with Fluzone® ID and 846 vaccinated with an investigational version of Fluzone® ID. The randomized, double-blind, active-controlled multicentre trial showed that the quadrivalent version of Fluzone® ID proved to be statistically non-inferior to the 2 vaccines both in terms of antibody responses and adverse effects in case of A and matched B strains, and even superior for the unmatched B strains.¹⁰² This further advance could overcome mismatched B strains in previous influenza vaccines.

Conclusion

The broadening of recommendations for receiving influenza vaccine, logistic and technical difficulties together with an unacceptably low influenza coverage make ID vaccines particularly attractive. ID vaccines are comparable and in some cases superior to IM vaccines in terms of immunogenicity, safety, reactogenicity, tolerability and cross-protection profiles, as well as in terms of patient preference, acceptance and vaccine selection.^103-108 Further advances, such as Fluzone® ID with alternative B strains and Quadrivalent Fluzone® ID or the possibility of self-administering the vaccines, make Fluzone® ID and Intanza® even more valuable.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.