of October 20, 2018.This information is current as

the Influenza Vaccine ModelProtective Immune Response: Lessons from Type I IFN as a Natural Adjuvant for a

David Tough, Isabella Donatelli and Filippo BelardelliVenditti, Imerio Capone, Isabelle Seif, Edward De Maeyer,Pucchio, Paola Sestili, Enrico De Vincenzi, Massimo Enrico Proietti, Laura Bracci, Simona Puzelli, Tiziana Di

http://www.jimmunol.org/content/169/1/375doi: 10.4049/jimmunol.169.1.375

2002; 169:375-383; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/169/1/375.full#ref-list-1

, 17 of which you can access for free at: cites 41 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2002 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on October 20, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

by guest on October 20, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Type I IFN as a Natural Adjuvant for a Protective ImmuneResponse: Lessons from the Influenza Vaccine Model1

Enrico Proietti,2* Laura Bracci,* Simona Puzelli,* Tiziana Di Pucchio,* Paola Sestili,*Enrico De Vincenzi,* Massimo Venditti,* Imerio Capone,* Isabelle Seif,† Edward De Maeyer,†

David Tough,‡ Isabella Donatelli,* and Filippo Belardelli*

The identification of natural adjuvants capable of selectively promoting an efficient immune response against infectious agentswould represent an important advance in immunology, with direct implications for vaccine development, whose progress isgenerally hampered by the difficulties in defining powerful synthetic adjuvants suitable for clinical use. Here, we demonstrate thatendogenous type I IFN is necessary for the Th1 type of immune response induced by typical adjuvants in mice and that IFN itselfis an unexpectedly powerful adjuvant when administered with the human influenza vaccine, for inducing IgG2a and IgA pro-duction and conferring protection from virus challenge. The finding that these cytokines, currently used in patients, are necessaryfor full expression of adjuvant activity and are sufficient for the generation of a protective immune response opens new perspec-tives in understanding the basis of immunity and in vaccine development. The Journal of Immunology, 2002, 169: 375–383.

T he immune-promoting activity of any given vaccinationstrategy is determined not only by the presence of therelevant antigenic components in the vaccine formulation,

but also by the addition of suitable adjuvants capable of activatingand promoting an efficient immune response against the infectiousagents (1, 2). A desirable feature of an adjuvant is that it shouldspecifically enhance the immune response to the vaccine Ag withwhich it is coadministered, without causing toxic effects. Cur-rently, special attention is being given to adjuvants capable of ef-ficiently promoting a Th1 type of immune response, which is con-sidered the best correlate of a protective immune response againstinfections (3). However, the most powerful Th1-promoting adju-vants exhibit some toxicity, which limits their clinical use. Asthese adjuvants induce the production of several cellular factors,the characterization of the natural mediator(s) essential for theirimportant biologic activity not only would increase our knowledgeof the mechanisms responsible for a protective immune response,but could also lead to novel and more effective strategies in vac-cine development.

Type I IFNs are cytokines endowed with multiple biologicalactivities (4). Although low levels of type I IFN are detected underphysiological conditions (5), its production is markedly enhancedduring infections (4). For a long time, the importance of the effectsof type I IFN on the immune system remained poorly considered(6). In recent years, however, some reports have shown that thesecytokines affect the differentiation, survival, and function of im-

mune cells, including T cells (7–15) and dendritic cells (DCs)3

(16–20) and efficiently enhance a primary Ab response (21). In arecent study it was shown that immunization of mice with chicken�-globulin in the presence of type I IFN results in the generationof a potent primary immune response, characterized by an isotypeswitching toward IgG2a Abs (21). In this study a defective pro-duction of IgG2a Abs was observed in type I IFN receptor knock-out (IFN-IR KO) mice immunized in the presence of CFA. Al-though this observation could suggest a role of type I IFN in theCFA-induced immune response, it was unclear whether the mostcurrently used adjuvants induced type I IFN and acted through theproduction of these cytokines for promoting a potentially protec-tive Th1 immune response. Moreover, as the effects of type I IFNon the immune response remained controversial, with some au-thors emphasizing the importance of the immunosuppressive ac-tivities of these cytokines (22), it was essential to evaluate whethertype I IFN, when administered at the time of the vaccine injection,could act as an effective natural adjuvant in an experimental settingin which a human vaccine and the relevant infectious agent couldbe used for testing vaccine efficacy. In the present study we usedIFN-IR KO mice for investigating whether endogenous type I IFNitself is an essential mediator in the immune response induced afterimmunization with a reference protein Ag in the presence ofsome of the most currently used adjuvants. Moreover, we haveevaluated the adjuvant activity of type I IFN compared withtypical adjuvants by using human influenza vaccine as a model.We found that endogenous type I IFN is the main mediator forthe promotion of Th1-type immune responses by a wide rangeof adjuvants. Furthermore, type I IFN itself was an unexpect-edly powerful vaccine adjuvant for achieving immune protec-tion against influenza virus. These findings can open new per-spectives for vaccine development.

*Department of Virology, Istituto Superiore di Sanita, Rome, Italy; †Institut Curie,Centre Universitaire, Unite Centre National de la Recherche Scientifique, Unité Mixtede Recherche 146, Orsay, France, and ‡The Edward Jenner Institute for VaccineResearch, Compton, Newbury, Berkshire, United Kingdom

Received for publication November 14, 2001. Accepted for publication May 6, 2002.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by grants from the Italian Ministry of Health(Cytokines as Vaccine Adjuvants), the Italian Project on AIDS, and the Italian As-sociation for Cancer Research.2 Address correspondence and reprint requests to Dr. Enrico Proietti, Laboratory ofVirology, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161, Rome, Italy.E-mail address: proietti@iss.it

3 Abbreviations used in this paper: DC, dendritic cells; DTH, delayed-type hypersen-sitivity; HA, hemagglutinin; HAI, hemagglutination inhibition; IFN-IR KO, type IIFN receptor knockout; i.n., intranasal, intranasally; poly(I:C), polyinosinic-polycyti-dylic acid.

