Impact of Recombinant Adenovirus Serotype 35 Priming versus ...

INFECTION AND IMMUNITY, Apr. 2008, p. 1709–1718 0019-9567/08/$08.00⫹0 doi:10.1128/IAI.01614-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Vol. 76, No. 4

Impact of Recombinant Adenovirus Serotype 35 Priming versus Boosting of a Plasmodium falciparum Protein: Characterization of T- and B-Cell Responses to Liver-Stage Antigen 1䌤 Ariane Rodrı´guez,1 Jaap Goudsmit,1,2 Arjen Companjen,1 Ratna Mintardjo,1 Gert Gillissen,1 Dennis Tax,1 Jeroen Sijtsma,1 Gerrit Jan Weverling,1 Lennart Holterman,1 David E. Lanar,3 Menzo J. E. Havenga,1 and Katarina Radosˇevic´1* Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands1; Center of Poverty-Related Communicable Diseases, Academic Medical Center, Amsterdam, The Netherlands2; and Division of Malaria Vaccine Development, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910-75003 Received 6 December 2007/Returned for modification 21 December 2007/Accepted 14 January 2008

Prime-boost vaccination regimens with heterologous antigen delivery systems have indicated that redirection of the immune response is feasible. We showed earlier that T-cell responses to circumsporozoite (CS) protein improved significantly when the protein is primed with recombinant adenovirus serotype 35 coding for CS (rAd35.CS). The current study was designed to answer the question whether such an effect can be extended to liver-stage antigens (LSA) of Plasmodium falciparum such as LSA-1. Studies with mice have demonstrated that the LSA-1 protein induces strong antibody response but a weak T-cell immunity. We first identified T-cell epitopes in LSA-1 by use of intracellular gamma interferon (IFN-␥) staining and confirmed these epitopes by means of enzyme-linked immunospot assay and pentamer staining. We show that a single immunization with rAd35.LSA-1 induced a strong antigen-specific IFN-␥ CD8ⴙ T-cell response but no measurable antibody response. In contrast, vaccinations with the adjuvanted recombinant LSA-1 protein induced remarkably low cellular responses but strong antibody responses. Finally, both priming and boosting of the adjuvanted protein by rAd35 resulted in enhanced T-cell responses without impairing the level of antibody responses induced by the protein immunizations alone. Furthermore, the incorporation of rAd35 in the vaccination schedule led to a skewing of LSA-1-specific antibody responses toward a Th1-type immune response. Our results show the ability of rAd35 to induce potent T-cell immunity in combination with protein in a prime-boost schedule without impairing the B-cell response. cytokine and chemokine milieu for the expansion of antigenspecific immune responses (3, 59). The immunogenicity of rAd, together with the availability of a highly efficient and scalable production platform (17, 21), has fuelled interest in pursuing Ad vectors as a vaccine platform. The preexisting neutralizing immunity limits the use of highly prevalent serotypes, such as Ad serotype 5 (Ad5) (2, 10, 11, 14, 19, 28, 31, 42, 57, 58, 60); however, the development of novel vectors based on low-seroprevalence serotypes that are not influenced by the preexisting immunity toward Ad5, such as Ad35 (1, 4, 60), open new avenues for the use Ad vectors as vaccine vehicles. We demonstrated recently in a nonhuman primate study that a prime-boost immunization regimen combining the rAd35 vector coding for the Plasmodium falciparum circumsporozoite (CS) protein and adjuvanted RTS,S was superior to either modality alone in the induction of T-cell immunity (56). In the current study, we investigated whether this effect can be extended to other Plasmodium falciparum antigens, in particular to liver-stage antigen 1 (LSA-1). LSA-1, an antigen that is specifically expressed in the hepatic stage of P. falciparum life cycle, is a promising malaria vaccine candidate. The LSA-1 contains B- and T-cell epitopes that are immunogenic during the course of natural infection (18). Hill et al. revealed the potential importance of LSA-1 as a vaccine candidate by demonstrating that an LSA-1-derived peptide binds to the HLA B53 molecule, a haplotype associated with resistance to severe malaria (22, 23). Other studies with humans have also associ-

