Download - Characterization ofin VivoPrimary and Secondary CD8+T Cell Responses Induced by Recombinant Influenza and Vaccinia Viruses

CELLULAR IMMUNOLOGY 173, 96–107 (1996)ARTICLE NO. 0255

Characterization of in Vivo Primary and Secondary CD8/ T CellResponses Induced by Recombinant Influenza and Vaccinia Viruses

KENICHIRO MURATA,* ADOLFO GARCIA-SASTRE,†,1 MORIYA TSUJI,* MAURICIO RODRIGUES,*DOLORES RODRIGUEZ,‡ JUAN RAMON RODRIGUEZ,‡ RUTH S. NUSSENZWEIG,*

PETER PALESE,† MARIANO ESTEBAN,‡ AND FIDEL ZAVALA*,2

*Department of Medical and Molecular Parasitology, New York University School of Medicine, New York, New York 10010;†Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029; and ‡Centro Nacional

de Biotecnologia, CSIC, Campus Universidad Autonoma, 28049 Madrid, Spain

Received March 4, 1996; accepted May 27, 1996

Major advances have been made in the last few yearswith regard to the characterization of the intracellularWe characterized the in vivo primary and secondarymechanisms involved in antigen processing, and themurine CD8/ T cell responses induced by immuniza-basic features which define the molecular interactiontion with influenza and vaccinia viruses, which werebetween peptide antigens and class I MHC moleculesengineered to express the same H-2Kk- and H-2Kd-re-

stricted epitopes. Our results show that the induction have been elucidated. The knowledge provided by theseand magnitude of the primary CD8/ T cell response investigations together with the development of newclosely depends on the viral dose used for immuniza- methodological approaches has further enhanced ourtion, while it is not affected by the route of immuniza- understanding of the basic biological features of CD8/tion. The induction of secondary CD8/ T cell responses T cells, particularly as it relates to antigen recognitionappears to be highly restricted, as suggested by the and signal transduction. There is, however, a surpris-lack of in vivo expansion of antigen-specific CD8/ T ing scarcity of information regarding fundamental as-cells after repeated immunization with the same virus. pects of the in vivo activity of these cells. For instance,In contrast, a 20- to 30-fold increase in the frequency there are few systematic and quantitative studiesof antigen-specific CD8/ T cells could be induced after aimed at evaluating parameters as basic as dose depen-combined immunization with recombinant influenza dency and relevance of the route of immunization inand vaccinia viruses. These findings may provide the the induction of primary CD8/ T cell responses. Fur-basis for the development of new prophylactic and

thermore, little is known regarding the conditions un-therapeutic strategies to prevent or control intracellu-der which secondary CD8/ T cell responses can be in-lar infections and certain malignancies. q 1996 Academicduced against epitopes derived from different infec-

Press, Inc.tious agents.

The limited availability of information regardingthese aspects of the in vivo activity of CD8/ T cells is,INTRODUCTIONin part, due to the lack of simple quantitative assayswhich permit the simultaneous evaluation and compar-CD8/ T lymphocytes represent one of the most im-ison of CD8/ T cell responses, generated in large num-portant immune effector mechanisms against intracel-bers of experimental animals. Recently, a novel ELIS-lular infections produced by viruses, parasites, and bac-POT3 assay which permits the rapid measurement ofteria. These T cells recognize microbe-derived peptidesthe frequency of epitope-specific CD8/ T cells has beenwhich are presented on the surface of infected cells.developed (2, 3). While comparable to the standard lim-The peptides are generated by intracellular processing,iting dilution assay with regard to sensitivity and speci-bound to class I MHC molecules, and then transportedficity, this assay allows the simultaneous evaluation ofto the cell surface (1).a large number of samples and is easy to perform.

Using this assay, we investigated and reexamined1 On leave of absence from the Department of Biochemistry andMolecular Biology, Faculty of Medicine, University of Salamanca,Salamanca, Spain.