The Journal of Immunology

Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00

by guest on October 20, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Materials and MethodsMice

IFN-IR KO C3H/HeN mice were generated at the Institut Curie (Orsay,France) as follows. The mutated allele of the original IFN-IR KO strain(IFN-�� R0/0A129) (23) was transferred into the C3H/HeN background.Briefly, a male IFN-�� R0/0A129 mouse was crossed with C3H/HeN fe-males. F1 females were crossed with C3H/HeN males. Backcross progenywere crossed with C3H/HeN mice for nine generations. From the 10thgeneration backcross, brother/sister mating for 10 generations produced theIFN-IR KO mice that were used for comparative studies with identicalbackground control C3H/HeN mice. In vitro aged peritoneal macrophagesfrom IFN-IR KO C3H/HeN mice were not responsive to 800 U/ml type IIFN, as revealed by measuring inhibition of vesicular stomatitis virus yieldusing procedures described previously (5, 24), while IFN-treated peritonealmacrophages from control mice showed a 3 log10 inhibition of virus yield.C57BL/6 mice were purchased from Charles River (Calco, Italy). Micewere housed in the facilities of the Department of Virology at IstitutoSuperiore di Sanita and were used at the age of 7–8 wk. Wild-type andIFN-IR KO mice were kept under specific pathogen-free conditions. Allwork with animals conformed to European Community guidelines.

Adjuvants

IFA and CFA (Sigma, St. Louis, MO) were each mixed with Ag solutionat a 1/1 (v/v) ratio and emulsified, using two glass syringes and luer lockconnectors, until a stable emulsion was formed. Alum (aluminum hydrox-

ide gel, Sigma) was dissolved in the Ag solution at a ratio 1/20 (w/v), andthe pH was adjusted to 6.5. After 1-h incubation at room temperature, thesolution was centrifuged, and the pellet resuspended in the previous vol-ume in saline. CpG was synthesized by Roche Diagnostic (Milan, Italy)with a phosphorothioate backbone (sequence: 5�-TsGsAsCsTsGsTsGsAsAsCsGsTsTsCsGsAsGsAsTsGsA-3�). Two hundred micrograms ofCpG was dissolved in 1 ml of a solution containing 200 �g OVA. Polyi-nosinic-polycytidylic acid (poly(I:C); Sigma) was dissolved in saline at aconcentration of 10 mg/ml, and 0.15 mg was injected i.p. in mice. MF59(an emulsion consisting of 5% (v/v) squalene, 0.5% (v/v) Tween 80, and0.5% (v/v) Span 85 in H2O) was provided by Chiron Vaccines (Siena,Italy) (25). MF59 was mixed with Ag solution at a 1/1 (v/v) ratio and wasemulsified by pipetting.

IFN titration

The biological activity of serum IFN was determined as described previ-ously (26). One of the units, as expressed in the text, is the equivalent offour IFN reference units.

Antigens

OVA (grade V, Sigma) was dissolved in 0.15 M NaCl and filter-sterilizedbefore injection. The subunit influenza vaccine Agrippal, used for 1999/2000 vaccination campaign (supplied by Chiron), was prepared from in-fluenza virus A/Beijing X127, a reassortant of influenza viruses A/Beijing/262/95 (H1N1) and A/PR/8/34 (H1N1). The hemagglutinin (HA)

FIGURE 1. Induction of type I IFN by adjuvants and its role in the Ab response to OVA. a, Detection of IFN activity in the serum of mice injectedwith different adjuvants. C3H/HeN mice were inoculated intradermally with 50 �l of each adjuvant mixed with saline. Poly(I:C) was injected i.p. After24, 48, and 72 h, three mice from each group were bled, and sera were tested for IFN activity. Values represent the mean of three sera � SE. b and c, Controland IFN-IR KO C3H/HeN mice were injected intradermally with OVA, OVA plus IFA, OVA plus CFA, OVA plus CpG, or OVA plus alum, as indicated.Two booster injections with OVA alone were performed 10 and 17 days after the first immunization. Saline-treated mice were used as negative controls.Sera were collected on day 25. Data represent the mean � SE of specific Ab titers of three sera for each experimental group, tested in duplicate. �, p �0.005; ��, p � 0.05 (vs IFN-IR KO mice). NS, not significant. Open bars, control mice; filled bars, IFN-IR KO mice.

376 TYPE I IFN AS SYSTEMIC AND MUCOSAL ADJUVANT

by guest on October 20, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

concentration in the subunit vaccine, calculated by single radial diffusion,was 470 �g/ml.

Interferon

High titer IFN-�/� (2 � 107 U/mg protein) was prepared in the C243-3 cellline following a method adapted from Tovey et al. (26). IFN was concen-trated and partially purified by ammonium sulfate precipitation and dialysisagainst PBS as described previously (21).

Immunizations

OVA (10 �g/mouse) with or without adjuvants was injected intradermallyin a volume of 50 �l/mouse. Ten and 17 days after the first treatment, micewere boosted with OVA alone. For delayed-type hypersensitivity (DTH)assays, mice were injected with OVA and adjuvant on days 0, 10, and 17.For systemic immunization, 100 �l influenza vaccine (150 �g HA/ml)mixed with 100 �l saline, type I IFN, or adjuvant were injected i.m. intothe mouse thigh. For mucosal immunization, mice were anesthetized andinstilled into alternate nostrils in dropwise fashion with 50 �l of a solutioncontaining 25 �l vaccine (470 �g HA/ml) and 25 �l saline, type I IFN, oradjuvant. Vaccination was performed on days 0 and 14.