The development of vaccination strategies against malaria receives high priority worldwide. Heterologous prime-boost vaccination regimen is a highly promising approach due to the high immunogenicity it induces compared to that seen for homologous immunizations (15). In addition, the immune response can be synergistically improved or even redirected when combining different types of vaccines (15, 34, 35). A number of studies have indicated that recombinant adenoviral (rAd) vectors are highly suitable as a component of a heterologous prime-boost regimen with other types of vaccines, such as DNA (32, 53, 54), poxvirus vectors (9, 20), or Mycobacterium bovis BCG (46, 55), leading to enhanced immunogenicity and protection in relevant models. rAd vectors are particularly suited for the induction of strong CD8⫹ T-cell responses due to intracellular expression of the transgene built in their genome and efficient routing of expressed protein toward the class I presentation pathway. rAd vectors have been shown to elicit rapid, strong, and persistent immune responses against a number of different antigens (3, 11, 14, 48, 61). The exceptional immunogenicity of rAd vectors is probably due to their ability to activate cells of the immune system and create a favorable

* Corresponding author. Mailing address: Crucell Holland BV, Archimedesweg 4-6, 2333 CN Leiden, The Netherlands. Phone: 31 (0)71 5199100. Fax: 31 (0)71 5199800. E-mail: Katarina.Radosevic @crucell.com. 䌤 Published ahead of print on 22 January 2008. 1709

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ated LSA-1-specific proliferative, cytokine, and antibody responses with resistance to reinfection with malaria parasite (7, 12, 26, 30, 33). Recently, an adjuvanted recombinant LSA-1 protein vaccine was tested in a phase I/IIa clinical trial. Although the preclinical studies with mice demonstrated that this protein vaccine induces a potent antibody response and a detectable CD4⫹ T-cell response (6, 24), no protection against experimental challenge was observed for LSA-1-vaccinated volunteers (16). A reason for this failure to protect may be the poor induction of CD8⫹ T-cell responses, a key factor for protection against preerythrocytic malaria. Therefore, a redirection of the immune response against the LSA-1 antigen may improve the efficacy of this vaccine. In the current study, we aimed to determine whether the low cellular immunity induced with the recombinant LSA-1 protein (6, 24) can be complemented with an rAd35 vector carrying the fully matching LSA-1 sequence, either as a priming or as a boosting vaccine. MATERIALS AND METHODS rAd35.LSA-1, recombinant LSA-1 protein, and peptides. The rAd35 vector for the LSA-1 gene (LSA-NRC sequence), optimized for expression in mammalian cells, was generated as described elsewhere (21). The recombinant LSA-1 protein (LSA-NRC sequence) was produced and purified as previously described (24). The 15-mer peptides overlapping by 11 amino acids spanning the recombinant LSA-1 (6) were synthesized (GenScript, Piscataway, NJ), dissolved in dimethyl sulfoxide at 10 mg/ml, and stored at ⫺80°C until use. Mice and immunizations. BALB/c female mice, 6 to 8 weeks old, were purchased from Harlan (Zeist, The Netherlands) and kept at the Animal Facility of Crucell Holland BV under specific-pathogen-free conditions. The Institutional Committees for Animal Care and Use reviewed and approved the mouse experiments. For the dose-finding study, mice (five per group) were immunized with 108, 109, or 1010 viral particles (VP) of rAd35.LSA-1 or with 1010 VP of an Ad35 vector without the transgene (Ad35.Empty). Two weeks after immunization, spleen cells and sera were collected for determination of LSA-1-specific T-cell and antibody responses. For the prime-boost experiments, mice (five per group) were immunized at 0, 3, and 6 weeks. Heterologous regimens involved one vaccination with 1010 VP of rAd35.LSA-1 followed by two injections of 10 ␮g of adjuvanted LSA-1 protein or two injections of adjuvanted protein followed by an Ad35 vaccination. Homologous regimens involved immunizations with either rAd35.LSA-1 (single injection) or adjuvanted LSA-1 protein (three injections). The LSA-1-specific T-cell and antibody responses were determined 2 weeks after the last immunization. As negative controls, mice (five per group) were immunized with Ad35.Empty or adjuvant alone. For administration of rAd35.LSA-1 or Ad35.Empty, mice were immunized intramuscularly (i.m.) with the required vector dose in a total volume of 100 ␮l of phosphate-buffered saline (PBS) containing 5% sucrose (50 ␮l/quadriceps of hind legs). Recombinant LSA-1 protein was administered subcutaneously in a volume of 100 ␮l upon adjuvation with 70% (vol/vol) Montanide ISA 720 (Seppic Inc., Paris, France). LSA-1-specific T-cell response. T-cell epitope mapping was performed 2 weeks after the last immunization as described elsewhere (41). For epitope mapping, splenocytes from mice immunized with rAd35.LSA-1 (five per group) or Ad35.Empty (three per group) were pooled and stimulated with different pools of overlapping 15-mer peptides covering the whole sequence of the LSA-1 recombinant protein. Peptide pools inducing gamma interferon (IFN-␥) production were identified by flow cytometry using intracellular cytokine staining in combination with surface staining of CD4 and CD8 markers (ICS). Induction of LSA-1-specific IFN-␥ production upon homologous and heterologous vaccination regimens was determined using the enzyme-linked immunospot (ELISPOT) assay 2 weeks after immunizations. Ninety-six-well multiscreen plates (Millipore, Bedford, MA) coated overnight with rat anti-mouse IFN-␥ (Pharmingen, San Diego, CA) were washed three times with Dulbecco’s PBS (Life Technologies, Gaithersburg, MD) containing 0.25% Tween 20 (DPBS–Tween) and blocked with D-PBS containing 5% fetal bovine serum for 2 h at 37°C. Splenocytes were prepared in R10 medium and plated in duplicate at