3 Abbreviations used: ELISPOT, enzyme-linked immunospot; CS,2 To whom correspondence and reprint requests should be ad-dressed at Department of Medical and Molecular Parasitology, New circumsporozoite; NP, nucleoprotein of influenza A virus; ME, ma-

laria epitope; PYCS, CS protein of Plasmodium yoelii; pfu, plaque-York University School of Medicine, 341 East 25th Street, New York,NY 10010. forming unit.

960008-8749/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

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97IN VIVO CD8/ T CELL RESPONSES

the induction of primary and secondary CD8/ T cell Recombinant Virusesresponses induced by immunization of mice with re-

The recombinant influenza virus ME, expressing incombinant influenza and vaccinia viruses. These liveits hemagglutinin molecule the class I MHC-restrictedvectors were engineered to express the same (3–5)NEDSYVPSAEQI epitope of the P. yoelii CS protein,MHC class I-restricted epitopes, including the H-2Kd-was generated as previously described (3, 4). This re-restricted epitope (SYVPSAEQI) of the circumsporo-combinant influenza virus was derived from influenzazoite (CS) protein of Plasmodium yoelii, (6), and the H-A/WSN/33 virus, subtype H1N1. The recombinant vac-2Kd (TYQRTRALV)-restricted and the H-2Kk (SDYEG-cinia virus PYCS, derived from the WR strain, ex-RLI)-restricted epitopes of the nucleoprotein (NP) ofpresses the CS protein of P. yoelii in the thymidinethe influenza A virus (7, 8).kinase region of the viral genome, under regulationThe availability of different recombinant viruses ex-of the viral early–late promoter P 7.5, as previouslypressing the same epitopes provides the unique oppor-described (3, 4). The recombinant vaccinia virus NP-tunity of comparing their immunogenic properties. WeVAC, expressing the NP of the influenza A virus, waswill also be in a position to identify features of thegenerated as described (5). The inactivation of influ-primary and secondary CD8/ T cell responses whichenza virus ME by ultraviolet (UV) irradiation was per-may be of general validity, and those which depend onformed as follows: 1.5 ml of sample containing 106 pfuthe particular characteristics of each virus. Further-was exposed to irradiation using an 8-W germicidalmore, the identification of the parameters which influ-UV lamp (General Electric Co., Whippany, NJ) at aence the CD8/ T cell responses against different micro-distance of 3 inches while being gently stirred for 20organisms should help to characterize the mechanismssec. No infectious viruses were found in the irradiatedinvolved in the induction and regulation of the in vivosamples as measured by plaque assay on Madin–expansion of this T cell subset. Finally, these studiesDarby bovine kidney cells (11).are also of considerable importance, in view of the po-

tential use of attenuated live virus vectors as vaccinesImmunization Proceduresdesigned to induce CD8/ T cell-mediated protective im-

munity. Details of the different protocols used for immuniza-tion are described in the legends to the respective fig-

MATERIALS AND METHODS ures. Immunization using various routes was done asfollows: (i) Intranasal (i.n.) immunization was per-

Animals formed in anesthetized mice which were inoculatedwith the viral suspension into their nasal cavity; (ii)Six- to nine-week-old female BALB/c mice, pur-the intradermal immunization was performed afterchased from Biological Testing Branch/National Can-scarification of the skin of the tail with a needle; (iii)cer Institute (Frederick, MD), or (BALB/cJ 1 A/J) F1for the subcutaneous immunization, mice were injectedmice purchased from Jackson Laboratories (Bar Har-with the viral suspension into the base of the tail; (iv)bor, ME), were used for the experiments.the intramuscular immunization was performed by in-jecting the viral suspension into the thigh muscle of anSynthetic Peptides and Tissue Culture Conditionsanesthetized mouse; (v) The intravenous injection was

Peptide SYVPSAEQI, an H-2Kd-restricted CD8/ T administered into the tail vein; and (vi) for the intra-cell epitope of P. yoelii CS protein (6), was synthesized peritoneal immunization the viral suspension was in-in our laboratory as described. Peptide TYQRTRALV, jected into the peritoneal cavity.an H-2Kd-restricted CD8/ T cell epitope of the influ-enza A virus NP (7), was purchased from the Peptide ELISPOT AssayLaboratory, Inc. (Berkeley, CA). Peptide SDYEGRLI,an H-2Kk-restricted CD8/ T cell epitope of influenza A The ELISPOT assay for the detection of epitope-spe-

cific IFN-g-secreting cells was performed essentially asvirus NP (8), was generously provided by Dr. V Cerun-dulo, Oxford University. P815(444/A1.1) cells were described previously, with slight modifications (2, 3).

