An engineered Plasmodium falciparum C-terminal 19-kilodalton merozoite surface protein 1 vaccine candidate induces high levels of interferon-gamma production associated with cellular immune responses to specific peptide sequences in Gambian adults naturally exposed to malaria

Abstract

The 19-kDa C-terminal region of merozoite surface protein 1 (MSP1₁₉), a major blood stage malaria vaccine candidate, is the target of cellular and humoral immune responses in humans naturally infected with Plasmodium falciparum. We have previously described engineered variants of this protein, designed to be better vaccine candidates, but the human immune response to these proteins has not been characterized fully. Here we have investigated the antigenicity of one such variant compared to wild-type MSP1₁₉-derived protein and peptides. Gambian adults produced both high T helper type 1 (Th1) [interferon (IFN)-γ] and Th0/Th2 [interleukin (IL)-13 and sCD30] responses to the wild-type MSP1₁₉ and the modified protein as wells as to peptides derived from both forms. Response to the modified MSP1₁₉ (with three amino acid substitutions: Glu27Tyr, Leu31Arg and Glu43Leu) relative to the wild-type, included higher IFN-γ production. Interestingly, some peptides evoked different patterns of cytokine responses. Modified peptides induced higher IL-13 production than the wild-type, while the conserved peptides P16 and P19 induced the highest IFN-γ and IL-13 and/or sCD30 release, respectively. We identified P16 as the immunodominant peptide that was recognized by cells from 63% of the study population, and not restricted to any particular human leucocyte antigen D-related (HLA-DR) type. These findings provide new and very useful information for future vaccine development and formulation as well as potential Th1/Th2 immunmodulation using either wild-type or modified protein in combination with their peptides.

Introduction

The 19-kDa C-terminal region of Plasmodium falciparum merozoite surface protein 1 (MSP1₁₉) is a major blood stage malaria vaccine candidate comprised of two epidermal growth factor (EGF)-like domains. An immune response to this protein may prevent or reduce malaria-related morbidity and mortality by inhibiting merozoite invasion of and development within erythrocytes, thus eliminating or reducing the parasite load [1]. In Phase I trials, immunization of malaria-naive human volunteers with either of two allelic forms of recombinant MSP1₁₉ induced high levels of antigen-specific CD4 T cell activation. Novel, conserved and allele-specific T cell epitopes able to induce cross-strain immune responses were identified in vaccinees [2]. Responses to many of these epitopes were also present in adults exposed to malaria [2], but the age-associated acquisition of immune responses to these epitopes was not assessed. A Phase II trial provided no evidence of clinical protection despite the induction of antibody to the somewhat larger MSP1₄₂ antigen used in the trial [3], suggesting that MSP1-based vaccines will need to be engineered to improve their induction of protective immune responses [4]. We have proposed that the function of antibody depends on its fine specificity; for example, in natural infection MSP1-specific antibody appears to act, at least in part, by inhibiting the protease-mediated cleavage of MSP-1 that is essential for invasion. However, some antibody blocks the binding of the inhibitory antibody, allowing MSP-1 cleavage and invasion to proceed [5,6]. To produce an antigen that binds and stimulates inhibitory but not blocking antibody, we introduced amino acid changes into the MSP1₁₉ sequence [7]. An example of one such protein has three substitutions (Glu27Tyr, Leu31Arg and Glu43Leu). However, the effect of such changes on the cellular response needs clarification; for example, the presence of new CD4 T cell epitopes in the modified MSP1₁₉ should improve the vaccine. MSP1₁₉ induces poor CD4 T cell responses, due probably to poor processing of its highly compact and disulphide-bonded structure by antigen-presenting cells (APC) [8–10]. Identification and inclusion of epitopes recognized by malaria-immune individuals will increase the likelihood that vaccine-induced responses will be boosted by natural infection. Using wild-type and modified proteins as well as peptides derived from them, we wished to identify immunodominant peptides as well as to assess CD4 cell activation and APC processing in samples derived from semi-immune Gambians. In this study, IFN-γ, IL-13 and sCD30 T cell responses to recombinant MSP1₁₉ and synthetic peptides were assessed.

Materials and methods

Volunteers

Forty-three adult donors exposed to malaria but asymptomatic were recruited from Brefet village, The Gambia. Blood samples were collected; packed cell volume (PCV) and microscopy measurements were used to determine their haemoglobin levels and parasitaemia. Blood samples were then processed to prepare plasma as well as peripheral blood mononuclear cells (PBMC), which were used to measure immunological parameters. Studies were approved by the Joint Gambia Government/MRC Ethics Committee. Individual informed consent was obtained from all subjects.

