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Th1 immune response to Plasmodium falciparum circumsporozoite protein is 7
boosted by adenovirus vectors 35 and 26 with homologous insert 8
9
Katarina Radošević1*
, Ariane Rodriguez1^
, Angelique A.C. Lemckert
1^†, Marjolein van 10
der Meer1, Gert Gillissen
1, Carolien Warnar
1, Rie von Eyben
1, Maria Grazia Pau
1 and 11
Jaap Goudsmit1,2
12
13
1Crucell Holland B. V., Leiden, The Netherlands and
2Center of Poverty-related 14
Communicable Diseases, Academic Medical Center, Amsterdam, The Netherlands
15
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Running Title: A 3-component heterologous prime-boost vaccination for malaria 17
18
^ Both authors contributed equally 19
† Present address: Batavia Bioservices B.V., Leiden, The Netherlands 20
* Corresponding Author. Mailing address: Crucell Holland BV, Archimedesweg 4-6, 21
2333 CN Leiden, The Netherlands. Phone: 31 (0)71 5199286. Fax: 31 (0)71 5199800. 22
E-mail: [email protected]. 23
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.00311-10 CVI Accepts, published online ahead of print on 8 September 2010
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ABSTRACT 1
The most advanced malaria vaccine, RTS,S, is comprised of an adjuvanted portion of the 2
Plasmodium falciparum circumsporozoite (CS) protein, fused to and admixed with the 3
hepatitis B surface antigen. This vaccine confers short term protection against malaria 4
infection with an efficacy of about 50% and induces particularly B-cell and CD4+ T-cell 5
responses. In the present study the hypothesis is tested that Th1 immune response to CS 6
protein, in particular the CD8+ T-cell response, which is needed for strong and lasting 7
malaria immunity, is boosted to sustainable levels using the Ad35.CS/Ad26.CS 8
combination. In this study we evaluated immune responses induced with vaccination 9
regimens based on an adjuvanted yeast-produced complete CS protein followed by two 10
recombinant low seroprevalent adenoviruses expressing P. falciparum CS antigen, 11
Ad35.CS (subgroup B) and Ad26.CS (subgroup D). Our results show that (i) the yeast-12
produced adjuvanted full-length CS protein is highly potent in inducing high CS-specific 13
humoral responses in mice, but poor T-cell response (ii) the Ad35.CS vector boosts the 14
IFNγ+ CD8+ T-cell response induced by the CS protein immunization and shifts the 15
immune response towards the Th1 type and (iii) a three-component heterologous 16
vaccination comprised of a CS protein prime, followed by boosts with Ad35.CS and 17
Ad26.CS, elicits an even more robust and sustainable IFNγ+ CD8+ T-cell response as 18
compared to one or two component regimens. The Ad35.CS/Ad26.CS combination 19
boosted particularly the IFNγ+ and TNFα+ T cells, confirming the shift of the immune 20
response from the Th2 to Th1 type. 21
These results support the notion of first immunizations of infants with an adjuvanted CS 22
protein vaccine, followed by a booster Ad35.CS/Ad26.CS vaccine at later age, to induce 23
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lasting protection against malaria for which the Th1 response and immune memory is 1
required. 2
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INTRODUCTION 1
Almost forty years after the feasibility of vaccination against malaria was first 2
demonstrated by means of irradiated sporozoites (9), a vaccine modality that efficiently 3
induces long-lived protective immunity remains elusive. The most advanced CS-based 4
malaria vaccine candidate to date is RTS,S, a vaccine based on a fragment of 5
Plasmodium falciparum circumsporozoite (CS) protein, fused to and admixed with 6
hepatitis B surface protein. In adults, RTS,S with adjuvant AS02 has consistently 7
conferred 40% protection against malaria infection upon sporozoite challenge (53). Even 8
though RTS,S/AS02 induces high level CS-specific antibody responses, the induced T-9
cell responses are weak (21). As Th1 response, and in particular IFNγ and CD8+ T cells, 10
are associated with protection, novel adjuvant systems were developed with the aim to 11
improve the induced T-cell response while maintaining potent levels of CS-specific 12
antibody responses. One of these novel adjuvant systems, AS01, demonstrated its 13
suitability in mice as it improved CS-specific CD4+ T-cell responses and led to induction 14
of CD8+ T cells (31). Non-human primate studies also demonstrated that RTS,S with 15
AS01 adjuvant induces strong CS-specific antibody responses as well as higher mean 16
frequencies of IFNγ- and TNFα-producing CD4+ T cells compared to the RTS,S with 17
AS02 adjuvant. However, the induction of CD8+ T cells was not confirmed in this non-18
human primate study (31). In humans, RTS,S/AS01 has shown to induce high titers of 19
CS-specific antibodies, higher numbers of Th1 CD4+ T cells compared to RTS,S/AS02, 20
but no CS-specific CD8+ T cells (22). Though, RTS,S/AS01 was able to afford 50% 21
protection against malaria infection in adults upon sporozoite challenge (22) and 53% 22
efficacy against disease in children between the ages of 5 to 17 months (5). These results, 23
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albeit far of being optimal, supported the progress of RTS,S/AS01 to phase III clinical 1
trial testing in early 2009, enrolling children at the age of 6 weeks to 17 months at 2
multiple sites in Sub-Saharan Africa. It is anticipated that RTS,S/AS01 will be the first 3