Ninety-six-well nitrocellulose plates (Multiscreen HA,used as target cells for the detection of H-2Kd-restrictedCD8/ T cells (9), while LM-1 cells were used for the Milipore Corp., Bedford, MA) were coated with 75 ml

of PBS containing 10 mg per milliliter of anti-mousedetection of H-2Kk-restricted CD8/ T cells (10). Thesetarget cells were cultured in DMEM-high glucose IFN-g mAb R4 (purchased from American Type Cul-

ture Collection (ATCC), Rockville, MD). After over-(Gibco, Grand Island, NY), supplemented with 10%FCS (Hyclone Lab. Inc., Logan, UT) and HAT supple- night incubation at room temperature, the wells were

washed with DMEM-high glucose containing 5% FCSment (Sigma Chemical Co., St. Louis, MO). For culturemedium in the ELISPOT assay, we used DMEM-high and incubated with DMEM-high glucose containing

10% FCS for more than 1 hr at 377C. Triplicates ofglucose containing 10% FCS and 1% EL-4 supernatantas described (2, 3). twofold dilution series of spleen cells, starting at 5 1


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105 cells per well, were placed into the mAb-coated shown). In the case of vaccinia virus, infection of micewith doses larger than 5 1 107 pfu produced severewells and cocultured with 105 cells per well of irradi-

ated target cells, coated with the synthetic peptide con- disease and mortality.taining the CD8/ T cell epitope, at a final concentrationof 1 mM, or target cells alone, as control. The plates The Route of Immunization Does Not Significantlywere incubated in a CO2 incubator (5% CO2, at 377C) Affect the Induction or Magnitude of thefor 24 to 28 hr. Subsequently, the plates were exten- Primary CD8/ T Cell Responsesively washed with PBS–Tween (0.05%), and 0.1 ml of2.5 mg per milliliter of solution of biotinylated anti- Next, we analyzed the effect of the route of immuni-

zation on the induction of CD8/ T cells. In these experi-mouse IFN-g mAb XMG1.2 (PharMingen, San Diego,CA) was added to each well. After overnight incubation ments, mice were immunized with 5 1 107 pfu of vac-

cinia virus or 5 1 103 pfu of influenza virus using sixat 47C, the plates were washed with PBS–Tween and0.1 ml of a 1:800 dilution of streptavidine peroxidase different routes of administration: intranasal, subcuta-

neous, intradermal, intramuscular, intraperitoneal,(KPL, Gaithersburg, MD) was added to each well. After1 hr of incubation at room temperature, the plates were and intravenous. The number of CD8/ T cells was de-

termined 10 days after immunization. At this time, wewashed with PBS–Tween and then with PBS alone.The spots were developed by adding a solution of 50 detected the highest number of CD8/ T cells regardless

of the route of immunization.mM Tris, at pH 7.5, containing 1 mg per milliliter ofthe substrate 3,3 *-diaminobenzidine tetrahydrochlo- As shown in Fig. 2A, immunization of mice with vac-

cinia virus, by different routes, induced similar num-ride dihydrate and 5 ml per 10 ml of 30% H2O2 to eachwell. The spots were counted with the aid of a stereomi- bers of CD8/ T cell in all groups. These results may

not be surprising in view of the capacity of vacciniacroscope, and the results are expressed as the meannumber of IFN-g-secreting cells per 106 spleen cells. virus to infect and replicate in different cell types and

tissues. It was, however, unexpected that the route ofimmunization by influenza virus did not significantlyRESULTSaffect the induction of CD8/ T cells (Fig. 2B). Unlikevaccinia, influenza viruses are thought to productivelyThe Induction and Magnitude of the CD8/ T Cellinfect only epithelial cells of the respiratory tract (12).Response Is Determined by the Viral DoseThe induction and magnitude of the CD8/ T cell re-Used for Immunizationsponse generated after subcutaneous immunizationwith this virus were also found to depend on the viralIn order to determine the effect of different immuniz-