Determination of serum Abs

To measure OVA- or influenza-specific Ab levels, standard direct ELISAswere performed. Ninety-six-well, flat-bottom microtiter plates (Immulon4HBX, Dynatech, Chantilly, VA) were coated with 100 �l of a 1 �g/ml(for total anti-OVA IgG detection) or 4 �g/ml (for anti-OVA IgG2a andIgG1 detection) of a solution of OVA or with 3.6 �g HA/ml influenza

vaccine. The following dilutions of peroxidase-conjugated secondary Abswere used for anti-OVA Ab detection: anti-mouse IgG (Fc-specific), (Sig-ma), 1/1,000; anti-mouse IgG2a (Cappel Research Products, Durham, NC),1/200; and anti-mouse IgG1 (Cappel Research Products, Durham, NC),1/400. For influenza-specific ELISA, peroxidase-conjugated secondaryAbs were used as follows: anti-mouse IgG (H � L chain; Pierce, Rockford,IL), 1/75,000; anti-mouse IgG2a (Cappel), 1/200; anti-mouse IgG1 (Cap-pel), 1/400; and anti-mouse IgA (Kirkegaarde & Perry, Guilford, U.K.),1/1,500. Ortho-phenylenediamine (Sigma) was used as enzymatic sub-strate, and plates were read in a microplate autoreader at 490 nmwavelength.

Serum hemagglutination inhibition (HAI) titers were measured accord-ing to standard procedures (27, 28).

Spleen cell proliferation assay against OVA

Preparation of spleen cell suspensions and [3H]thymidine uptake assaywere performed as described previously (29). The spleen cell concentrationwas 5 � 105 in 0.2 ml/well of 10% FCS RPMI medium containing differentconcentrations of OVA (0, 50, 100, and 200 �g/ml).

OVA-specific DTH

Thirty-five days after the first injection, control and IFN-IR KO mice werechallenged with OVA (20 �g/50 �l) intradermally into the right footpad,while the left footpad was injected with saline as a control. Foot swellingwas measured with a microcaliper 48 h later. Data represent the mean ofOVA-challenged minus saline-challenged (contralateral) foot size of threemice per group.

FIGURE 2. OVA-specific cellular immune response in control and IFN-IR KO mice immunized with OVA and different adjuvant preparations. a,Control and IFN-IR KO C3H/HeN mice were injected intradermally with OVA, OVA plus IFA, OVA plus CFA, OVA plus CpG, or OVA plus alum, asindicated. Two booster injections with OVA alone were performed at days 10 and 17. Saline-treated mice were used as negative controls. Thirty-two daysafter the first immunization, spleen cells were tested in a standard proliferation assay using OVA as stimulation Ag. Data represent the mean � SE of[3H]thymidine incorporation of three individual spleen cell suspensions, tested in triplicate, after incubation with 100 �g/ml OVA. �, p � 0.04 vs IFN-IRKO mice. NS, not significant. b, Control and IFN-IR KO C3H/HeN mice were injected intradermally with OVA, OVA plus IFA, OVA plus CFA, OVAplus CpG, or OVA plus alum, as indicated. Two booster injections with OVA alone or mixed with adjuvants were performed on days 10 and 17.Saline-treated mice were used as negative controls. Thirty-five days after the first immunization mice were challenged with OVA for evaluating the DTHresponse. Data represent the mean � SE of specific foot swelling of three mice for each group. �, p � 0.003 vs IFN-IR KO mice. Open bars, control mice;filled bars, IFN-IR KO mice.

377The Journal of Immunology

by guest on October 20, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Virus and virus challenge

The original H1N1 A/Beijing/262/95 influenza virus (supplied by NationalInstitute for Biological Standards and Controls, Hertfordshire, U.K.) wasadapted to mouse after seven blind intranasal (i.n.) passages. The virus titerwas 64 HAU/or 1.1 � 107 PFU/ml. The LD50 corresponded to a dilutionof 1/1000. For virus challenge, anesthetized mice were instilled i.n. with 50�l of a virus suspension containing 10 LD50.

Statistical analysis

Data were analyzed by the Wilcoxon rank-sum test.

ResultsInduction of type I IFN in mice injected with adjuvants and itsrole in the adjuvant-induced humoral and cellular immuneresponses against OVA

We first investigated whether injection of mice with currently usedadjuvants, IFA, CFA, alum, or CpG oligonucleotides (CpG), couldinduce IFN and whether the integrity of the type I IFN system wasessential for the immune-promoting activity after immunizationwith a protein Ag (OVA). C3H/HeN mice were treated with thedifferent adjuvants or poly(I:C) as a reference type I IFN inducer.Biologically active IFN was detected in the sera of mice 24 and48 h after the injection of CFA or IFA. Mice injected with CpGshowed increasing serum IFN levels between 24 and 72 h, whereas

no serum IFN activity was detectable in alum-injected mice (Fig.1a).

To assess the role of type I IFN in the biological activity of theseadjuvants, control and IFN-IR KO C3H/HeN mice were immu-nized with OVA with or without the different adjuvants. As ex-pected, control mice immunized with the Ag together with adju-vants exhibited a marked increase in the overall OVA-specific Abresponse compared with animals immunized with the Ag alone(Fig. 1, b and c). IFA and CFA acted as effective adjuvants for bothIgG1 and IgG2a subclasses (typically associated with Th2 and Th1types of Ab response, respectively), whereas CpG and alum spe-cifically enhanced IgG2a (Th1 type) and IgG1 (Th2 type) re-sponses, respectively. Of interest, IFN-IR KO mice showed amarked defect in the generation of anti-OVA Abs when CFA,IFA, or CpG was used as adjuvant. This defect was especiallypronounced with regard to the IgG2a Abs (Fig. 1, b and c). Inmice immunized with alum as adjuvant, a weak Ab response,with prevalence of IgG1 Abs, was detected in both control andIFN-IR KO mice.