Antigens

Wild-type MSP1₁₉ as well as a variant containing three amino acid substitutions were expressed in Pichia pastoris and then purified [11]. Both wild-type and modified sequence (containing Glu27Tyr, Leu31Arg and Glu43Leu) are based on MSP-1 residues 1526–1621 (Accession number P04933) with serine at position 3 and in a potential N-glycosylation site replaced by alanine. The proteins were used at 10 µg/ml. Peptides consisting of 20-mers each overlapping the adjacent peptide by 10 residues and spanning the entire sequence of both the wild-type and the triple residue variant were designed by Proimmune (Oxford, UK) and used in cultures at 25 µg/ml (Table 1).

Table 1.

The panel of wild-type and modified 20-mers overlapping merozoite surface protein 1 (MSP1₁₉) peptides

		Amino acid substitutions in the primary sequence
		Position(s)
Peptides code	Sequence	Single or multiple amino acid	Wild-type residue	Variant residue
P13	NISQH QCVKK QCPQN SGCFR
P13M	NIAQH QCVKK QCPQY SGCFR	3	Ser	Ala
P14	QCPQN SGCFR HLDER EECKC
P14M	QCPQY SGCFR HLDER EYCKC	27	Glu	Tyr
P15	HLDER EECKC LLNYK QEGDK
P15M	HLDER EYCKC RLNYK QEGDK	27+31	Glu+Leu	Tyr+Arg
P16	LLNYK QEGDK CVENP NPTCN
P16M	RLNYK QEGDK CVLNP NPTCN	31+43	Leu+Glu	Arg+Leu
P17	CVENP NPTCN ENNGG CDADA
P17M	CVLNP NPTCN ENNGG CDADA	43	Glu	Leu
P18	ENNGG CDADA KCTEE DSGSN
P19	KCTEE DSGSN GKKIT CECTK
P20	GKKIT CECTK PDSYP LFDGI
P21	PDSYP LFDGI FCSS SN
Triple mutant protein		27+31+43	Glu+Leu+Glu	Tyr+Arg+Leu

Human leucocyte antigen (HLA) class II typing

Thirty individuals were typed for class II molecules using the low-resolution polymerase chain reaction–sequence-specific primers (PCR–SSP), technique as described elsewhere [12], with minor modifications. The loci under investigation included HLA-DRB1, HLA-DRB3, HLA-DRB4 and HLA-DQB1.

IFN-γ-producing CD4 T cells identified by enzyme-linked immunospot assay (ELISPOT)

PBMC were separated from venous blood by density gradient centrifugation on Ficoll (Pharmacia, Oslo, Norway) at 580 g in a bench-top centrifuge for 20 min. Cells were washed in RPMI-1640 medium (Sigma, St Louis, USA) and then resuspended in the same medium supplemented with 2 mm l-glutamine (Life Technologies), 100 IU/ml penicillin, 100 µg/ml streptomycin (Life Technologies) and 10% heat-inactivated (30 min at 56°C) human AB serum (Sigma).

PBMC, 2 × 10⁵ per well, were cultured at 37°C in a humid CO₂ incubator with 25 µg/ml of the MSP1₁₉ peptides or 10 µg/ml of the MSP1₁₉ proteins representing both wild-type and modified forms. As controls, 2·5 µg/ml of phytohaemagglutinin (PHA) and 10 µg/ml purified protein derivative of Mycobacterium tuberculosis (PPD RT49; State Serum Institute, Copenhagen, Denmark), were used.

Supernatants were collected on day 3 and kept for cytokine determination. Twenty U/ml IL-2 (Proleukine) was added to cultures on days 3 and 7, then cells were washed and collected on day 10 and transferred to ELISPOT plates [Millipore MAIPS45 polyvinylidene difluoride (PVDF)-backed plates]. Cell activation was performed overnight in the presence of the MSP1₁₉ protein and peptides; the ELISPOT assay was performed using the IFN-γ ELISPOT kit (Mabtec, Stockholm, Sweden) following the manufacturer's recommendations. Briefly, 0·4 million PBMC/well were cultured in 96-well flat-bottomed nitrocellulose plates (MAIP 45S; Millipore, Molsheim, France) coated with anti-IFN-γ (Mabtech, Nadra, Sweden). Plates were washed six times with phosphate-buffered saline (PBS) with 0·05% Tween 20 (Sigma, Irvine, CA, USA) and incubated at room temperature for 2 h with the corresponding biotinylated second antibodies, followed by washing as described above and 2 h incubation with a streptavidin–alkaline phosphatase conjugate. After washing, a precipitable alkaline phosphatase substrate (Bio-Rad, Hercules, CA, USA) was added. Individual lymphokine-producing cells were enumerated by a computerized system and expressed as spot-forming units (SFU) per million PBMC.