INFECT. IMMUN. 5 ⫻ 105 cells/well and 2 ⫻ 105 cells/well in 100-␮l reaction volumes containing 1 ␮g/ml peptides. Following 18 h of incubation at 37°C with 10% CO2, the plates were washed with D-PBS–Tween and incubated for 1.5 h with a biotinylated rat anti-mouse IFN-␥ (Pharmingen, San Diego, CA). Plates were washed and incubated for 1.5 h with streptavidin-alkaline phosphatase (Southern Biotechnology Associates, Birmingham, AL). Upon final washing, specific staining was developed with nitroblue tetrazolium–5-bromo-4-chloro-3-indolyl-phosphate chromogen (Pierce, Rockford, IL), stopped by washing with tap water, air dried, and analyzed using an Aelvis ELISPOT reader (Aelvis GmbH). For pentamer staining, an H-2Kd/YYIPHQSSL allophycocyanin-labeled pentamer (LSA-1 pentamer) (Proimmune, Oxford, United Kingdom) was used to stain the antigen-specific CD8⫹ T cells. Mice were bled via tail cut every week upon the last immunization. Mouse blood was collected in RPMI 1640 containing 40 U/ml heparin. After washing the cells with RPMI, red blood cells were lysed in 0.017 M Tris-0.14 M NH4Cl buffer. Upon centrifugation, the remaining cells were washed with PBS containing 0.1% bovine serum albumin (BSA) (PBS-0.1% BSA) and passed through a 30-␮m preseparation filter (Miltenyi Biotec, Auburn, CA). Cells were incubated with 7.5 ␮l of allophycocyaninlabeled LSA-1 pentamer for 15 min in the dark at room temperature. After being washed, cells were incubated with a fluorescein isothiocyanate-labeled antiCD8␣ monoclonal antibody (BD Pharmingen, San Diego, CA) for 30 min. Finally, the cells were washed and fixed in 0.25 ml PBS containing 2.5% paraformaldehyde and 1% fetal bovine serum. Samples were analyzed by two-color flow cytometry with a FACSCalibur instrument (BD Pharmingen, San Diego, CA). Gated CD8⫹ T lymphocytes were analyzed for staining with the LSA-1 pentamer. The CD8⫹ T lymphocytes from naı¨ve mice, utilized as negative controls, exhibited on average fewer than 0.1% pentamer-positive cells. LSA-1-specific antibody response. The LSA-1-specific antibody response was determined using enzyme-linked immunosorbent assay as previously described (24). Ninety-six-well microtiter plates (Maxisorp; Nunc) were coated overnight at 4°C with 1 ␮g/ml of LSA-1 protein in PBS. Plates were washed and blocked with PBS containing 3% BSA and 0.05% Tween 20 for 1 h at 37°C. After the plates were washed, 1:100-diluted individual serum samples were added to the wells and serially twofold diluted. Plates were incubated for 2 h at room temperature. Plates were washed again and incubated with biotin-labeled anti-mouse immunoglobulin G (IgG) (Dako, Denmark) and afterwards with horseradish peroxidase-conjugated streptavidin (Pharmingen San Diego, CA) for 30 min each at 37°C. For detection of the IgG subclasses, samples were incubated with horseradish peroxidase-labeled anti-mouse IgG1 or IgG2a antibodies (Southern Biotech, Birmingham, AL). Finally, the plates were washed and 100 ␮l of 3-3,5,5⬘tetramethylbenzidine (TMB) substrate (Lucron Bioproducts, Gennep, The Netherlands) was added to each well. After 10 min, the reaction was stopped by adding 50 ␮l/well of 1 M HCl. The optical density was measured at 450 nm using a Bio-Tek reader (Bio-Tek Instruments, Winooski, VT). Statistical analysis. Statistical analyses were performed using SPSS version 12.0.1. Experimental groups were compared using analyses of variance and post hoc testing according to the Bonferroni adjustment (the negative controls were omitted from analyses to increase stringency). In the case of the IgG2a/IgG1 ratios, t testing was used to compare experimental groups. The difference was considered significant when the P value was ⬍0.05.