licensed malaria vaccine, provided its efficacy is confirmed in the phase III trial. 4
Albeit our understanding about the correlate(s) of protection for malaria is limited, there 5
is ample evidence that circumsporozoite (CS) protein-specific antibodies, CD8+ T cells 6
and Th1 cytokines, and in particular IFNγ, play a central role in controlling the pre-7
erythrocytic and early liver stages of malaria (19, 20, 34, 46, 56). Adenoviral vectors are 8
particularly suited for induction of IFNγ-producing CD8+ T cells required to combat 9
malaria infection (32, 42), due to intracellular expression of a transgene inserted in the 10
vector genome and efficient routing of expressed protein towards the class I presentation 11
pathway. Recently, we have demonstrated the advantage of utilizing two recombinant 12
adenoviral vectors derived from distinct serotypes, Ad35.CS and Ad5.CS, in a 13
heterologous prime-boost regimen in mice and non-human primates (45). This 14
heterologous prime-boost regimen elicited high CS-specific IFNγ+ T-cell response as 15
well CS-specific Th1 type antibodies able to bind malaria parasites. Though the Ad5-16
based vectors are very potent vaccines, the high prevalence of pre-existing immunity 17
towards Ad5 in the human population hampers their immunogenicity and clinical utility 18
(8, 37). Low seroprevalence of Ad5 neutralizing antibodies in infants of 6 months to 1.5 19
years of age offers an opportunity to administer Ad5-based vaccines to this population 20
without antibodies interfering and neutralizing the vaccine efficacy (41), however, 21
acceptance of this approach by regulatory agencies remains elusive. Novel vaccine 22
vectors based on rare low seroprevalent Ad serotypes have an advantage of not being 23
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hampered by anti-Ad5 immunity while inducing a strong immune response (1, 4, 28, 32, 1
40). 2
Within the current study we evaluate whether vaccination with Ad35.CS and Ad26.CS 3
can enhance the CS-specific immune response induced by a yeast-produced full-length 4
CS protein vaccine, and in particular, whether the combined vaccination sustainably 5
potentiate the Th1 responses necessary for protection against malaria. The Ad35.CS 6
vaccine candidate is currently being evaluated in a Phase 1 clinical study, in partnership 7
with the National Institute of Allergy and Infectious Diseases, and so far has shown safe. 8
Ad35-based vaccine candidates against other infectious diseases, i.e. tuberculosis and 9
HIV, have also been clinically evaluated, and demonstrated safe and immunogenic. 10
Recently, an Ad26-vectored vaccine against HIV was also been clinically assessed in a 11
Phase I study, which showed that a 3-dose regimen of this HIV candidate vaccine is safe 12
and immunogenic. Based upon encouraging results, the clinical testing of the 13
combination of Ad35- and Ad26-based vaccines against malaria and HIV is in 14
preparation. 15
16
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MATERIALS AND METHODS 1
Vector and protein construction, production and purification. 2
E1/E3-deleted, replication-incompetent Ad26 and Ad35 vectors expressing the same P. 3
falciparum CS gene were generated in E1-complementing PER.C6® cells and purified 4
using CsCl gradients as previously described (1, 16). Viral particles (vp) were quantified 5
by high-performance liquid chromatography (HPLC). The P. falciparum CS gene is a 6
synthetic, mammalian-codon optimized insert encoding a CS protein based on the EMBL 7
protein sequence CAH04007, and truncated for the last 14 amino acids at the C-terminus. 8
The N-terminus of this CS protein is a consensus assembled by alignment of various 9
sequences present in the GenBank, while the repeat region and the C-terminus are based 10
on the sequence of the 3D7 P. falciparum clone. The CS repeat region consisted of 27 11
NANP repeats, a cluster of 3 NVDP and one separate NVDP. CS protein of identical 12
sequence as in the adenovectors has been produced in Hansenula polymorpha RB11 13
clone by ARTES Biotechnology GmbH (Germany). A C-terminal His-tag sequence was 14
introduced into the construct to facilitate Ni-column purification of the CS protein from 15
the culture supernatant. 16
17
Characterization of the yeast-produced CS protein 18
The yeast-produced CS protein was analyzed by CS-specific Western blot and 19
InstantBlue staining, which demonstrated the identity and purity of the CS protein (more 20
than 80% pure) (Figure 1a). For the Western blot, rabbit polyclonal antibody against P. 21
falciparum CS (MRA-24, MR4/ATCC) was used in combination with goat-anti-rabbit 22
IgG conjugated to horseradish peroxide (HRP, Biorad) and enhanced chemiluminescence 23
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(ECL+, GE healthcare) to detect CS expression. The InstantBlue staining was performed 1
according to protocol provided by the manufacturer (Expedeon). 2
A dose of the yeast-produced CS protein for prime-boost immunogenicity studies was 3
selected using immunization of BALB/c mice (n = 5 per group) with increasing dosages 4
of CS protein (5 µg, 10 µg or 25 µg), formulated with the Montanide ISA 720 (Seppic, 5
France) at a 30:70 volume-based ratio, at 3-weeks intervals. The CS-specific humoral 6
response were assessed using ELISA, which demonstrated that the yeast-produced CS 7
protein induces maximal CS-specific antibody responses already at the lowest tested dose 8
(5 µg) and after two immunizations (Figure 1b). The induced IgG response consisted 9