As expected, CFA, IFA, and CpG also enhanced T cell priming.Thus, spleen cells derived from control mice immunized withOVA in the presence of CFA, IFA, or CpG showed a higher invitro proliferative response to OVA compared with splenocytes

FIGURE 3. Adjuvant effect of type I IFN in C57BL/6 mice vaccinated i.m. with influenza (FLU) vaccine. a, FLU-specific IgG titers of mice immunizedi.m. with FLU vaccine, FLU vaccine plus type I IFN, or saline as a control. Mouse sera were collected 14 days after the first immunization (upper graph)or 14 days after the second immunization (lower graph). b, Survival time of mice injected i.m. with FLU vaccine, FLU vaccine plus type I IFN, or salineas a control and challenged with 10 LD50 of FLU virus after one (upper graph) or two (lower graph) immunizations. Virus challenge was performed 38days after the last immunization. c, HAI titers and analysis of FLU-specific Ab isotype in mice immunized i.m. with FLU vaccine alone or FLU vaccinemixed with type I IFN. Mouse sera were collected 14 days after the first immunization (upper graph) or 14 days after the second immunization (lowergraph). Data represent the mean � SE of specific Ab titers of three sera for each experimental group, tested in duplicate. �, p � 0.001; ��, p � 0.05 (vsmice treated with FLU vaccine alone). NS, not significant. F, saline-treated mice; ‚ or open bars, mice treated with FLU vaccine; E or filled bars, micetreated with FLU vaccine plus IFN.

378 TYPE I IFN AS SYSTEMIC AND MUCOSAL ADJUVANT

by guest on October 20, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

from mice immunized with the Ag alone; no significant increase inlymphocyte proliferation was observed in animals immunized inthe presence of alum (Fig. 2a). Importantly, as for the Ab response,greatly reduced priming for lymphocyte proliferation was detectedin IFN-IR KO mice immunized with the Ag in association withCFA, IFA, or CpG. Furthermore, while control mice immunizedwith the Ag in the presence of adjuvants showed a clear-cut DTHresponse, no significant response was detected in IFN-IR KO mice(Fig. 2b). These results indicate that endogenous type I IFN isessential for the promotion of both IgG2a Ab responses (a typicalhumoral hallmark of the Th1 type of immune response) and T cellresponses by a variety of adjuvants.

Adjuvant activity of type I IFN in the influenza vaccine model:importance of dose, time of administration, and specificity

To evaluate the adjuvant activity of type I IFN, we used a com-mercially available influenza vaccine obtained from an H1N1 in-fluenza virus circulating in 1999–2000 (A/Beijing/262/95) and, asan infectious agent, the corresponding virus.

A single injection of vaccine together with IFN into C57BL/6mice resulted in a clear-cut seroconversion of all the animals. Incontrast, only a limited portion (6 of 14) of mice inoculated withthe vaccine alone seroconverted, showing Ab titers lower thanthose of animals treated with vaccine and IFN (Fig. 3a, top panel).

FIGURE 4. Type I IFN administration modalities for achieving optimal adjuvant effects and comparison with other adjuvants. a, Dose-response of theIFN adjuvant effect on Ab titers. C57BL/6 mice were injected i.m. on days 0 and 14 with different concentrations of type I IFN (2 � 103, 2 � 104 and2 � 105 U/mouse) mixed with influenza (FLU) vaccine. Fourteen days after the last immunization, sera were collected and analyzed for FLU-specific totalIgG or IgG2a Ab response. Data represent the mean � SE of specific Ab titers of five sera for each experimental group, tested in duplicate. b, Dose-responseeffect of different concentrations of type I IFN coinjected with FLU vaccine on weight loss and survival of mice challenged with FLU virus. Forty-five daysafter the second injection, mice, immunized as described in a, were challenged i.n. with 10 LD50 of FLU virus. Data represent the mean weight course ofinfected mice and the percentage of surviving mice of the total number of animals. There were five mice per group. c, Effect of repeated administrationof type I IFN in mice immunized with FLU vaccine. C57BL/6 mice were injected i.m. with saline, FLU vaccine alone (15 �g/mouse), or FLU vaccine mixedwith IFN (2 � 105 U). This last group was split into two arms that received two additional daily injections of saline or type I IFN at the same site of thefirst inoculation. Fourteen days later mice were bled, and sera were tested for total IgG and IgG2a FLU-specific Abs. Data represent the mean � SE ofspecific Ab titers of three sera for each experimental group, tested in duplicate. d, Importance of coinjection of type I IFN adjuvant with the vaccine.C57BL/6 mice were injected i.m. with type I IFN (2 � 105 U) 1 or 2 days before or 1 or 2 days after FLU vaccine administration with respect to micetreated with FLU vaccine mixed with IFN or with vaccine alone or saline as control. When administered alone, type I IFN was injected at the same siteof vaccine inoculation. Data represent the mean � SE of specific Ab titers of five sera for each experimental group, tested in duplicate. e, Comparison ofthe adjuvant effect of type I IFN on mouse survival with that of other adjuvant preparations. On days 0 and 14 C57BL/6 mice were given two i.m. injectionsof FLU vaccine alone or FLU vaccine mixed with alum, IFN (2 � 105 U), MF59, or CFA as adjuvants or with saline as a control, as described in Materialsand Methods. Thirty-nine days after the last immunization mice were challenged i.n. with 10 LD50 of FLU virus. Data represent the percentage of survivingmice of the total number of mice (there were five mice for each experimental group). Mean survival time � SE are indicated in brackets. �, p � 0.01 vsall the experimental groups not marked by the asterisk. White bars, FLU-specific total IgGs; dark bars, FLU-specific IgG2a; F, saline-treated mice; f, micetreated with FLU vaccine alone; �, mice treated with FLU vaccine plus IFN (2 � 103 U/mouse); �, mice treated with FLU vaccine plus IFN (2 � 104

U/mouse); E, mice treated with FLU vaccine plus IFN (2 � 105 U/mouse).

379The Journal of Immunology

by guest on October 20, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

This weak Ab response was not associated with any protectionfrom influenza virus infection, since all the animals died within 10days after challenge. In contrast, all the mice injected with thevaccine and type I IFN were protected from virus infection (Fig.3b, top panel). After two injections with the vaccine alone, themajority of mice were still not protected from virus challenge (Fig.3b, bottom panel). Two immunizations with the vaccine mixedwith IFN resulted in a homogeneous increase in Ab titers in all themice (Fig. 3a, bottom panel).