ing doses on the induction and magnitude of the CD8/ dose (data not shown).In view of these findings and considering the resultsT cell response, mice were immunized with different

nonlethal amounts of vaccinia and influenza viruses. of recent studies indicating that CD8/ T cells were in-duced by immunization with large numbers of forma-The inoculation of mice with the vaccinia virus was

done by intraperitoneal injection, while the influenza lin-fixed influenza virus (13) or purified recombinantviral protein (14), we investigated the possibility thatvirus was administered by the intranasal route.

As described elsewhere, vaccinia and influenza vi- processing of the viral proteins alone, in the absenceof viral replication, could be responsible for the induc-ruses were engineered to express the same class I

MHC-restricted epitope (SYVPSAEQI) originally iden- tion of CD8/ T cells by subcutaneous influenza virusadministration. We therefore determined the immuno-tified in the CS protein of P. yoelii (6). The frequency

of CD8/ T cells induced by immunization with these genicity of influenza viruses which were exposed sec-onds to UV irradiation, a treatment which abrogatedviruses was measured in the spleen of immunized mice

using the ELISPOT assay. This assay was performed the capacity of these viruses to replicate. In this experi-ment we measured the CD8/ T cell response to theusing spleen cells obtained 10–14 days after immuni-

zation. Immune spleen cells were incubated with P815 SYVPSAEQI plasmodial epitope and also to the TYQ-RTRALV epitope of the NP of influenza virus (7). Thetarget cells coated with the synthetic peptide SYVPSA-

EQI and placed in 96-well filtration plates precoated results shown in Fig. 3 clearly indicate that intranasaland subcutaneous immunizations induce a comparablewith anti-IFN-g mAb (2, 3). The presence of IFN-g

spots was revealed after 24–28 hr, as described in de- CD8/ T cell response to the SYVPSAQEI epitope, whilea lower anti-TYQRTRALV CD8/ T cell response is in-tail in earlier publications (2, 3).

As shown in Figs. 1A and 1B, the number of CD8/ duced by subcutaneous as compared to i.n. immuniza-tion. More importantly, these experiments demon-T cells induced by the immunization closely correlates

with the doses of vaccinia or influenza virus adminis- strate that induction of CD8/ T cell responses to bothepitopes requires fully infectious virus, as UV irradia-tered to the mice. Immunizing doses of influenza virus

greater than 105 pfu per mouse did not increase the tion of the viruses abolishes their capacity to induceCD8/ T cells.frequency of antigen-specific CD8/ T cells (data not



FIG. 1. Dose dependence of primary CD8/T cell responses against influenza and vaccinia viruses. Groups of three BABL/c mice wereimmunized ip with various doses of vaccinia virus PYCS (A) or i.n. with various doses of influenza virus ME (B). Spleen cells from immunizedmice were obtained 10 days later and used as effector cells in the ELISPOT assay. The figure shows the number of IFN-g-secreting cellswhen immune spleen cells were incubated with P815 target cells alone (0) or with P815 cells previously coated with the SYVPSAEQIpeptide (/). Results are expressed as average {SE of duplicate cultures.

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FIG. 2. Effect of the route of immunization on primary CD8/ T cell responses. Groups of three mice were immunized with 5 1 107 pfuof vaccinia virus PYCS (A) or with 5 1 103 pfu of influenza virus ME (B), using different routes: i.n., id, sc, im, iv, and ip. Spleen cells fromthe immunized mice were obtained 10 days later and used as effector cells in the ELISPOT assay. The figure shows the number of IFN-g-secreting cells when immune spleen cells were incubated with P815 target cells alone (0) or with P815 cells previously coated with theSYVPSAEQI peptide (/). Results are expressed as average {SE of triplicate cultures.