predominantly of IgG1 antibodies, indicating the Th2 type response (Figure 1C). 10
Analysis of the CS-specific cellular immunity using ELISPOT revealed poor induction of 11
IFNγ+ T-cells for all doses (data not shown). 12
13
Animals and vaccinations regimens. 14
Our study seek to evaluate whether vaccination with Ad35.CS and Ad26.CS can enhance 15
the CS-specific immune response induced by a protein-based vaccine (eventually RTS,S), 16
as potential vaccination strategy for malaria. Since the RTS,S vaccine was not available, 17
for these studies we have used a yeast-produced full-length CS protein vaccine. 18
All animal experiments were approved in advance by an independent Ethical Review 19
Board. Six to eight week-old female BALB/c mice were purchased from Harlan (Zeist, 20
The Netherlands) and kept at the institutional animal facility under specific-pathogen-free 21
conditions during the experiment. 22
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To evaluate the immunogenicity of the heterologous CS protein/Ad prime-boost 1
regimens, BALB/c mice (n = 8 per group) were primed at week 0 with 5 µg adjuvanted 2
CS protein and boosted at week 4 with 109 vp Ad35.CS. The optimal immunization doses 3
of Ad.CS for immunization were selected from earlier dose response experiments (data 4
not shown). Another group of mice (n = 8) received a homologous prime-boost regimen 5
of 5 µg adjuvanted CS protein. As negative control group, BALB/c mice (n = 6) were 6
injected at week 0 with adjuvant montanide ISA720 and at week 4 with 109 vp 7
Ad35.Empty (adenovector without insert; indicated as sham immunization group). 8
To evaluate the 3-component heterologous prime-boost, BALB/C mice (n = 8) were 9
immunized at week 0 with 5 µg adjuvanted CS protein, boosted at week 4 with 109 vp 10
Ad35.CS and at week 8 with 1010
vp Ad26.CS. Comparator groups of BALB/C mice (n = 11
8 per group) started immunization at week 4 with 5 µg adjuvanted CS protein and were 12
boosted after 4 weeks (at week 8) with either 109 vp Ad35.CS or 5 µg adjuvanted CS 13
protein. As a negative control group, mice (n = 3) received the adjuvant montanide 14
ISA720 at week 0, 109 vp rAd35.Empty at week 4 and 10
10 vp rAd26.Empty at week 8. 15
16
CS-specific T-cell assays. 17
CS-specific cellular immune responses in vaccinated mice were assessed using 18
interferon-γ (IFNγ) ELISPOT assay, intracellular cytokine staining in combination with 19
surface staining of CD4 and CD8 markers (ICS), as described previously elsewhere (4, 20
44), and cytometric bead array (CBA) assay. 21
For the stimulation of splenocytes in the ELISPOT and ICS, a peptide pool consisting of 22
11-amino acids overlapping 15-mer peptides spanning the whole sequence of the P. 23
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falciparum CS protein was used. The pool contained a highly immunodominant CD8+ T-1
cell epitope (NYDNAGTNL; H-2Kd), which is responsible for the main part of measured 2
responses in the ELISPOT and the CD8+ responses in the ICS. This was confirmed with 3
an experiment wherein the splenocytes were stimulated with the 9-mer peptides, which 4
generated virtually identical responses as the peptide pool (data not shown). For the 5
ELISPOT, ninety-six-well multiscreen plates (Millipore, Bedford, MA) were coated 6
overnight with 100 µl/well of 10 µg/ml anti-mouse IFN-γ (BD Pharmingen, San Diego, 7
CA) in endotoxin-free Dulbecco’s PBS (D-PBS). The plates were then washed three 8
times with D-PBS containing 0.05% Tween-20 (D-PBS/Tween), blocked for 2 h with D-9
PBS containing 5% FBS at 37oC, and rinsed with RPMI 1640 containing 10% FBS. 10
Splenocytes from individual mice were stimulated with the CS peptide pool for 18 h at 11
37oC. Following incubation the plates were washed six times with D-PBS/Tween and 12
once with distilled water. The plates were then incubated with 2 µg/ml biotinylated anti-13
mouse IFN-γ (BD Pharmingen, San Diego, CA) for 2 h at room temperature, washed six 14
times with D-PBS/Tween, and incubated for 2 h with a 1:500 dilution of streptavidin-15
alkaline phosphatase (Southern Biotechnology Associates, Birmingham, AL). Following 16
six washes with D-PBS/Tween and one with PBS, the plates were developed with nitro 17
blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate chromogen (Pierce, Rockford, 18
IL), reaction was stopped with tap water, air dried, and read using an ELISPOT reader 19
(Aelvis GmbH). Spot-forming units (SFU) per 106 cells were calculated. In the case of 20
the ICS, splenocytes from individual animals were stimulated with the CS peptide pool or 21
cultured with medium alone. All cultures
contained monensin (GolgiStop; BD 22
Biosciences) as well as 1 µg/ml anti-CD49d (BD Biosciences). The cultured cells
were 23
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stained with monoclonal antibodies specific for cell surface molecules (CD4 and CD8). 1
After fixing with Cytofix/Cytoperm solution (BD Biosciences), cells were permeabilized 2
and stained
with antibodies specific for mouse IFNγ. Approximately 200,000 to 3
1,000,000 events were collected per sample. The background level of cytokine staining
4
was typically lower than 0.01% for CD4+ T cells and lower than 0.05% for CD8+ T cells. 5
The T-helper response induced by the different vaccination regimens was evaluated using 6
Cytometric bead array (CBA) assay. Splenocytes from individual mice were stimulated 7
with 5 µg/ml yeast-produced CS protein. After 48 h of incubation at 37°C, supernatants 8