RESULTS T-cell epitope mapping upon rAd35.LSA-1 immunization. To generate immunological tools for characterization of the immune responses induced by different vaccination regimens, we performed T-cell epitope mapping. Identification of CD4⫹ and CD8⫹ T-cell epitopes was performed for BALB/c (H-2d) mice 2 weeks after immunization with rAd35.LSA-1 and using 15-mer peptides arranged in a matrix as described previously (6, 41). The different steps involved in the epitope mapping are depicted in Fig. 1, in which the identified CD8⫹ T-cell epitope is used as an example. The pooled splenocytes from immunized mice were stimulated with row (R) and column (C) peptide pools from the LSA-1 peptide matrix, and IFN-␥ production by T cells was measured using ICS (Fig. 1A). The interception of positive R and C peptide pools allowed the identification of potential T-cell-reactive peptides (Fig. 1B). Further confirmation of identified CD4 and CD8 15-mer pep-

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FIG. 1. Example of epitope mapping in BALB/c mice immunized with rAd35.LSA-1. Groups of mice were immunized i.m. with rAd35.LSA-1 or Ad35.Empty. (A) Two weeks after the immunization, the frequencies of IFN-␥-producing cells among splenocytes stimulated with R and C 15-mer peptide pools was determined using ICS. The dotted line indicates the background level, i.e., the average frequency of IFN-␥-producing cells in splenocytes from Ad35.Empty-immunized mice. (B) Potentially reactive CD4 and CD8 15-mer peptides (corresponding to those shown in panel A) were selected from the interception of the positive R and C peptide pools. This peptide pool matrix table has been modified from reference 6. (C) Confirmation of the positive 15-mer peptides was performed in a second immunization experiment using ICS. None, no peptide added. (D and E) The predicted 9-mer peptide (D) and the corresponding LSA-1 pentamer (E) were generated and tested in subsequent immunization experiments. SFU, spot-forming units.

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tide(s) was performed in a second immunization experiment using ICS (Fig. 1C). In our study, we identified two CD8⫹ T-cell-reactive overlapping peptides, SENERGYYIPHQSSL (peptide 50, amino acids 199 to 213; LSA-NRC sequence) and RGYYIPHQSSL PQDN (peptide 51, amino acids 203 to 217), and two CD4⫹ T-cell-reactive overlapping peptides, QVNKEKEKFIKSLFH (peptide 101, amino acids 403 to 417) and EKEKFIKSLFH IFDG (peptide 102, amino acids 407 to 421). In the ICS assay, the CD4 responses were relatively low compared to the CD8 responses, while by the ELISPOT assay the difference was less pronounced (data not shown), confirming a previously published observation of the high sensitivity of the ELISPOT assay relative to that of the ICS for the detection of antigen-specific CD4 responses (41). Based on the overlapping sequence of the BALB/c-reactive CD8⫹ T-cell 15-mer peptides and theoretical prediction using SYFPEITHI (www.syfpeithi.de), the YYIPHQSSL 9-mer peptide and the corresponding LSA-1 pentamer were generated and their specificities confirmed in subsequent ELISPOT (Fig. 1D) and FACS (Fig. 1E) experiments, respectively. The T-cell epitopes identified in our study have been described earlier for the LSA-1 antigen in other studies (5, 6, 40, 52). Dose finding for rAd35.LSA-1. In order to determine the dose of rAd35.LSA-1 to be used in the prime-boost immunizations, mice were immunized with an increasing vector dose of 108, 109, and 1010 VP. Two weeks after the immunization, IFN-␥ production by splenocytes from individual mice stimulated with the identified CD4-reactive 15-mer peptides (peptide 101, QVNKEKEKFIKSLFH; peptide 102, EKEKFIKSL FHIFDG) and CD8-reactive 15-mer peptides (peptide 50, SE NERGYYIPHQSSL; peptide 51, RGYYIPHQSSLPQDN) was assayed using the ELISPOT assay. As shown in Fig. 2, IFN-␥ T-cell responses were induced in a vector dose-dependent manner. CD4⫹ and CD8⫹ T cells were detected at VP doses of 109 and 1010. We selected 1010 VP as the immunization dose, since at this dose the highest numbers of IFN-␥producing cells were detected. T-cell epitope mapping upon prime-boost immunizations. In order to address whether heterologous prime-boost vaccination regimens would expand the repertoire of CD4- and CD8reactive peptides compared to what was seen for homologous regimens, we performed an additional T-cell epitope mapping 2 weeks after the last immunization. Homologous vaccination regimens consisted of immunizations with either rAd35.LSA-1 (one immunization) or adjuvanted LSA-1 protein (three immunizations). Heterologous regimens consisted of either one vaccination with rAd35.LSA-1 followed by two immunizations with adjuvanted LSA-1 protein or two vaccinations with adjuvanted protein followed by one rAd35.LSA-1 immunization. Splenocytes from rAd35.LSA-1-immunized mice exhibited IFN-␥ production upon stimulation with peptide pools R4/C8 and R7/C1, corresponding to CD8-reactive 15-mer peptides 50 and 51, respectively (Fig. 3), thus confirming results obtained in the initial epitope mapping experiments (Fig. 1). Accordingly, low numbers of IFN-␥-producing CD4⫹ T cells were detected upon stimulation with peptide pools R3/C4 and R5/ C10 (Fig. 3), corresponding to CD4-reactive 15-mer peptides 101 and 102, respectively (Fig. 1). Upon the same stimulation,