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FIG. 3. Lack of CD8/ T cell response to immunization with UV-irradiated influenza virus. Influenza virus ME was UV irradiated for20 sec. A dose of irradiated virus equivalent to 104 pfu before the UV treatment was used to immunize groups of three mice intranasallyor subcutaneously. Control mice were immunized with the same dose of untreated virus. Spleen cells from immunized mice were obtained11 days later and used as effector cells in the ELISPOT assay. The figure shows the number of IFN-g-secreting cells detected after immunespleen cells were incubated with P815 target cells alone (0) or with P815 cells coated with either the SYVPSAEQI or the TYQRTRALVpeptide. Results are expressed as average {SE of triplicate cultures.

Lack of a Secondary CD8/ T Cell Response after Strong Secondary CD8/ T Cell Responses AreInduced after Priming and Boosting with DifferentPriming and Boosting with the Same VirusViruses Expressing the Same Epitope

In order to characterize the conditions for the induc- Because recombinant vaccinia and influenza virusestion of secondary CD8/ T cell responses, we determined express the same epitope, we asked whether successivethe frequency of epitope-specific T cells after repeated immunization with these vectors would induce a sec-immunization with the same virus. Mice were immu- ondary CD8/ T cell response. In these experiments, anized ip with 5 1 107 pfu of vaccinia virus or i.n. with group of mice was first primed with the influenza virus5 1 103 pfu of influenza virus. Twenty days after the and 21 days later boosted with vaccinia virus. Anotherfirst immunizing dose, a second identical viral dose was group was immunized with the same viruses but inadministered, and 10 days later, the frequency of the the reverse sequence, i.e., primed with vaccinia andCD8/ T cells was determined by the ELISPOT assay. boosted with influenza virus. Results shown in Fig. 5As shown in Fig. 4, mice immunized twice with vaccinia clearly indicate that priming with influenza and boost-virus had essentially the same number of CD8/ T cells ing with vaccinia virus induce a strong secondary CD8/as those which received a single immunizing dose. The T cell response, as seen by the striking expansion ofnumber of specific CD8/ T cells detected in spleens of the number of antigen-specific CD8/ T cells, which ismice inoculated twice with influenza virus was also 20–30 times higher than the number of cells detectedsimilar to that observed in mice which received a single in a primary CD8/ T cell response.immunizing dose. The small differences observed be- We found that the sequence in which the respectivetween mice immunized once or twice with the same vectors are used for priming and boosting appears tovector were not detected consistently in different exper- be crucial, since we consistently observed that primingiments. Clearly, both viral systems failed to induce sec- with vaccinia virus followed by boosting with influenzaondary CD8/ T cell responses, since the frequency of virus induced a significantly lower frequency of pep-the CD8/ T cells did not increase after repeated expo- tide-specific CD8/ T cells than the reverse protocol.

The magnitude of this secondary response could not besure of the mice to the same virus.


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FIG. 4. Repeated immunization with the same virus fails to induce a secondary CD8/ T cell response. Groups of three mice wereimmunized once or twice ip with 5 1 107 pfu per mouse of vaccinia virus PYCS (Vacc.), or i.n. with 5 1 103 pfu per mouse of influenzavirus ME (Infl.), as indicated in the figure. The booster dose was administered 20 days after priming. Spleen cells from immunized micewere obtained 10 days after the second immunizing dose and used as effector cells in the ELISPOT assay. The figure shows the numberof IFN-g-secreting cells detected after incubation of immune spleen cells with P815 target cells alone (0) or with P815 cells coated withthe SYVPSAEQI peptide (/). Results are expressed as average {SE of triplicate cultures.

enhanced by changing the route of administration of pressing the influenza virus NP, following the sameprotocol of the experiment shown in Figs. 4 and 5. Thethe booster or by increasing the immunizing dose by a

factor of 100 (data not shown). The characteristics of primary and secondary CD8/ T cell responses againstthe H-2Kk-restricted NP epitope followed essentiallythe secondary responses observed in these experiments

were essentially the same whether the booster dose the same pattern as that observed for the H-2Kd-re-stricted SYVPSAEQI epitope (data not shown). We sub-was administered 21 days (Figs. 4 and 5) or 7 weeks