Qualitative analysis of the Ig response at 2 wk after the firstimmunization with IFN revealed high titers of IgG2a Abs, notdetectable in mice injected with the vaccine alone (Fig. 3c, toppanel). Two weeks after the second immunization, mice immu-nized with the vaccine together with IFN showed higher levels ofserum IgA than animals injected with vaccine alone. Moreover,only sera from IFN-treated mice exhibited detectable HAI titers(Fig. 3c, bottom panel).

The adjuvant effect of type I IFN on the Ab response to influ-enza vaccine (total Igs and IgG2a) was dose dependent (Fig. 4a)and paralleled a dose-dependent protection from virus challenge(Fig. 4b). Of interest, in mice immunized with the vaccine togetherwith the highest dose of IFN (2 � 105 U), there was completeprotection, as evaluated not only by the survival data, but also bythe lack of any virus-induced decrease in mouse weight. Miceinjected with 2 � 104 U IFN survived after virus challenge, but atransient decrease in body weight after infection was observed.Only a low level of protection was detected in animals immunizedwith the vaccine together with the lowest dose of IFN (2 � 103 U;Fig. 4b). Additional IFN injections on days 1 and 2 markedly en-hanced the Ab response (total IgGs and IgG2a) with respect tolevels found in mice only coinjected with IFN and vaccine on day0 (Fig. 4c). Notably, mice subjected to a single IFN injection either

before or after vaccine inoculation developed much lower Ab titersthan animals coinjected with the vaccine and the cytokine (Fig. 4d)and were not protected from virus challenge (data not shown). Theadjuvant potency of type I IFN in protecting mice from a lethalchallenge with influenza virus was comparable to that obtainedwith two of the best currently available adjuvants (CFA andMF59), while alum was ineffective (Fig. 4e).

Further comparative experiments using control or IFN-IR KOC3H/HeN mice allowed us to verify the specificity of the IFNeffect and the role of endogenous type I IFN in this vaccine model.As a reference adjuvant, we used MF59, recently considered thebest adjuvant for influenza vaccine in humans (25). At all timepoints, the adjuvant effect of exogenous type I IFN was clearlydetected in control mice, but not in IFN-IR KO animals (Fig. 5).After a first immunization, while induction of total Igs and IgG1Abs was similar in animals immunized with either MF59 or IFN asadjuvants, only IFN-treated mice showed high levels of serumIgG2a Abs (Fig. 5a, top). Of interest, control mice immunized withthe vaccine alone showed detectable levels of IgA Abs, whichwere not revealed in IFN-IR KO mice, suggesting that endogenousIFN played a role in the induction of IgA in the early phase of theimmune response. After the second immunization, there was anincrease in Ab production in all vaccinated control mice. Interest-ingly, while IgG1 levels were higher in mice treated with MF59,considerable IgG2a titers were only detected in IFN-treated ani-mals (Fig. 5, bottom). Likewise, IFN-IR KO mice immunized withMF59 as an adjuvant did not show any level of IgG2a Abs, furtherindicating a selective role of type I IFN for IgG2a induction. No-tably, no adjuvant effect on IgA Abs was observed in IFN-IR KOmice immunized in the presence of either type I IFN or MF59 (Fig.5, bottom), suggesting that type I IFN is important for a sustainedIgA production.

FIGURE 5. Influenza (FLU)-specific Ab isotype analysis in control and IFN-IR KO mice immunized i.m. with FLU vaccine, alone or mixed with typeI IFN as adjuvant. Control and IFN-IR KO C3H/HeN mice were injected i.m. on days 0 and 14 with FLU vaccine alone, FLU vaccine plus type I IFN,or FLU vaccine plus MF59 adjuvant. Thirteen days after the first and 19 days after the second immunization, sera were collected and analyzed forFLU-specific Ab response. Data represent the mean � SE of specific Ab titers of five sera for each experimental group, tested in duplicate. �, p � 0.002;��, p � 0.05 (vs IFN-IR KO mice). NS, not significant. �, control mice; f, IFN-IR KO mice.

380 TYPE I IFN AS SYSTEMIC AND MUCOSAL ADJUVANT

by guest on October 20, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Type I IFN as an unusually powerful mucosal adjuvant ofinfluenza vaccine

Identification of mucosal adjuvants is an important task of vaccineresearch, since induction of protective mucosal immunity is crucialfor achieving local immune protection at the pathogen entry site. Ina first set of experiments we immunized C57BL/6 mice by givingtwo i.n. administrations, 14 days apart, of influenza vaccine aloneor mixed with type I IFN; Ab levels were measured 2 wk after eachimmunization (Fig. 6a). A general increase in Ab production (es-pecially IgG2a) was detectable in IFN-treated animals after thefirst immunization. Two weeks after the second immunization,there was a further increase in Ab titers in IFN-treated mice com-pared with animals injected with the vaccine alone. Notably, at thistime point, an impressive increase in IgG2a and IgA titers (1000-and 100-fold, respectively) was observed in animals immunizedwith the vaccine mixed with IFN compared with mice injectedwith vaccine alone. Mice immunized with IFN as an adjuvant alsoshowed higher levels of secretory pulmonary IgA than control an-imals. Of interest, all mice given the IFN-adjuvanted vaccine i.n.were protected from influenza virus infection, as revealed by bothsurvival values and lack of decrease in mouse weight after chal-lenge, while only a partially protective effect was found in animalsimmunized with vaccine alone (Fig. 6b). In a similar immunizationexperiment in IFN-IR KO and control C3H/HeN mice, type I IFN

proved to be superior to MF59 in inducing IgG2a and IgA incontrol animals at both time points, while MF59 was more effec-tive in inducing IgG1 Abs after two immunizations (Fig. 6c, bot-tom). As expected, no significant Ab response for all Ig subclasseswas observed in IFN-IR KO animals immunized i.n. with IFN asadjuvant. In contrast, MF59 was still capable of inducing IgG1Abs in IFN-IR KO mice, but the induction of IgG2a and IgA waslargely abrogated compared with the response detected in controlanimals (Fig. 6c).