were harvested and analyzed for the presence of the Th1 (IFNγ, TNFα, IL-2), Th2 (IL-4, 9
IL-6, IL-10) and Th17 (IL-17) cytokines using the Mouse Th1/Th2/Th17 Cytokine Kit 10
according to protocol provided by the manufacturer (BD Biosciences). 11
12
CS-specific antibody assays. 13
CS-specific antibody responses were assessed by enzyme-linked immunosorbent assay 14
(ELISA) as previously described (33). Ninety-six-well microtiter plates (Maxisorp; 15
Nunc) were coated overnight at 4°C with 2 µg/ml of CS-specific (NANP)6C peptide in 16
0.05 M Carbonate buffer (pH 9.6). Plates were washed three times and blocked with PBS 17
containing 1% BSA and 0.05% Tween-20 for 1 h at 37°C. After the plates were washed 18
three times, 1:100-diluted individual serum samples were added to the wells and serially 19
twofold diluted in PBS containing 0.2% BSA and 0.05% Tween-20. Plates were 20
incubated for 2 h at 37°C. Plates were washed three times and incubated with biotin-21
labeled anti-mouse or anti-rabbit immunoglobulin G (IgG) (Dako, Denmark), followed 22
by horseradish peroxidase-conjugated streptavidin (Pharmingen San Diego, CA) for 30 23
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min each at 37°C. For detection of the IgG subclasses, samples were incubated with 1
horseradish peroxidase-labeled anti-mouse IgG1 or IgG2a antibodies (Southern Biotech, 2
Birmingham, AL). Finally, the plates were washed and 100 µl of o-phenylenediamine 3
dihydrochloride (OPD) substrate (Pierce, Rockford, IL) was added to each well. After 10 4
min, the reaction was stopped by adding 100 µl/well of 1 M H2SO4. The optical density 5
was measured at 492 nm using a Bio-Tek reader (Bio-Tek Instruments, Winooski, VT). 6
The ELISA units were calculated relative to the OD curve of the serially diluted standard 7
serum, with one ELISA unit corresponding to the serum dilution at 50% of the maximum 8
of the standard curve. The IgG2a/IgG1 ratio was determined using titer values of IgG1 9
and IgG2a antibodies, which are expressed as a reverse of serum dilution. 10
11
Statistical analyses. 12
Comparisons of geometric mean immune responses were performed by student t-test after 13
logarithmic transformation to account for two test groups. Comparisons of geometric 14
mean immune responses were performed by analyses of variance (ANOVA) with Tukey 15
adjustments after logarithmic transformation to account for multiple comparisons. In all 16
cases, p-values lower than 0.05 were considered significant. 17
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RESULTS 1
2
Immunogenicity of CS protein prime followed by Ad35.CS boost. 3
CS-specific humoral response induced in BALB/c mice with an CS protein prime and 4
Ad35.CS boost at two weeks post immunization was assessed by ELISA assay (Figure 5
2A and B) while the cellular immune responses were measured using IFNγ ELISPOT 6
(Figure 2C) and ICS (Figure 2D). The homologous prime-boost regimen with the CS 7
protein elicited a very potent CS-specific IgG response. The levels of the antibody 8
response elicited by the heterologous CS protein/Ad35.CS regimen were comparable to 9
that seen for the homologous CS protein prime-boost regimen (P > 0.05 comparing CS-10
specific IgG levels with ANOVA). Beside the total CS-specific IgG levels, we 11
determined the IgG2a/IgG1 ratio to obtain indications of the type of T-helper responses 12
induced by the different prime-boost regimens (Figure 2B). The homologous CS protein 13
prime-boost regimen elicited primarily IgG1 antibody responses indicating a more Th2 14
type immune response while replacing the protein boost with Ad35.CS boost resulted in a 15
more pronounced induction of IgG2a antibodies indicating shift towards a Th1 type 16
response (P < 0.05 comparing IgG2a/IgG1 ratios with ANOVA). 17
Evaluation of the CS-specific T-cell responses using ELISPOT (Figure 2C) and ICS 18
(Figure 2D) assays showed that the homologous CS protein regimen evoked a poor but 19
measurable CS-specific T-cell response. The inclusion of Ad35.CS as a boost to the CS 20
protein prime resulted in significantly increased levels of CS-specific IFNγ-producing 21
CD8+ T-cells (P < 0.05 comparing CS-specific CD8+ T-cell levels with ANOVA). This 22
correlated to the more Th1 type response for the CS protein/Ad35.CS regimens as 23
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determined by CS-specific IgG2a/IgG1 ratio. It should be noted that IFNγ+ CD4+ 1
response might have been underestimated using the stimulation with the 15-mer peptides, 2
as we observed in another study (39). Stimulation of splenocytes with the CS-protein in 3
the current study did show higher CD4+ responses, however, the background in the assay 4
was unacceptably high (data not shown). 5
6
Immunogenicity of a three-component heterologous prime-boost regimen. 7
The combination of the yeast-produced CS protein with the Ad35.CS in a heterologous 8
prime-boost regimen results in the induction of high level of IFNγ+ CD8+ T cells, 9
maintained high level of CS-specific IgG response and the antibody response was shifted 10
towards the Th1 type. We next investigated whether a prime-boost regimen comprised of 11
the three components, CS protein, Ad35.CS and Ad26.CS, might result in an even more 12
robust and sustained Th1 immune response. Our earlier experiments demonstrated that 13
the Ad35.CS/Ad26.CS combination induces significantly higher immune responses than 14
the Ad35.CS/Ad35.CS combination (data not shown) and, therefore, the homologous 15