after priming (Fig. 6). sequently measured the secondary CD8/ T cell re-sponse against the H-2Kk-restricted and the H-2Kd-re-Finally, as for the primary CD8/ T cell response, the

induction of the secondary response did not appear to stricted epitopes using (BALB/cJ 1 A/J)F1 mice, whichbear both of these H-2 molecules. As shown in Fig. 8,be affected by the route of delivery of the priming or

the boosting dose. As shown in Fig. 7, priming with the CD8/ T cell response against the H-2Kd-restrictedSYVPSAEQI epitope followed the same pattern ob-influenza virus followed by a booster with vaccinia vi-

rus resulted in identical secondary responses, regard- served after immunization of BALB/c mice. Measuringthe CD8/ T cell response against the H-2Kk-restrictedless of the routes used for immunization.epitope we found essentially the same result; i.e., mice

CD8/ T Cell Responses against a Different Epitope, primed with influenza virus and boosted with vacciniaby Mice of Different Genetic Backgrounds expressing the same H-2Kk-restricted epitope gener-

ated a strong secondary CD8/ T cell response. ThisThe above-described characteristics of the secondaryresponse was significantly lower in mice primed withCD8/ T cell response are not limited to a particularthe vaccinia virus and boosted with the influenza virus.epitope or strain of mice. This was determined by ana-

lyzing the CD8/ T cell response of C3H/HeOuJ mice DISCUSSIONagainst the H-2Kk-restricted SDYEGRLI epitope of theNP of the influenza virus. Mice were immunized with The main purpose of the experiments described in

this paper was to define the basic parameters involvedinfluenza virus and a recombinant vaccinia virus ex-



FIG. 5. Secondary CD8/ T cell responses after priming and boosting with different viruses expressing the same epitope. Groups of threemice were immunized either i.n. with 104 pfu of influenza virus ME (Infl.) or ip with 5 1 107 pfu of vaccinia virus PYCS (Vacc.), followingthe sequence shown in the figure. The second immunization with the heterogenous virus was administered 21 days after priming. Spleencells from the immunized mice were obtained 13 days after the second immunization and used as effector cells in the ELISPOT assay. Thefigure shows the number of IFN-g-secreting cells detected after immune spleen cells were incubated with P815 target cells alone (0) orwith P815 cells coated with the SYVPSAEQI peptide (/). Results are expressed as average {SE of triplicate cultures.

in the induction of primary CD8/ T cell responses shown that the in vitro or in vivo treatment with anti-CD8 but not with anti-CD4 antibodies completely abro-against influenza and vaccinia viruses, and to deter-

mine the conditions under which antigen-specific CD8/ gates the response detected in the ELISPOT assay (3).For the ELISPOT assay, we used target cells coatedT cells are optimally expanded in vivo, generating

strong secondary responses. with synthetic peptides rather than target cells in-fected with viruses. These peptides represent well-de-The CD8/ T cell responses evaluated in this study

were induced against well-characterized epitopes, fined class I MHC-restricted epitopes which, as shownin previous work, are recognized only by CD8/ T cellsknown to be recognized only by CD8/ T cells. These

responses were measured using a recently developed (3, 7, 8). Therefore, the T cell responses measured inthe ELISPOT assay were only those specific for thequantitative ELISPOT assay to determine the number

of antigen-specific CD8/ T cells present in the spleens epitope represented by the synthetic peptide, eliminat-ing the cytotoxic effect that NK, gd, and CD4/ T cellsof immunized mice. This assay is based on the detection

of IFN-g produced by individual epitope-specific CD8/ may have upon virus-infected target cells (15–18).The effect of the immunizing dose on the CD8/ T cellT cells after incubation with peptide-pulsed target cells

which express only class I MHC molecules. In a previ- response was examined, and we observed that, withina certain range, the magnitude of the response to vac-ous study we have shown that the ELISPOT assay is

more sensitive than the standard chromium release cinia and influenza viruses closely correlated with thesize of the viral inoculum. Infection with larger dosesassay and that it provides quantitative data on T cell

precursors which are entirely comparable with those of infectious viruses, i.e., 106 pfu of influenza virus,made the mice visibly sick and did not increase theobtained by the limiting dilution analysis (2). Further-

more, we have shown that under the experimental con- number of epitope-specific CD8/ T cells. Some of thecharacteristics of the CD8/ T cell response, induced byditions in which this assay is performed, it detects only