DiscussionAlthough type I IFNs are the most used cytokines in patients, theirclinical use as modulators of the immune response has receivedpoor consideration (6). Recently, some studies have described neweffects of type I IFN on DC differentiation and function (16–20),while other reports have emphasized the importance of immuno-suppressive activity of these cytokines (22). The most remarkablefinding reported in the present study is the demonstration that typeI IFN, coadministered with a human vaccine (influenza), repre-sents an unexpectedly powerful adjuvant, inducing a Th1 type ofimmune response and protection against virus challenge. The find-ing that widely used adjuvants, such as IFA, CFA, and CpG, in-duce the expression of type I IFN together with the demonstrationof clear-cut defects in the production of IgG2a Abs and in the

FIGURE 6. Powerful adjuvant effect of type I IFN when administered i.n. with influenza (FLU) vaccine. a, Analysis of FLU-specific HAI titers, serumAb isotype, and broncho-alveolar lavage (BAL) IgA of C57BL/6 mice immunized i.n. with FLU vaccine alone or mixed with type I IFN. Mice were instilledi.n. on days 0 and 14 with 50 �l FLU vaccine, alone or mixed with type I IFN (4 � 104 U). Sera were collected 14 days after the first immunization (leftgraph). Fourteen days after the second immunization (right graph), mice were sacrificed, and blood samples and BAL were taken for Ig analysis. Datarepresent the mean � SE of specific Ab titers of five samples for each experimental group, tested in duplicate. ��, p � 0.002 vs FLU vaccine alone. NS,not significant. �, Vaccine alone; f, vaccine plus IFN. b, Survival time of C57BL/6 mice immunized with two i.n. administrations of FLU vaccine aloneor mixed with type I IFN (4 � 104 U) or saline as a control and challenged with 10 LD50 of FLU virus 38 days thereafter. Data represent the mean weightcourse (�SE) of infected mice and the percentage of surviving mice with respect to the total number of animals. There were five mice per group. F,Saline-treated mice; E, mice instilled i.n. with FLU vaccine; �, mice instilled i.n. with FLU vaccine plus IFN. c, Control and IFN-IR KO C3H/HeN micewere instilled i.n. on days 0 and 14 with FLU vaccine alone, FLU vaccine plus type I IFN, or FLU vaccine plus MF59 adjuvant. Thirteen days after thefirst (upper panels) and 19 days after the second (lower panels) immunization, sera were collected and analyzed for FLU-specific Ab response. Datarepresent the mean � SE of specific Ab titers of five samples for each experimental group, tested in duplicate. �, p � 0.004 vs IFN-IR KO mice. NS, notsignificant. �, control mice; f, IFN-IR KO mice.

381The Journal of Immunology

by guest on October 20, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Ag-specific T cell response (T cell proliferation in vitro and DTHresponse in vivo) specifically occurring in IFN-IR KO mice dem-onstrate that the endogenous IFN system is essential for full ex-pression of a Th1-type immune response. Intriguingly, defectiveproduction of IgG1 Abs was observed in IFN-IR KO mice whenalum, an adjuvant promoting a Th2 type of immune response, wasinjected together with OVA. Thus, we can assume that endogenousIFN may play a different role in shaping the immune response,depending on the type of adjuvant and Ag. Recently, Van Hudenand colleagues have reported that IFN-IR KO mice exhibit a de-fective IgG2a response with respect to control animals when im-munized with �-galactosidase together with CpG as adjuvant, sug-gesting that type I IFN is required to mount an adaptive responseto immunostimulatory DNA (30). Notably, our results obtained inthe influenza vaccine model show that type I IFN was unexpect-edly effective in inducing rapid seroconversion in all the animals,characterized by selective induction of high levels of IgG2a evenafter a single immunization, resulting in full protection from viruschallenge.

IgG2a Abs are characteristic of the response to virus infection,are often protective, and demonstrate neutralizing activity (31).Thus, we may assume that in the course of a virus infection theexpression of type I IFN and generation of IgG2a Abs are linkedevents of biological relevance for the subsequent generation ofprotective immunity.

When given i.m. as adjuvant, type I IFN was far superior toalum and was equivalent to CFA, considered one of the most pow-erful adjuvants in animal models, and to MF59, a new adjuvantrecently considered the best candidate for anti-influenza vaccina-tion in elderly individuals (32). Of interest, while these typicaladjuvants have been used at the maximal tolerated dose, IFN dos-ages higher than those used in our experiments could result in evenmore potent adjuvant activity. Notably, two subsequent treatmentswith IFN on days 1 and 2 further increased the level of IgG2a Ab,while a single IFN injection at a time different from that of vaccineadministration was ineffective. Thus, the optimal adjuvant activityis obtained under conditions mimicking the natural response toviral infections, often resulting in long term immunity, where con-siderable levels of type I IFN are produced at early times aftervirus contact with specific host cells, such as the so-called naturalIFN-producing cells (also named pDC2 or plasmocytoid DCs),considered professional cells for producing high IFN levels in re-sponse to viral challenge (33, 34).

One of the major issues in vaccine research is the definition ofstrategies for inducing mucosal immunity and IgA production,which are important for immune protection against infectiousagents transmitted through the respiratory system (35). In this re-gard it is of special interest that i.n. injection of influenza vaccinewith IFN was particularly effective in inducing serum IgG2a andIgA Abs and full protection from virus challenge. Type I IFN wassuperior to MF59 in inducing IgG2a and IgA Abs when used as ani.n. adjuvant. This study reports the first evidence indicating thattype I IFN is important for IgA production and for the establish-ment of mucosal immunity. Recent studies have shown that the i.n.administration of type I IFN represents an effective delivery sys-tem for inducing therapeutic effects with these cytokines (36).However, the mechanisms of action are still unclear (37). Thus, thepresent finding showing that the i.n. administration of IFN is un-usually effective in enhancing vaccine efficacy and inducing im-mune correlates of protection encourages further studies for un-derstanding whether type I IFN can represent a crucial factor inbreaking tolerance and inducing mucosal immunity.