adenovector combination Ad35.CS/Ad35.CS was not included as a booster vaccine in the 16
current study. A group of mice received a prime with adjuvanted CS protein and a boost 17
with Ad35.CS followed by a second boost with Ad26.CS (three-component heterologous 18
prime-boost). A comparator group of mice received a prime with adjuvanted CS protein 19
followed by an Ad35.CS boost. At two weeks post the final boost immunization, mice 20
receiving the three-component heterologous prime-boost regimen showed a significantly 21
higher levels of CS-specific IFNγ-producing CD8+ T-cells compared to the mice 22
receiving the CS protein prime and Ad35.CS boost regimen (Figure 3A; P > 0.05 23
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comparing CS-specific IFNγ-producing CD8+ T-cell levels with ANOVA). At eight 1
weeks post the final boost immunization, the IFNγ+ CD8+ T-cell response induced by the 2
three-component prime-boost regimen was still significantly higher compared to the CS 3
protein/Ad35.CS regimen (Figure 3B; P > 0.05 comparing CS-specific IFNγ-producing 4
CD8+ T-cell levels with ANOVA). Importantly, at both time points the levels of CS-5
specific IgG responses induced by the three-component prime-boost regimen were 6
comparable to that seen for the CS protein/Ad35.CS regimen (Figure 3B and D; P > 0.05 7
comparing CS-specific IgG levels with ANOVA). The IgG2a/IgG1 ratio of CS-specific 8
antibodies induced with the CS protein/Ad35.CS/Ad26.CS vaccine regimen was 9
comparable to the ratio induced with the CS protein/Ad35.CS immunization (data not 10
shown). 11
12
Cytokine profile induced by the different vaccination regimens 13
The total number of CS-specific CD4+ T cells expressing two or more immune markers, 14
being Th1 cytokines IFNγ, TNFα, IL2, and activation marker CD40L, induced upon 15
immunization with RTS,S, has been associated with protection to malaria infection in the 16
human challenge model (22). We investigated cytokine profile breadth induced in CS-17
specific T cells with three component malaria vaccine, CS protein/Ad35.CS/Ad26.CS, 18
and compared it to the cytokine profiles induced with CS-protein/CS protein or CS 19
protein/Ad35.CS regimen. Two weeks after the final boost immunization, expression 20
levels of the Th1 (IFNγ, TNFα, IL-2), Th2 (IL-4, IL-6, IL-10) and Th17 (IL-17) 21
cytokines were determined using the cytometric bead array (CBA) assay upon 48 hrs in 22
vitro stimulation of splenocytes with the CS protein. The CBA assay with protein 23
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stimulation provides a blueprint of the type of T helper cells that have been induced with 1
the vaccination regiment. All vaccination regimens, except for the sham, induced the 2
tested cytokines, with an exception of IL-4 which was not detected (data not shown) 3
(Figure 4). The CS protein/Ad35.CS/Ad26.CS regimen induced significantly higher 4
levels of IFNγ and TNFα compared to either the CS protein or the CS protein/Ad35.CS 5
regimen (Figure 4; P < 0.05 comparing cytokine levels with ANOVA). The levels of 6
other cytokines (IL-2, IL-6, IL-10 and IL17) were comparable for all immunization 7
regimens (Figure 4; P > 0.05 comparing cytokine levels with ANOVA). 8
Summarizing, these data confirm that a prime-boost regimen comprising of the three 9
components, CS protein, Ad35.CS and Ad26.CS, results in a robust and broad Th1 type 10
immune response. 11
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DISCUSSION 1
Immunizations with a CS protein vaccine elicit potent antibody responses, but poor 2
cellular responses. In this study we demonstrated that vaccination with the CS protein 3
followed by Ad35.CS vector in a heterologous prime-boost regimen results in 4
enhancement of IFNγ+ CD8+ T-cell responses. The boosting with Ad35.CS did not 5
hamper the level of CS-specific humoral response induced with the protein vaccination, 6
but shifted the Ig isotypes towards a Th1 type of response. In addition, we established 7
that a heterologous prime-boost regimen comprising a CS protein prime followed by 8
boosts with Ad35.CS and Ad26.CS elicits strong CS-specific Th1 type responses, with a 9
durable enhancement of the IFNγ+ CD8+ T-cells and potent antibody responses. 10
The ongoing Phase III trial of RTS,S, a CS-protein based vaccine adjuvanted with a 11
AS01 adjuvnat, represents a breakthrough for malaria vaccine development. The vaccine 12
induces primarily antibody responses and has been shown to partially protect young 13
children and infants in malaria-endemic areas, reducing the risk of clinical episodes of 14
malaria by 53 percent over an eight-month follow-up period (5). Whilst RTS,S gives a 15
better chance of surviving to the most vulnerable part of the population, it is clear that 16
more must be done in order to develop vaccines that will provide greater and more 17
sustainable levels of protection to fully eradicate malaria. 18
IFN-γ+ CD8+ T cells have been associated with protection against liver stage malaria 19
parasites as they inactivate and eliminate intracellular parasites through IFN-γ induced 20
production of nitric oxide and through cell-mediated cytotoxicity (6, 12, 43, 47). 21
Therefore, it is widely accepted that persistent protective immunity against malaria likely 22
requires both high levels of Th1 type immune responses targeting the pre-erythrocytic 23
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stage of the malaria parasites. Such a complex immunity is not easily achieved by single 1