IFN-g-secreting CD8/ T cells. In fact, it has been influenza and vaccinia viruses, are likely to differ from


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FIG. 6. Secondary CD8/ T cell responses to booster inoculations given 7 weeks after priming. Groups of three mice were immunizedi.n. with 5 1 103 pfu of influenza virus ME (Infl.) or ip with 3 1 107 pfu of vaccinia virus PYCS (Vacc.), following the immunization sequencesshown in the figure. The second immunization was administered 7 weeks after priming. Spleen cells from the immunized mice were obtained10 days after the second immunization and used as effector cells in the ELISPOT assay. The figure shows the number of IFN-g-secretingcells detected after immune spleen cells were incubated with P815 target cells alone (0) or with P815 cells previously coated with theSYVPSAEQI peptide (/). Results are expressed as average {SE of triplicate cultures.

those observed following infection with persistent or The CD8/ T cell response induced by intranasal andsubcutaneous immunization with influenza virus waslatent viruses. In fact, the initial inoculum with a per-

sistent strain of lymphocytic choriomeningitis virus also analyzed with regard to a second epitope, derivedfrom the NP of this virus. Both routes of immunization(LCMV) influences the CD8/ T cell response in a very

different way, since large viral doses are known to in- generated CD8/ T cells against the TYQRTRALV se-quence, corresponding to an epitope of NP of the influ-duce T cell unresponsiveness (19).

Primary CD8/ T cell responses against vaccinia and enza virus. Importantly and regardless of the routeused for immunization, the induction of CD8/ T cellsinfluenza viruses can be induced regardless of the route

of immunization. Furthermore, we did not detect sig- requires viral replication, as demonstrated by the in-ability of UV-inactivated virus to induce a detectablenificant differences in the number of antigen-specific

CD8/ T cells induced by using different routes of immu- CD8/ T cell response. This finding strongly suggeststhat even when administered subcutaneously, somenization. The successful induction for CD8/ T cells by

different immunization routes is not surprising in the level of transcription of viral genes must occur in cellscapable of inducing CD8/ T cells. Dendritic cells maycase of vaccinia virus, because of its capacity to infect

and replicate in different organs and cell types. A simi- be involved in the induction of these CD8/ T cells, sincethey are highly efficient antigen-presenting cells,lar result was, however, unexpected for immunizations

with influenza virus. This virus may be capable of in- widely distributed in different tissues, and shown tobe susceptible to in vitro infection by influenza virusesfecting different cell types, but generates productive

infections almost exclusively in epithelial cells of the (20). These findings are highly relevant for vaccine de-velopment based on recombinant vectors, as they dem-respiratory tract (12). It appears that the intranasal

dosage (5 1 103 pfu) used to infect mice represents a onstrate that priming of CD8/ T cells is not restrictedby the route of immunization, and that disseminatedlow threshold resulting in minimal replication in the

lungs and thus the CD8/ T cell response is comparable viral infection does not appear to be a necessary condi-tion for optimal induction of antigen-specific CD8/ Tto that observed by other routes of immunization.



FIG. 7. Secondary CD8/ T cell responses are not affected by the route of immunization. Groups of three mice were immunized with 51 103 pfu of influenza virus ME (Infl.) or with 3 1 107 pfu of vaccinia virus PYCS (Vacc.), using the sequence and injection routes indicatedin the figure. The second challenge was administered 21 days after the first challenge. Spleen cells from the immunized mice were obtained11 days after the second immunization and used as effector cells in the ELISPOT assay. The figure shows the number of IFN-g-secretingcells detected after immune spleen cells were incubated with P815 target cells alone (0) or with P815 cells previously coated with theSYVPSAEQI peptide (/). Results are expressed as average {SE of triplicate cultures.

cells. These observations are in agreement with recent In contrast to the lack of secondary responses, afterrepeated immunizations with the same virus, a largereports indicating that Semliki forest (21), canary pox