An ELISPOT response was positive if the χ² comparison of the numbers of lymphokine positive cells in the test well and the control well based on a binomial distribution at 2 × 10⁵ cells per well yielded a P-value < 0·05 [13].

Measurement of antibody to MSP1₁₉

Enzyme-linked immunosorbent assay (ELISA) was used to measure antibodies to MSP1₁₉ in the plasma samples. Briefly, 96-well flat-bottomed well ELISA plates (Immunlon-4; Dynatech Laboratories, Chantilly, VA, USA) were coated with 1 µg/ml of MSP1₁₉ in carbonate buffer and incubated overnight at 4°C. The plates were washed with PBS-Tween 20 (0·05%), flicked dry, and 150 µl of 1% bovine serum albumin in PBS (PBS–BSA) added as blocking solution. Plates were incubated at room temperature for 3 h, flicked dry, and then serum samples were added. Sera were diluted in PBS–BSA at 1:1000. Serial dilutions of purified human immunoglobulin (Ig)G and anti-human IgG-POD were used as standards on each plate to assess the relative concentrations of antibody to MSP1₁₉. A pool of sera from adults positive for malaria infection was used as a positive control. A pool of sera of European tourists with no previous exposure to malaria was used as a negative control. The mean ± 3 standard deviations (s.d.) of the negative control value was used as the cut-off to define ‘responders’. The plates were incubated overnight at 4°C, washed six times as above and horseradish peroxidase (HRP) goat anti-human IgG (Dako A/S, Glostrup, Denmark) or HRP rabbit anti-human IgG1, or anti-human IgG3 (The Binding Site, Birmingham, UK) for the subtype study, were added at a dilution of 1:5000 in PBS–BSA. The plates were incubated for 3 h at room temperature under slow agitation and washed six times. The reaction was developed by adding 100 µl tetramethyl-benzidine (TMB; Sigma, Poole, UK) and stopped with 100 µl 2 m H₂SO₄, and then the plates were read at 450 nm.

Measurement of cytokines

The concentration of different T helper type 1 (Th1)/Th2 cytokines in the supernatants collected on days 3–4 from ELISPOT culture was determined. IFN-γ, IL-13 and sCD30 levels were determined using ELISA kits from Biosource (IFN-γ and IL-13; BioSource Europe, Fleurus, Belgium) and MedSystems (sCD30; MedSystems Diagnostics GmbH, Vienna, Austria), following the manufacturer's instructions.

Statistical analysis

A χ² test was used to compare proportion and the Wilcoxon test was used to compare values in response to MSP1₁₉ proteins or peptides. Calculations were made using statistics software spss version 17. The results were considered significant for P < 0·05.

Results

Responses of Gambian adults to MSP1₁₉ proteins

We investigated the T cell responses to wild-type and modified MSP1₁₉ proteins in Gambian semi-immune adults. T cells secreting IFN-γ in response to either wild-type or modified proteins were found in 11 of 43 (27%) and 13 of 43 (30%) donors, respectively. The difference was not significant. The numbers of IFN-γ-secreting T cells were equal in response to the two MSP1₁₉ proteins (average ± s.d.: 654 ± 189 and 721 ± 299 SFU/10⁶ PBMC for wild-type and mutant, respectively). However, higher IFN-γ secretion was observed in response to the mutant compared to the wild-type MSP1₁₉ protein (average ± s.d.: 4500 ± 256 and 3186 ± 267, P = 0·015) (Fig. 1a). The levels of sCD30 and IL-13 were slightly higher in response to wild-type compared to modified protein; but the differences were not significant (Fig. 1b,c).

Fig. 1.

Cytokine production by peripheral blood mononuclear cells (PBMC) in response to merozoite surface protein 1 (MSP1₁₉) proteins. PBMC from 43 adults were cultured for 3 days with MSP1₁₉ proteins and assessed for their cytokine production and sCD30 release. T helper type 1 (Th1) [interferon (IFN)-γ] (a) and Th0/Th2 [interleukin (IL)-13 and sCD30] (b,c) were measured using enzyme-linked immunosorbent assay (ELISA). Results are presented as average ± standard deviation. (a) A significantly higher amount of IFN-γ was produced by PBMC in response to modified (hatched bar, right) MSP1₁₉ compared to wild-type protein (hatched bar, left) (4500 ± 256 and 3186 ± 267, P = 0·015). (b,c) In contrast, the same amounts of interleukin (IL)-13 and sCD30 were produced in response to mutant (hatched bars right) and wild-type protein (hatched bars, left).