With regard to the role of endogenous type I IFN in the immuneresponse to influenza vaccine, our results indicate that the induc-

tion of IgG2a and IgA Abs is mostly mediated by type I IFN itself,since no or poor production of these Abs was observed in IFN-IRKO mice. Early studies had shown that low levels of spontaneoustype I IFN can be responsible for the natural antiviral state ofmacrophages (5) as well as for host-mediated restriction of tumorgrowth in mice transplanted with syngeneic IFN-resistant tumorcells (38), supporting the concept that even basal IFN levels canplay important in vivo roles. Thus, we may argue that even in theabsence of specific IFN induction, the defective response to the i.n.immunization of IFN-IR KO mice with influenza vaccine is indic-ative of the importance of type I IFN basal levels in the generationof a specific humoral immune response.

Identification of new adjuvants is an urgent need for vaccinedevelopment and especially for subunit vaccines, which are poorlyimmunogenic. The use of adjuvants effective in animal models isoften restricted by safety concerns. The finding that type I IFNs,cytokines with a long record of clinical use (39, 40), are necessaryand sufficient to induce a protective immune response to vaccinesis not only important for the comprehension of the mechanismsunderlying the immune response to infections, but can also exhibitpractical implications for new strategies in vaccine development.As differences in the type I IFN-mediated regulation of the Th1responses between mice and humans have been reported (41), thepossible transference to humans of data obtained in mouse modelsshould be regarded with caution. Selected clinical trials are neededto establish whether type I IFN can represent valuable natural ad-juvants for human vaccines.

AcknowledgmentsWe thank Dr. I. Gresser for helpful discussion. We are grateful to AnnaFerrigno and Cinzia Gasparrini for excellent secretarial assistance.

References1. Liu, M. A. 1998. Vaccine developments. Nat. Med. 4:515.2. Hilleman, M. R. 1998. Six decades of vaccine development: a personal history.

Nat. Med. 4:507.3. Singh, M., and D. O’Hagan. 1999. Advances in vaccine adjuvants. Nat. Biotech-

nol. 17:1075.4. Belardelli, F. 1995. Role of interferons and other cytokines in the regulation of

the immune response. APMIS 103:161.5. Belardelli, F., F. Vignaux, E. Proietti, and I. Gresser. 1984. Injection of mice with

antibody to interferon renders peritoneal macrophages permissive for vesicularstomatitis virus and encephalomyocarditis virus. Proc. Natl. Acad. Sci. USA 81:602.

6. Belardelli, F., and I. Gresser. 1996. The neglected role of type I interferon in theT-cell response: implication for its clinical use. Immunol. Today 17:369.

7. Romagnani, S. 1992. Induction of Th1 and Th2 responses: a key role for the“natural” immune response? Immunol. Today 13:379.

8. Tough, D. F., P. Borrow, and J. Sprent. 1996. Induction of bystander T cellproliferation by viruses and type I interferon in vivo. Science 272:1947.

9. Rogge, L., L. Barberis-Maino, M. Biffi, N. Passini, D. H. Presky, U. Gubler, andF. Sinigaglia. 1997. Selective expression of an interleukin-12 receptor componentby human T helper 1 cells. J. Exp. Med. 185:825.

10. Von Hoegen, P. 1995. Synergistic role of type I interferons in the induction ofprotective cytotoxic T lymphocytes. Immunol. Lett. 47:157.

11. Palmer, K. J., M. Harries, M. E. Gore, and M. K. Collins. 2000. Interpheron-�(IFN-�) stimulates anti-melanoma cytotoxic T lymphocyte (CTL) generation inmixed lymphocyte tumour cultures (MLTC). Clin. Exp. Immunol. 119:412.

12. Sun, S., X. Zhang, D. F. Tough, and J. Sprent. 1998. Type I interferon-mediatedstimulation of T cells by CpG DNA. J. Exp. Med. 188:2335.

13. Marrack, P., J. Kappler, and T. Mitchell. 1999. Type I interferons keep activatedT cells alive. J. Exp. Med. 189:531.

14. Matikainen, S., T. Sareneva, T. Ronni, A. Lehtonen, P. J. Koskinen, I. Julkunen.1999. Interferon-� activates multiple STAT proteins and upregulates prolifera-tion-associated IL-2R�, c-myc, and pim-1 genes in human T cells. Blood 93:1980.

15. Belardelli, F., M. Ferrantini, S. M. Santini, S. Baccarini, E. Proietti,M. P. Colombo, J. Sprent, and D. F. Tough. 1998. The induction of in vivoproliferation of long-lived CD44hi CD8� T cells after injection of tumor cellsexpressing IFN-�1 into syngeneic mice. Cancer Res. 58:5795.

16. Paquette, R. L., N. C. Hsu, SM. Kiertscher, A. N. Park, L. Tran, M. D. Roth, andJ. A. Gaspy. 1998. Interferon-� and granulocyte-macrophage colony-stimulatingfactor differentiate peripheral blood monocytes into potent antigen-presentingcells. J. Leukocyte Biol. 64:358.

382 TYPE I IFN AS SYSTEMIC AND MUCOSAL ADJUVANT

by guest on October 20, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

17. Luft, T., K. C. Pang, E. Thomas, P. Hertzog, D. N. J. Hart, J. Trapani, andJ. Cebon. 1998. Type I IFNs enhance the terminal differentiation of dendriticcells. J. Immunol. 161:1947.

18. Radvanyi, L. G., A. Banerjee, M. Weir, and H. Messner. 1999. Low levels ofinterferon-� induce CD86 (B7.2) expression and accelerates dendritic cell mat-uration from human peripheral blood mononuclear cells. Scand. J. Immunol. 50:499.