vaccine modalities as demonstrated by the low number of malaria vaccine regimens in 2
advanced clinical trials (38). 3
Adenoviral vectors are known to induce high levels of antigen-specific IFN-γ+ CD8+ T 4
cells (52). The combination of adenovectors with other vaccine types has proven highly 5
efficient in eliciting strong and sustainable T-cell immunity as well as humoral responses 6
(7, 14, 26, 48-50). Indeed within the current study we show that the priming with an 7
adjuvanted yeast-produced CS protein followed by the Ad35.CS boost results in the 8
induction of higher CS-specific IFN-γ+ CD8+ T-cell responses compared to exclusively 9
protein-based vaccine regimen. Importantly, while the overall CS-specific IgG levels 10
were not affected compared to the responses induced with an entirely CS protein 11
vaccination regimen, the CS protein/Ad35.CS regimen elicited a more Th1 type response. 12
These results corroborated our earlier findings in which prime-boost regimens comprised 13
of Ad35 vaccine vectors expressing CS or LSA-1, and RTS,S or a LSA-1 protein vaccine 14
resulted in potent Th1 type T-cell responses and high level humoral responses (44, 51). 15
Previously we reported on the heterologous prime-boost regimen utilizing the Ad35.CS 16
and Ad5.CS vaccine vectors that elicited high levels of CS-specific IFN-γ producing T 17
cells in both mice and non-human primates (45). These results demonstrated the potential 18
of adenovector-based heterologous prime-boost regimens to induce the type of immunity 19
required to combat malaria. Because of the high Ad5 seroprevalence in the human 20
population, considerable effort has been directed towards the development of novel low 21
seroprevalent adenoviral vaccine vectors that are able to circumvent anti-Ad5 immunity 22
and are highly immunogenic (1, 4, 13, 17, 25, 35, 55). The Ad26-based vaccine vector 23
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has been shown to be a particular interesting vector considering the ability to induce 1
immune responses in mice (1), non-human primates (27, 28) and humans (3), and 2
particularly suited as a boost to other adenovector-based vaccines that utilize different 3
adenovirus serotypes. Given the wide diversity of adenoviruses in nature many different 4
serotypes are potentially available. In our study, the inclusion of the Ad26.CS boost to the 5
CS protein/Ad35.CS prime-boost regimen elicited an overall higher and more sustainable 6
CS-specific IFNγ+ CD8+ immune response as compared to the homologous or the 2-7
component heterologous prime-boost regimens. 8
The recent association of Th1 cytokine expressing CD4+ T cells, induced with RTS,S 9
vaccine, with protection against malaria infection in the human challenge model has 10
reinforced the view that induction of a broad immune response of Th1 type is required for 11
development of efficient malaria vaccines (22). Induction of balanced pro-inflammatory 12
and regulatory immune responses is also a key factor determining the outcome of malaria 13
infection. Failure to develop an effective pro-inflammatory response might result in 14
unrestricted parasite replication, whereas failure to control this response can lead to the 15
development of severe immunopathology (10). Boosting of the CS protein vaccine with 16
the Ad35.CS and, in particular, with the Ad35.CS/Ad26.CS combination strongly 17
enhanced the levels of Th1 cytokines IFNγ and TNFα, while the levels of Th1 cytokine 18
IL-2, Th2 cytokines IL-6 and IL-10 and Th17 cytokine IL-17 were comparable to the 19
levels induced with the CS protein vaccine alone. This result indicated the capacity of the 20
three-component regimen to stimulate an overall balanced cytokine response, with a 21
strong shift towards the Th1 responses as compared to the homologous CS protein 22
regimen inducing primarily a Th2 biased response. While the role for Th1 type response 23
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in protection against malaria has been well documented (6, 12, 19, 20, 43, 47), to our 1
knowledge there are no reports concerning the role of Th17 cells in malaria infection. 2
However, there is mounting evidence that IL-17 might be relevant for protection against 3
parasitic infections, as it has been indicated by the induction of this cytokine in response 4
to infection with a number of parasites such as Eimeria maxima (18), Nippostrongglyus 5
brasiliensis (59) and Leishmania donovani (36). For instance, production of IL-17 and 6
IL-22 in humans was shown to have a strong and independent association with protection 7
the Kala Azar disease, caused by L. donovani (36). Other studies have also indicated a 8
role for Th17 cells in protection against other pathogens such as, Mycobacterium 9
tuberculosis (2, 23, 24), Streptococcus pneumoniae (29, 30, 58), Helicobactor pylori (11, 10
54), and Influenza virus (15, 57). In the current study, albeit no significant difference was 11
observed in the mean level of the IL-17 cytokines between different groups, the 12
adenovector containing regimens induced more uniform IL-17 responses as compared to 13
the protein immunization. 14
The limited and short lived protection induced with the CS protein vaccine points in two 15
directions of improvement using Ad.CS-based vaccines. One by administering Ad35.CS 16
vaccine as a priming vaccine to the CS protein vaccine, to strongly increase Th1 cellular 17
responses, as described in Stewart et al (51). Second, as demonstrated in the current 18