Responses of Gambian adults to MSP1₁₉ peptides

We investigated CD4 T cell responses to MSP1₁₉ peptides in the Gambian adults to identify immunodominant peptides. Cultured ELISPOT was performed using PBMC of 43 adults to determine the frequency of IFN-γ-secreting T cells in response to MSP1₁₉ peptides derived from both wild-type and modified proteins (Table 1). Fourteen 20-mer peptides were used in this study. Nine peptides were based on the wild-type sequence (P13–P21) and five were based on the mutant (P13M–P17M).

Only 11 of 43 (26%) donors had T cells secreting IFN-γ in response to P13, while P16 induced this response in 27 of 43 (63%) donors (Fig. 2a). No significant differences were found when we compared the number of responders to wild-type or the corresponding mutant peptides. As shown in Fig. 2b, the highest frequency of IFN-γ-secreting T cells was detected with peptides P16 and P21 (average ± s.d.: 531 ± 598 and 400 ± 689 SFU/10⁶ PBMC). P16 appears to contain an immunodominant epitope. The release of IFN-γ (Th1), IL-13 and sCD30 (Th0/Th2) cytokines in response to MSP1₁₉ peptides is shown in Fig. 3. While there was no difference in IFN-γ release in response to either wild-type or mutant peptides, the immunodominant peptide P16 induced the higher IFN-γ release when compared with most of the other peptides (P13, P13M, P15, P17, P18, P19 and P20) (Fig. 3a). Interestingly, IL-13 production showed a different pattern to that of IFN-γ production in response to peptides with either the wild-type or the mutant sequences. Higher IL-13 release was obtained with mutant peptides P13M, P15M and P17M compared to the corresponding wild-type peptides P13, P15 and P17 (P13M versus P13, P = 0·025; P15M versus P15, P = 0·001; P17M versus P17, P = 0·03) (Fig. 3b). Significantly higher amounts of IL-13 were induced by P19 compared with other peptides [P19 versus P13M (P = 0·033); P19 versus P14 (P = 0·046); P19 versus P14M (P = 0·003); P19 versus P15M (P < 0·001); P19 versus P16 (P = 0·030); P19 versus P16M (P = 0·003); P19 versus P17 (P < 0·0001); P19 versus P17M (P = 0·019); P19 versus P18 (P = 0·002) and P19 versus P21 (P = 0·019)] (not shown).

Fig. 2.

(a) Response by interferon (IFN)-γ production to merozoite surface protein 1 (MSP1₁₉) peptides in 43 Gambian malaria-exposed donors. Donors with IFN-γ responses to peptides significantly different from background (P < 0·05) were scored positive. Results are presented as percentage of IFN-γ positive responders to each MSP1₁₉ wild-type (unfilled bars) or mutant peptide (hatched bars). No differences in the number of responders were found when we compared wild-type and modified peptides. (b) Frequencies of IFN-γ-secreting T cells in response to wild-type (unfilled bars) and mutant (hatched bars) MSP1₁₉ peptides. Results are presented as the average spot-forming units (SFU)/10⁶ peripheral blood mononuclear cells (PBMC) ± standard deviation. IFN-γ-secreting T cells were produced most frequently in response to peptides P16 and P21 (531 ± 598 and 400 ± 689 SFU/10⁶ PBMC, respectively). No differences were found for the frequency of IFN-γ-secreting T cells in response to wild-type and mutant peptides.

Fig. 3.

Cytokine production by peripheral blood mononuclear cells (PBMC) in response to merozoite surface protein 1 (MSP1₁₉) peptides. PBMC from 43 adults were cultured for 3 days with MSP-1₁₉ peptides. Their cytokine production was assessed as well as their sCD30 release. T helper type 1 (Th1) [interferon (IFN)-γ] and Th0 and Th2 [interleukin (IL)-13 and sCD30] markers were measured using enzyme-linked immunosorbent assay (ELISA). Results are presented as mean ± standard deviation. (a) Significantly higher amounts of IFN-γ were produced by P16 stimulation compared with other peptides [P16 versus P13 (P* < 0·001); P16 versus P13M (P** = 0·02); P16 versus P15 (P^# = 0·02); P16 versus P17 (P^## = 0·008); P16 versus P18 (P^§ = 0·005); P16 versus P19 (P^§§ = 0·017); P16 versus P20 (P^& = 0·045)]. (b) MSP1₁₉ mutant peptides induced a higher IL-13 release compared to the wild-type [P13M versus P13 (P* = 0·025); P15M versus P15 (P** = 0·001); P17M versus P17 (P*** = 0·03)]. (c) For sCD30 mutant and wild-type peptides showed the same pattern of response, the highest sCD30 release was obtained with P19.