19. Santini, S. M., C. Lapenta, M. A. Logozzi, S. Parlato, M. Spada, T. Di Pucchio,and F. Belardelli. 2000. Type I interferon as a powerful adjuvant for monocyte-derived dendritic cell development and activity in vitro and in Hu-PBL-scid mice.J. Exp. Med. 191:1777.

20. Gallucci, S., M. Lolkema, and P. Matzinger. 1999. Natural adjuvant: endogenousactivators of dendritic cells. Nat. Med. 11:249.

21. Le Bon, A., G. Schiavoni, G. D’Agostino, I. Gresser, F. Belardelli, andE. Proietti. 2001. Type I interferons potently enhance humoral immunity and canpromote isotype switching by stimulating dendritic cells in vivo. Immunity 14:461.

22. Zagury, D., A. Lachgar, V. Chams, L. S. Fall, J. Bernard, J. F. Zagury, B. Bizzini,A. Gringeri, E. Santagostino, J. Rappaport, et al. 1998. Interferon � and Tatinvolvement in the immunosuppression of uninfected T cells and C-C chemokinedecline in AIDS. Proc. Natl. Acad. Sci. USA 95:3851.

23. Muller, U., U. Steinhoff, L. Reis, S. Hemmi, J. Pavlovic, R. M. Zinkernagel, andM. Aguet. 1994. Functional role of type I and type II interferons in antiviraldefense. Science 264:1918.

24. Gessani, S., F. Belardelli, P. Borghi, D. Boraschi, and I. Gresser. 1987. Corre-lation between the lipopolysaccharide response of mice and the capacity of mouseperitoneal cells to transfer an antiviral state: role of endogenous interferon I.J. Immunol. 139:1991.

25. Cataldo, D. M., and G. Van Nest. 1997. The adjuvant MF59 increases the im-munogenicity and protective efficacy of subunit influenza vaccine in mice. Vac-cine 15:1710.

26. Tovey, M. G., J. Begon-Lours, and I. Gresser. 1974. A method for the large scaleproduction of potent interferon preparations. Proc. Soc. Exp. Biol. Med. 146:809.

27. Mancini, G., I. Donatelli, C. Rozera, G. Arangio Ruiz, and S. Butto. 1985. An-tigenic and biochemical analysis of influenza “A” H3N2 viruses isolated frompigs. Arch. Virol. 83:157.

28. Gaush, C. R., and T. F. Smith. 1968. Replication and plaque assay of influenzavirus in an established line of canine kidney cells. Appl. Microbiol. 16:588.

29. Proietti, E., G. Greco, B. Garrone, S. Baccarini, C. Mauri, M. Venditti, D. Carlei,and F. Belardelli. 1998. Importance of cyclophosphamide-induced bystander ef-fect on T cells for a successful tumor eradication in response to adoptive immu-notherapy in mice. J. Clin. Invest. 101:429.

30. Van Huden, J. H., C. H. Tran, D. A. Carson, and E. Raz. 2001. Type I interferonis required to mount an adaptive response to immunostimulatory DNA. Eur.J. Immunol. 31:3281.

31. Coutelier, J. P., J. T. Van der Logt, F. W. Heessen, G. Warnier, and J. Van Snick.1987. IgG2a restriction of murine antibodies elicited by viral infections. J. Exp.Med. 165:64.

32. Podda, A. 2001. The adjuvanted influenza vaccines with novel adjuvants: expe-rience with the MF59-adjuvanted vaccine. Vaccine 19:2673.

33. Siegal, F. P., N. Kadowaki, M. Shodell, P. A. Fitzgerald-Bocarsly, K. Shah,S. Ho, S. Antonenko, and Y. J. Liu.1999. The nature of the principal type Iinterferon-producing cells in human blood. Science 284:1835.

34. Cella, M., D. Jarrossay, F. Facchetti, O. Alebardi, H. Nakajima, A. Lanzavecchia,and M. Colonna. 1999. Plasmacytoid monocytes migrate to inflamed lymphnodes and produce large amounts of type I interferon. Nat. Med. 5:919.

35. Czerkinsky, C., F. Anjuere, J. R. McGhee, A. George-Chandy, J. Holmgren,M. P. Kieny, K. Fujiyashi, J. F. Mestecky, V. Pierrefite-Carle, C. Rask, et al.1999. Mucosal immunity and tolerance: relevance to vaccine development. Im-munol. Rev. 170:197.

36. Tovey, M. G., J. F. Meritet, J. Guymarho, and C. Maury. 1999. Mucosal cytokinetherapy: marked antiviral and antitumor activity. J. Interferon Cytokine Res. 19:911.

37. Bocci, V. 1999. The oropharyngeal delivery of interferons: where are we andwhere do we need to go? J. Interferon Cytokine Res. 19:859.

38. Gresser, I., F. Belardelli, C. Maury, M. T. Maunoury, and M. G. Tovey. 1983.Injection of mice with antibody to interferon enhances the growth of transplant-able murine tumors. J. Exp. Med. 158:2095.

39. Gutterman, J. U. 1994. Cytokine therapeutics: lessons from interferon alpha.Proc. Natl. Acad. Sci. USA 91:1198.

40. Pfeffer, L. M., C. A. Dinarello, R. B. Herberman, B. R. Williams, E. C. Borden,R. Bordens, M. R. Walter, T. L. Nagabhushan, P. P. Trotta, and S. Pestka. 1998.Biological properties of recombinant �-interferons: 40th anniversary of the dis-covery of interferons. Cancer Res. 58:2489.

41. Farrar, D. J., J. D. Smith, T. L. Murphy, S. Leung, G. R. Stark, and K, M,Murphy. 2000. Selective loss of type I interferon-induced STAT4 activationcaused by a minisatellite insertion in mouse Stat2. Nat. Immunol. 1:65.

383The Journal of Immunology

by guest on October 20, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from