(22), and fowl pox (23) viruses, which do not undergo expansion of the number of antigen-specific CD8/ Tcells occurred in mice primed and boosted with differ-significant replication in mice, are capable of efficiently

inducing CD8/ T cell responses. ent viruses expressing the same epitope. This strongsecondary CD8/ T cell response was particularly evi-Our results indicate that priming and boosting with

the same virus fail to induce a secondary CD8/ T cell dent when mice were primed with influenza virus andboosted with vaccinia virus. The number of antigen-response, as shown by the lack of expansion of CD8/

T cells. These results are in agreement with earlier specific CD8/ T cells increased by 20–30 times com-pared to that induced by a single immunization, reach-findings, revealing that repeated immunization with

the same influenza virus failed to increase the cytotoxic ing estimated frequencies as high as 1/200–1/120splenocytes. This strong secondary CD8/ T cell re-activity of spleen cells against influenza virus-infected

target cells (24). This differs, however, from recent ob- sponse was not linked to a particular epitope sequence,since it could be induced against the SYVSAPEQI epi-servations suggesting that repeated immunization

with either a recombinant salmonella (25) or a recombi- tope in BALB/c mice and also against the SDYEGRLIepitope in C3H/HeOuJ and (BALB/cJ 1 A/J)F1 mice.nant Semliki forest virus (21) induces a secondary re-

sponse. Taken together these findings appear to indi- Of considerable interest was the finding that themagnitude of this secondary CD8/ T cell response wascate that the capacity for inducing secondary CD8/ T

cell responses may vary among different intracellular entirely dependent on the order of immunization withthe two recombinant viruses. Thus, priming with themicroorganisms. Still, it must be determined whether

the enhanced cytotoxic activity observed after repeated recombinant vaccinia followed by boosting with the in-fluenza virus resulted in a much lower secondary CD8/immunization with some of these microbial agents is

the result of an increased number of antigen-specific response than the reverse protocol.In a previous study we reported that protective im-CD8/ T cells or is due to cytotoxic T cells with an in-

creased interaction with target cells (26). munity against malaria parasites could be induced only


106 MURATA ET AL.

FIG. 8. Induction of secondary CD8/ T cell responses against H-2Kk- and H-Kd-restricted epitopes in (BALB/cJ 1 A/J)F1 mice. Groupsof three (BALB/cJ 1 A/J)F1 mice were immunized i.n. with 5 1 103 pfu of influenza virus ME (Infl.), ip with 5 1 107 pfu of vaccinia virusPYCS expressing the CS epitope SYVPSAEQI (Vacc. CS), or ip with 107 pfu of vaccinia virus NP-VAC expressing the NP epitope SDYEGRLI(Vacc. NP). The immunization protocol for each group is shown in the figure. The second immunization was administered 17 days afterpriming. Spleen cells from the immunized mice were obtained 10 days after the second immunization and used as effector cells in theELISPOT assay. The figure shows the number of IFN-g-secreting cells detected after immune spleen cells were incubated with target cellsalone (0) or with target cells previously coated with the respective peptide. As target cells, P815 cells were used for the detection of CD8/

T cells against SYVPSAEQI (H-2Kd-restricted epitope) and LM-1 cells for the detection of the CD8/ T cells against SDYEGRLI (H-2Kk-restricted epitope). Results are expressed as average {SE of triplicate cultures.

after combined immunization with influenza and vac- ACKNOWLEDGMENTScinia viruses and that the sequence of immunization

We thank Dr. Jerome L. Schulman for helpful suggestions andwas crucial since protective immunity could bediscussions. We also thank Chui Ng, John Hirst, Scott Kerns, andachieved only after priming with influenza virus fol- Ricardo Renvill for excellent technical assistance. This work was

lowed by a booster with recombinant vaccinia virus. supported by grants from the National Institutes of Health (AI-The data described here are consistent with these ear- 27458, AI-36526), Agency for International Development, and a

grant (A.G.-S.) from the Stony Wold-Herbert Fund.lier findings on protection and provide an immunologi-cal basis that explains the restrictions we described for

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