Overall, the peptides showed an interesting distinct Th1/Th2 pattern; for example, P16 induced higher IFN-γ production, characteristic of a Th1 response, while P19 induced the highest IL-13 and sCD30 production (Fig. 3c), characteristic of a Th2 response.

Correlation between antibody and T cell responses to MSP1₁₉ antigens

Because the CD4 T cell response is important for antibody production, we measured total IgG specific for the wild-type MSP1₁₉ antigen present in the plasma samples. We detected specific antibody in 20 of 42 (48%) donors tested. No correlation of antibody with parasitaemia, SFU/10⁶ PBMC or cytokine production could be found.

HLA-DR typing and responses to MSP1₁₉ peptides

A total of 30 individuals were typed for class II molecules using the low-resolution PCR–SSP technique. The loci investigated included HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DR5 and HLA-DQB1. Table 2 shows the allele frequencies in the study population. None of the study participants had DRB4 or DRB5 alleles. Eleven DRB1 alleles were found among the study participants: 19 of 30 (63%), 10 of 30 (33%) and seven of 30 (23%) donors carried the alleles DRB1*13, DRB1*11 and DRB1*10, respectively. The response to MSP1₁₉ peptides was heterogeneous and was not allele type-dependent; none of the peptides was restricted by a particular DRB1 allele. In this population, three DRB3 alleles were found: DRB3*01 (n = 2), DRB3*02 (n = 18) and DRB3*03 (n = 9). Individuals carrying the allele DRB3*01 responded only to peptides P14M and P15M, which are the modified forms of P14 and P15, respectively. Individuals carrying other DRB3 alleles responded to all peptides and none of the peptides was restricted by a particular DRB3 allele. All five DQB1 group-specific alleles were present in the study population. Individuals carrying the DQB1*02 allele did not respond to P13, P14 and P21 (located within the second EGF domain), all others responded to both wild-type and modified peptides. Interestingly, 50% of DQB1*02 participants responded to P13M, a modified form of P13 and 75% of DQB1*02 participants responded to P16. None of the peptides was restricted by a particular DQB1*05 or DQB1*06 allele.

Table 2.

Human leucocyte antigen D-related (HLA-DR) class II alleles detected in the study population and the percentage of individuals carrying specific alleles

Gene	Alleles	Number of individuals	Percentage (%)
DRB1	01	3	10
	03	6	20
	04	4	13
	07	1	3
	08	1	3
	09	2	7
	10	7	23
	11	10	33
	13	19	63
	14	2	7
	15	1	3
DQB1	02	4	13
	03	21	70
	04	4	13
	05	14	47
	06	6	20
DRB3	01	2	6
	02	18	60
	03	9	30

Discussion

The MSP1₁₉ triple mutant protein is a vaccine candidate designed to improve the specificity and efficacy of the antibody response induced. However, its ability to induce a CD4 T cell response and its magnitude compared to the response to the wild-type protein was unknown. It is possible that the amino acid substitutions affect the processing and presentation of peptides by APC or create or destroy T cell epitopes; such epitopes recognized by individuals naturally exposed to the parasite are of particular interest. Modifications of MSP1₁₉ that improve the quality of both the cellular and the humoral response to the protein may be particularly beneficial. We therefore investigated cellular immune responses to the modified MSP1₁₉ compared to the wild-type protein and identified immunodominant peptides in individuals naturally exposed to P. falciparum malaria.

As IFN-γ is the dominant lymphokine induced in response to MSP1₁₉ antigen in both vaccinated volunteers and adults naturally exposed to malaria [2], we first used an ELISPOT method to test for IFN-γ production from cells obtained from adult Gambian donors in response to stimulation with both the wild-type and modified proteins. We found that about one-third of the individuals responded to both proteins. This result is similar to that obtained in previous studies in The Gambia and Kenya when T cell proliferation in response to overlapping MSP1₁₉ peptides was measured [10,14]. Equal numbers of IFN-γ secreting T cells responded to both wild-type and modified protein in these samples from adults naturally exposed to malaria. IFN production by ELISPOT measurement in response to overlapping wild-type and mutant MSP1₁₉ peptides showed that the number of responders to overlapping wild-type and mutant MSP1₁₉ peptides varied from 26% (P13) to 63% (P16).