study, by administering the Ad35.CS/Ad26.CS combination as a booster vaccine (in 19
second year of life or even at school age) following an early-in-life protein CS vaccine, to 20
induce long lasting protection for which the Th1 type response and immune memory is 21
required. 22
23
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ACKNOWLEDGMENTS 1
We acknowledge financial support from the European Commission (FP6 grant for the 2
project PRIBOMAL). 3
4
5
6
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Bouzourene, and P. Michetti. 2009. Interleukin-17 is a critical mediator of 17
vaccine-induced reduction of Helicobacter infection in the mouse model. 18
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55. Vogels, R., D. Zuijdgeest, R. van Rijnsoever, E. Hartkoorn, I. Damen, M. P. 20
de Bethune, S. Kostense, G. Penders, N. Helmus, W. Koudstaal, M. Cecchini, 21
A. Wetterwald, M. Sprangers, A. Lemckert, O. Ophorst, B. Koel, M. van 22
Meerendonk, P. Quax, L. Panitti, J. Grimbergen, A. Bout, J. Goudsmit, and 23 M. Havenga. 2003. Replication-deficient human adenovirus type 35 vectors for 24
gene transfer and vaccination: efficient human cell infection and bypass of 25
preexisting adenovirus immunity. J Virol 77:8263-71. 26
56. Weiss, W. R., M. Sedegah, R. L. Beaudoin, L. H. Miller, and M. F. Good. 27
1988. CD8+ T cells (cytotoxic/suppressors) are required for protection in mice 28
immunized with malaria sporozoites. Proc Natl Acad Sci U S A 85:573-6. 29
57. Williman, J., E. Lockhart, L. Slobbe, G. Buchan, and M. Baird. 2006. The use 30
of Th1 cytokines, IL-12 and IL-23, to modulate the immune response raised to a 31
DNA vaccine delivered by gene gun. Vaccine 24:4471-4. 32
58. Zhang, Z., T. B. Clarke, and J. N. Weiser. 2009. Cellular effectors mediating 33
Th17-dependent clearance of pneumococcal colonization in mice. J Clin Invest 34
119:1899-909. 35
59. Zhugong Liu, H. L., Qian Liu, Gity Mousavi and William C. Gause 2008. The 36
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FASEB Journal 22. 39
40
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FIGURE LEGENDS 1
2
Figure 1. Characterization of the yeast-produced CS protein. (A) The yeast-produced 3
CS protein was analyzed by CS-specific Western blot and InstantBlue staining. (B) 4
BALB/c mice (n = 5 per group) were immunized s.c. three times with 5, 10 or 25 µg 5
montanide ISA720 adjuvanted CS protein at 3-weeks interval. CS-specific humoral 6
responses were assessed every 1.5 weeks up to 8 weeks after the initial immunization by 7
ELISA. Mean titers with 95% confidence interval are depicted. EU; ELISA units. (C) 8
IgG2a/IgG1 ratios upon measurement of CS-specific IgG2a and IgG1 responses 8 weeks 9
after the initial immunization. Bars represent geometric means IgG2a/IgG1 ratios 10
11
Figure 2. Immunogenicity of heterologous prime-boost regimen comprised of the 12
yeast-produced CS protein and Ad35.CS. BALB/c mice(n = 8 per group) were 13
immunized as indicated in the graphs. A negative control group received the adjuvant and 14
Ad35.Empty vector (sham). Two weeks after the boost immunization CS-specific 15
humoral immune responses were assessed by (A) CS-specific IgG responses using 16
ELISA and (B) IgG2a/IgG1 ratios upon measurement of CS-specific IgG2a and IgG1 17
responses. CS-specific CD8+ T-cell immune responses were assessed by (C) IFNγ 18
ELISPOT and (D) IFNγ ICS. Bars represent geometric means of (A) ELISA units (EU), 19
(B) IgG2a/IgG1 ratios, (C) spot forming units (SFU), or (D) percentage of IFNγ+ CD4+ 20
or IFNγ+ CD8+ positive cells. The background level of cytokine staining was typically 21
lower than 0.01% for the CD4+ T cells and lower than 0.05% for the CD8+ T cells. 22
23
24
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Figure 3. Immunogenicity of a three-component heterologous prime-boost regimen. 1
BALB/c mice(n = 8 per group) were immunized as indicated in the graphs. A negative 2
control group received the adjuvant and Ad.Empty vectors (sham). Two-weeks (A) and 3
eight-weeks (C) after the final boost immunization CS-specific IFNγ+ CD8+ T-cell 4
responses were assessed using ELISPOT. Two-weeks (B) and eight-weeks (D) after the 5
final boost immunization CS-specific humoral immune responses were assessed by IgG 6
ELISA. Bars represent geometric means of (A, C) spot forming units (SFU) or (B, D) 7
ELISA units (EU). 8
9
Figure 4. Cytokine profile induced by the different vaccination regimens. 10
BALB/c mice (n = 8 per group) were immunized as indicated in the graphs. A negative 11
control group received the adjuvant and Ad.Empty vectors (sham). Two-weeks after the 12
final boost immunization cytokine expression was assessed by CBA assay upon 48 hrs in 13
vitro stimulation of splenocytes with the CS protein. Bars represent geometric means of 14
pg/ml IFNγ, TNFα, IL-2, IL-6, IL-10 or IL-17 cytokine levels. In none of the immunized 15
mice measurable levels of IL-4 were detected. 16
17
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Figure 1
0 1 2 3 4 5 6 7 81
10
100
1000
10000
100000
10000005 ug
10 ug
25 ug
Prime Boost Boost
Weeks
EU
A B
20
30
40
50
60
80
100
C
0.0001
0.001
0.01
0.1
1
10
100
- + - + - + 5 ug 10 ug 25 ug
AdjuvantProtein
rati
o I
gG
2a/I
gG
1 Th1
Th2
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A
C
B
Figure 2
0
100
200
300
400
P < 0.05
IFN
- γγ γγ S
FU
/10
6 s
ple
no
cyte
s
0.0001
0.001
0.01
0.1
1
10
100 P < 0.05
rati
o IgG
2a/IgG
1
1
10
100
1000
10000
100000
N.S.