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FIG. 2. rAd35.LSA-1 dose response. Mice were immunized i.m. with 108 to 1010 VP of rAd35LSA-1 or 1010 VP of Ad35.Empty (negative control). Two weeks after the immunization, splenocytes were collected and stimulated with the identified LSA-1 CD4- and CD8reactive peptides. The IFN-␥-producing T cells were quantified using the ELISPOT assay. The bars represent the geometric means of spotforming units (SFU).

splenocytes from protein-immunized mice contained barely detectable IFN-␥-producing CD8⫹ or CD4⫹ cells. After the heterologous prime-boost vaccination regimens using rAd35.LSA-1 and adjuvanted LSA-1 protein, CD4⫹ and CD8⫹ IFN-␥-producing cells were detected upon stimulation of splenocytes with the peptide pools, although no additional CD4 or CD8 epitopes were identified over what was seen for the homologous vaccination regimens. It is interesting that in the rAd35-priming/protein-boosting regimen, the CD8 responses were comparable to the responses observed upon immunization with rAd35.LSA-1 alone, while the CD4 responses were clearly increased. Contrary to this, in the protein-priming/rAd35-boosting regimen the CD8 responses were increased, while the CD4 responses remained similar to what was seen for the rAd35.LSA-1 single immunization. Characterization of T-cell responses upon prime-boost immunizations. Production of IFN-␥ induced by the different vaccination regimens was assessed by use of ELISPOT assay upon stimulation of splenocytes with the two CD4-reactive 15-mer peptides and the CD8-reactive 9-mer peptide (YYIP HQSSL) at 2 weeks after immunizations. As shown in Fig. 4 and in agreement with the dose-finding data, immunization with rAd35.LSA-1 efficiently induced both IFN-␥ CD4⫹ and CD8⫹ T-cell responses, yet CD8⫹ T cells were more prominent. On the other hand, protein immunizations elicited barely detectable CD4ⴙ and CD8⫹ IFN-␥ responses. The combination of rAd35.LSA-1 and LSA-1 protein in heterologous prime-boost vaccination regimens resulted in the induction of high IFN-␥ responses, with a more balanced contribution of

FIG. 3. Epitope mapping after homologous and heterologous vaccinations. Groups of mice (eight per group) were immunized with either rAd35.LSA-1 (Ad35), adjuvanted LSA-1 protein (Protein), rAd35.LSA-1 followed by adjuvanted protein (Ad35/Prot), or adjuvanted protein followed by rAd35.LSA-1 (Prot/Ad35). Two weeks after immunization, splenocytes from groups of mice were collected, pooled, and stimulated with R and C peptide pools. The frequencies of IFN-␥-producing CD4⫹ and CD8⫹ cells were measured using ICS. The dotted line represents the background level, i.e., the average frequency of IFN-␥-producing cells in splenocytes from Ad35.Empty-immunized mice. None, no peptide added. 1713