The P. falciparum MSP1₁₉ sequence is highly conserved, largely with diversity at only four positions in the 96 amino acid residues [15] preceding the C-terminal glycosylphosphatidylinositol (GPI) anchor [16]. In the first EGF domain residue 14 is either Gln or Glu and in the second EGF domain residues 61, 70 and 71 are Lys, Asn, Gly or Thr, Ser and Arg, respectively. Starting with the Q-KNG variant, a panel of modified proteins has been designed in an attempt to improve the immunogenicity of MSP1₁₉[4]. Such modifications may alter or remove T cell epitopes. We found no difference in the prevalence of response to wild-type and modified protein or wild-type and modified peptide. The number of IFN-γ-secreting T cells was also similar following stimulation with either wild-type or modified peptides and proteins. Our results showed that the antigenic linear epitopes in MSP1₁₉ peptides and proteins were not altered by the chosen amino acid substitutions.

In a previous report, a higher T cell response as measured by proliferation was induced in individuals naturally exposed to malaria by certain modified MSP1₁₉ proteins compared to wild-type [17]. This result differs from ours and may be explained by differences such as the individuals included in the two studies (asymptomatic versus symptomatic), the nature of the wild-type and modified MSP1₁₉ protein, and in T cell proliferation assays used.

We also measured the Th1 (IFN-γ) and Th0/Th2 (IL-13 and sCD30) lymphokine release from T cells stimulated with wild-type and modified MSP1₁₉ proteins and peptides. We found a higher IFN-γ release with the modified MSP1₁₉ compared to wild-type protein. Higher levels of IFN-γ may increase resistance to malaria reinfection [18]. In contrast, wild-type and modified protein showed the same patterns of IL-13 and sCD30 production.

With respect to the responses to peptides, no differences were observed for IFN-γ and sCD30 release comparing wild-type and mutant peptides. Interestingly, individuals naturally exposed to malaria showed higher IL-13 release in response to mutant peptides compared to wild-type peptides. Mutant peptides that elicited a Th0 response through IL-13 may activate antibody production more efficiently [19].

Two conserved MSP1₁₉ peptides (P16 and P19) showed different patterns of cytokine response. The immunodominant peptide P16 and its modified form P16M showed the highest IFN-γ release among most MSP1₁₉ peptides, whereas the highest IL-13 production was obtained in response to P19. These differences may be very useful in immune manipulation through vaccination.

It is possible that amino acid substitutions may alter the kinetics of MSP1₁₉ processing and presentation by APC in association with HLA class II molecules. The importance of HLA class II molecules in protection or susceptibility to malaria infection has been reported in several studies [20–22]. HLA class II molecules can affect the level of antibody to malaria. We investigated the prevalence of the loci HLA-DRB1, HLA-DRB3, HLA-DR4, HLA-DR5 and HLA-DQB1 in our study population. None of the subjects included in the study carried HLA-DR4 and HLA-DR5 alleles. Eleven DRB1 alleles were found; DRB1*13 was more representative with 63% of individuals carrying the allele. The HLA-DRB1*04 associated with malaria anaemia [23] was carried by 13% of individuals. Responses of individuals to MSP1₁₉ peptides were not restricted to a particular HLA DRB1 and DQB1 allele.

In summary, we found strong Th1 and Th2 responses to wild-type and modified forms of MSP1₁₉ proteins and peptides in Gambian adults naturally exposed to malaria. No difference in term of number of responders with IFN-γ production was found in our study; however, the triple-variant MSP1₁₉ protein induced higher Th1 cytokine (IFN-γ) production than did the wild-type. In addition, we have identified the peptide P16 as immunodominant, being recognized by 63% of study participants and not restricted by particular DRB1, DRB3 and DQB1 alleles. Interestingly, the MSP1₁₉ peptides P16 and P19 induced different cytokine responses as P16 induced a higher Th1 response (IFN-γ) and P19 induced high levels of IL-13 and sCD30.

Altogether, these findings will be very useful in future vaccine development and studies of Th1/Th2 immunmodulation, using either wild-type or modified protein in combination with their peptides.

Disclosure

None.