EU
sham
sham
CS Prot
CS Prot
CS Prot
Ad35.CS
sham
sham
CS Prot
CS Prot
CS Prot
Ad35.CS
CS Prot
CS Prot
CS Prot
Ad35.CS
Th1
Th2
D
CS ProtCS Prot
CS ProtAd35.CS
0.00
0.25
0.50
0.75
1.00P < 0.05
CD4
CD8
% IFNγγ γγ+ C
D+ c
ells
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A
C
Figure 3
B
D
0
500
1000
1500
2000
2500 P < 0.05
IFN
- γγ γγ S
FU
/ 1
06 s
ple
no
cy
tes
1
10
100
1000
10000
100000N.S.
EU
0
500
1000
1500
2000
2500P < 0.05
IFN
- γγ γγ S
FU
/ 10
6 s
ple
no
cyte
s
1
10
100
1000
10000
100000N.S.
EU
shamsham
sham
CS Prot
Ad35.CS
CS ProtAd35.CS
Ad26.CS
shamsham
sham
CS Prot
Ad35.CS
CS ProtAd35.CS
Ad26.CS
sham
shamsham
CS Prot
Ad35.CS
CS Prot
Ad35.CSAd26.CS
sham
shamsham
CS Prot
Ad35.CS
CS Prot
Ad35.CSAd26.CS
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Figure 4
0
1000
2000
3000
4000
5000P < 0.05
P < 0.05
IFNγγ γγ (
pg
/ml)
0
100
200
300
400
500
600P < 0.05
TN
Fαα αα
(p
g/m
l)
0
100
200
300N.S.
IL-2
(p
g/m
l)
0
100
200
300
400
500
600
700
800N.S.
IL-6
(p
g/m
l)
0
100
200
300N.S.
IL-1
0 (
pg
/ml)
0
100
200
300N.S.
IL-1
7 (
pg
/ml)
sham
shamsham
CS Prot
CS Prot
CS Prot
Ad35.CSAd26.CS
CS Prot
Ad35.CS
sham
shamsham
CS Prot
CS Prot
CS Prot
Ad35.CSAd26.CS
CS Prot
Ad35.CS
sham
shamsham
CS Prot
CS Prot
CS Prot
Ad35.CSAd26.CS
CS Prot
Ad35.CS
Th1
Th2 Th17
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CLINICAL AND VACCINE IMMUNOLOGY, Feb. 2011, p. 353 Vol. 18, No. 21556-6811/11/$12.00 doi:10.1128/CVI.00554-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.
ERRATUM
The Th1 Immune Response to Plasmodium falciparum Circumsporozoite ProteinIs Boosted by Adenovirus Vectors 35 and 26 with a Homologous Insert
Katarina Radosevic, Ariane Rodriguez, Angelique A. C. Lemckert, Marjolein van der Meer,Gert Gillissen, Carolien Warnar, Rie von Eyben, Maria Grazia Pau, and Jaap Goudsmit
Crucell Holland B.V., Leiden, Netherlands, and Center for Poverty-Related Communicable Diseases,Academic Medical Center, Amsterdam, Netherlands
Volume 17, no. 11, pages 1687–1694, 2010. Page 1687, Abstract, the first sentence should read: “The most advanced malariavaccine, RTS,S, is comprised of a portion of the Plasmodium falciparum circumsporozoite (CS) protein, fused to and admixed withthe hepatitis B virus surface antigen, and an adjuvant.”
The third sentence should read: “In the present study, we tested the hypothesis that the Th1 immune response to CS protein,in particular the CD8� T-cell response, which is needed for strong and lasting malaria immunity, is boosted to sustainable levelsby adenovirus vectors 35 and 26 with a homologous insert (Ad35.CS/Ad26.CS).”
353