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FIG. 4. LSA-1-specific T-cell responses induced by homologous and heterologous immunization regimens. Groups of mice were immunized as described for Fig. 3. Two weeks after immunizations, the splenocytes from individual mice were stimulated with CD8- and CD4-reactive peptides, and the IFN-␥ production was measured using the ELISPOT assay. The bars represent the geometric means of spot-forming units (SFU). ⴱ, P ⬍ 0.05 (heterologous versus homologous groups).

CD4⫹ and CD8⫹ T cells. Compared to what was seen upon the single rAd35.LSA-1 immunization, the increase in the CD8 responses induced upon heterologous prime-boost immunizations did not reached statistical significance (rAd35-priming/ protein boosting P ⫽ 0.925; protein priming/rAd35 boosting P ⫽ 0.050). In the case of the CD4 responses, the increase was significant for rAd35-priming/protein-boosting regimen (P ⫽ 0.001) but the P value was just under the level of significance for the protein-priming/rAd35-boosting regimen (P ⫽ 0.050). Comparable to the results obtained with the epitope mapping, the ELISPOT assay data showed that the protein-priming/ rAd35-boosting regimen favors augmentation of CD8 responses, whereas rAd35 priming/protein boosting favors the CD4 responses. The kinetics of the CD8⫹ T-cell responses elicited by homologous and heterologous vaccination regimens was followed for 7 weeks after the last immunization by use of LSA-1 pentamer staining of circulating T cells. As illustrated in Fig. 5, vaccination regimens involving an administration of rAd35.LSA-1 just prior to the analysis, i.e., rAd35.LSA-1 single immunization (Fig. 5A) and heterologous protein-priming/rAd35-boosting regimen (Fig. 5B), exhibited the highest percentages of LSA-1 pentamer-positive CD8⫹ T cells. Compared to what was seen for the rAd35.LSA-1 single immunization, the percentage of LSA-1 pentamer-positive CD8⫹ T cells obtained in the protein-priming/rAd35-boosting regimen was significantly high (P ⬍ 0.001). The rAd35-priming/protein-boosting regimen (Fig. 5B) exhibited lower percentages of LSA-1 pentamerpositive CD8⫹ T cells, which were comparable to the percentages obtained 7 weeks after the immunization with rAd35.LSA-1

(Fig. 5A), indicating that no significant boost of CD8⫹ T-cell responses was elicited with protein immunizations. Finally, with the homologous protein vaccination regimen, no significant number of LSA-1 pentamer-positive CD8⫹ T cells was detected (Fig. 5A). These results support the finding that the protein-priming/rAd35-boosting regimen favors the augmentation of CD8 responses. Antibody responses induced upon prime-boost vaccination regimens. In order to assess a possible effect of heterologous vaccination on the LSA-1-specific antibody response, we measured the antibody responses induced by the different vaccination regimens at 2 weeks after immunizations. Enzyme-linked immunosorbent assay results revealed that vaccination with the adjuvanted recombinant LSA-1 protein induced high levels of antigen-specific IgG antibodies, whereas rAd35.LSA-1-immunized mice did not exhibit detectable LSA-1-specific IgG antibodies (Fig. 6A). Comparable to homologous protein vaccinations, the heterologous regimens exhibited high LSA-1specific IgG responses. In addition to the total LSA-1-specific IgG antibodies, we determined the levels of LSA-1-specific IgG1 and IgG2a isotypes. We calculated the IgG2a/IgG1 ratio as an indication of the type of T-helper responses elicited by the different vaccination regimens. As shown in Fig. 6B, LSA-1 protein immunizations induced predominantly IgG1 antibody responses, indicating a Th2-biased response. However, the combination of rAd35.LSA-1 and protein immunizations exhibited higher IgG2a/IgG1 ratios, indicating a more balanced Th1/Th2 type of response (rAd35 priming/protein boosting P ⫽ 0.018; protein priming/rAd35 boosting P ⫽ 0.050).

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FIG. 5. LSA-1 pentamer staining of CD8⫹ cells upon homologous and heterologous vaccinations. Groups of mice were immunized as described for Fig. 3. The pentamer staining of blood cells was performed weekly for 7 weeks after the last immunization. The percentages of LSA-1 pentamer-positive CD8⫹ cells after homologous (A) and heterologous (B) immunizations were determined using flow cytometry. The symbols indicate mean percentages of positive cells, while bars show standard errors of the means.

DISCUSSION

FIG. 6. LSA-1-specific antibody responses induced by homologous and heterologous immunization regimens. Groups of mice were immunized as described for Fig. 3. (A) Two weeks after immunizations, the level of LSA-1-specific total IgG was determined for sera from individual mice. (B) Additionally, the titers of LSA-1-specific IgG1 and IgG2a isotypes were determined and the IgG2a/IgG1 ratio was calculated. Bars represent the geometric means of the endpoint titers (A) or the ratios (B). ⴱ, P ⬍ 0.05 (heterologous versus homologous groups).

In this study, we demonstrate that combining the LSA-1 protein with the low-seroprevalence serotype rAd35 vector expressing the identical LSA-1 antigen in prime-boost immunizations efficiently enhanced the IFN-␥ T-cell responses. At the same time, combined regimens did not hamper the level of LSA-1-specific antibody response induced with the protein but rather skewed the Ig isotypes toward a Th1 type of response. Additionally, we found that the rAd35 vector when utilized as a priming agent complemented the protein vaccine in a manner different from that seen when it was used as a boosting agent. When rAd35 was used as a priming vaccine, it significantly enhanced the number of IFN-␥-producing CD4⫹ cells, while when used as a boosting modality, it had a more profound effect on IFN-␥-producing CD8⫹ cells. Epidemiological studies of the correlation between responses against LSA-1 and protection against malaria support the promise of this antigen as a malaria vaccine candidate (7, 12, 22, 23, 26, 30, 33). Evaluation of an adjuvanted LSA-1 protein vaccine in preclinical studies had shown the induction of potent antibody responses but undetectable CD8 responses and no protection in a phase I/IIa clinical trial (16). While antibodies have been correlated with protection from infection

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with the malaria parasite (25, 27), T cells are believed to play a critical role in the elimination of liver-stage parasites and protection against the disease (29, 36). Studies with mice indicate that a major mechanism of protection against liver-stage malaria is the production of IFN-␥ by activated CD8⫹ T cells, which induces infected hepatocytes to synthesize nitric oxide, a molecule with a potent antiparasitic activity (8, 13, 50, 51). Other mechanisms implicated in protection are cytotoxicity by activated cytotoxic T lymphocytes through the release of perforin and granzyme B and the induction of apoptosis by the cross-linking of Fas molecules expressed on the infected hepatocytes (8, 44). These findings emphasize the importance of developing preerythrocytic malaria vaccines and vaccination strategies that are able to induce not only antibody response but also a strong T-cell immunity. In this regard, heterologous prime-boost vaccination schedules are highly attractive, since they can expand and activate both arms of the immune system. Ad vectors have been extensively used in the development of vaccines against diseases where a strong T-cell response is required (3, 43, 45, 47). They are particularly attractive because of their intrinsic ability to induce a strong T-cell response characterized by high levels of IFN-␥ and cytotoxic activity (59). In our study, the analysis of the T-cell responses in the spleen by ELISPOT assay and ICS indeed showed that immunizations with rAd35.LSA-1 efficiently induce IFN-␥ T-cell responses, in contrast to the protein immunizations. The IFN-␥ response elicited upon rAd35.LSA-1 immunization was largely mediated by CD8⫹ T cells, while in the case of protein immunizations the IFN-␥ response was due to a few antigen-specific CD4⫹ T cells. In addition to the T-cell responses measured in the spleen, the pentamer staining revealed a high frequency of circulating LSA-1-specific CD8⫹ T cells associated with rAd35.LSA-1 immunization. The production of IFN-␥ by CD4⫹ but not by CD8⫹ T cells upon vaccination with adjuvanted LSA-1 protein has been reported previously for mice (6) and more recently for nonhuman primates (39). This finding is not surprising, since recombinant protein vaccines formulated with adjuvants have been shown to generally elicit humoral and CD4 responses but poor or no CD8⫹ T-cell responses (49). It should be mentioned that the number of CD4⫹ IFN-␥-producing T cells upon immunization with the adjuvanted LSA-1 protein that we measured was lower than found in the previous studies (6, 24). This discrepancy might be explained by the use of different adjuvants for the formulation of the LSA-1 protein and/or the use of protein instead of 15-mer peptides for the stimulation of splenocytes in the ELISPOT assay. Further analysis of the T-cell responses showed that the combination of the rAd35 vector and the adjuvanted protein in prime-boost vaccinations resulted in the enhancement of the IFN-␥ CD4⫹ and CD8⫹ T-cell responses compared to what was seen for either modality alone. Interestingly, the overall data on the T-cell responses pointed toward a different quality of Ad35 depending on its use as a priming or a boosting vaccine. Epitope mapping data clearly showed that Ad35 as a priming agent favors the enhancement of CD4 responses, whereas Ad35 as a boosting agent favors the enhancement of CD8 responses. This observation was corroborated by the results obtained in the ELISPOT assay and with pentamer staining. A difference in the effect of Ad35 when used as a priming or a boosting vaccine has been indicated in an earlier study in

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which prime-boost immunizations of nonhuman primates with rAd35.CS and adjuvanted RTS,S protein were performed (56). Although the complete sequences of the antigen expressed by the rAd35 vector and the adjuvanted protein were not completely matching (N-terminal truncation in the protein), the study showed that rAd35 priming/protein boosting was a superior regimen for inducing IFN-␥-producing CD4⫹ T cells. The data from our epitope mapping and ELISPOT assay showing the increase of IFN-␥-producing CD4⫹ T cells in the rAd35priming/protein-boosting regimen are in line with this finding. The analysis of the antibody responses showed that immunization with rAd35.LSA-1 did not induce any detectable anti-LSA-1 antibody response, while protein immunizations induced a strong IgG response. The absence of LSA-1-specific antibody responses in mice immunized with rAd35.LSA-1 was surprising, since rAd35 has been reported in other studies to induce antibody responses against the transgene expressed (37, 38). A possible explanation of our results is that the LSA-1 protein expression and the antigen processing upon immunization are not optimal for the induction of T-helper cells required to trigger anti-LSA-1 antibody responses, probably due to the modified sequence of the protein (24). In line with this hypothesis, it is noteworthy that a relatively high dose of rAd35 (1010 VP) was required to induce significant T-cell responses, in contrast to our previous results with other rAd35 vectors where a 1-log-lower dose (109 VP) was sufficient to induce an optimal T-cell response (37, 38, 41). The heterologous vaccination regimens elicited a level of anti-LSA-1 IgG responses comparable to that seen for the protein vaccinations. However, the analysis of the IgG subclasses revealed that heterologous vaccinations with rAd35 and protein induced levels of IgG2a higher than those seen for homologous protein vaccination. These data indicate that although the rAd35.LSA-1 immunization alone could not trigger an anti-LSA-1 antibody response, when combined with protein immunizations it did promote the cytokine milieu favoring IgG isotype switching toward IgG2a, thus skewing the immune responses toward the Th1 type. Altogether, our study strengthens the scientific evidence highlighting the value of rAd35 as a potent inducer of the cellular immune responses and Th1 skewing when used as either a priming or a boosting vaccine with a protein in a prime-boost immunization schedule. Although animal models may not exactly predict the immune responses in humans and thus always require confirmation in clinical trials, our work provides relevant information encouraging further research into vaccination strategies for malaria. REFERENCES 1. Abbink, P., A. A. Lemckert, B. A. Ewald, D. M. Lynch, M. Denholtz, S. Smits, L. Holterman, I. Damen, R. Vogels, A. R. Thorner, K. L. O’Brien, A. Carville, K. G. Mansfield, J. Goudsmit, M. J. Havenga, and D. H. Barouch. 2007. Comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups B and D. J. Virol. 81: 4654–4663. 2. Barouch, D. H., P. F. McKay, S. M. Sumida, S. Santra, S. S. Jackson, D. A. Gorgone, M. A. Lifton, B. K. Chakrabarti, L. Xu, G. J. Nabel, and N. L. Letvin. 2003. Plasmid chemokines and colony-stimulating factors enhance the immunogenicity of DNA priming-viral vector boosting human immunodeficiency virus type 1 vaccines. J. Virol. 77:8729–8735. 3. Barouch, D. H., and G. J. Nabel. 2005. Adenovirus vector-based vaccines for human immunodeficiency virus type 1. Hum. Gene Ther. 16:149–156. 4. Barouch, D. H., M. G. Pau, J. H. Custers, W. Koudstaal, S. Kostense, M. J. Havenga, D. M. Truitt, S. M. Sumida, M. G. Kishko, J. C. Arthur, B.

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