Liver-stage Antigen Research Articles

Immune responses toPlasmodium vivax pre-erythrocytic stage antigens in naturally exposed Duffy-negative humans: a potential model for identification of liver-stage antigens

Duffy antigen is the receptor used by Plasmodium vivax to invade erythrocytes. Consequently, individuals lacking Duffy antigen [Fy(-)] do not develop blood-stage infections. We hypothesized that naturally exposed Fy(-) humans may develop immune responses mainly to pre-erythrocytic stages and could be used to study acquired immunity to P. vivax and to identify liver-stage antigens. We report here that antibody and IFN-gamma responses to known sporozoite antigens were significantly induced by natural exposure in Fy(-) humans, whereas responses to blood-stage antigens were significantly induced in Fy(+) humans. IFN-gamma responses to sporozoite antigens were lower in Fy(+) than in Fy(-) humans, indicating that in Fy(+) humans blood-stage infections may have suppressed T cell responses to pre-erythrocytic stages. We evaluated the immune responses to 18 novel P. vivax homologs of P. falciparum sporozoite proteins identified from the P. vivax genome sequence. Eight proteins recalled IFN-gamma responses in P. vivax-exposed but not in unexposed individuals. Of these, 3 antigens elicited IFN-gamma responses in Fy(-) but not in Fy(+) individuals. These results suggest that differential immune responses observed in naturally exposed Fy(-) and Fy(+) individuals can be exploited to identify P. vivax stage-specific antigens.

Enhanced T cell-mediated protection against malaria in human challenges by using the recombinant poxviruses FP9 and modified vaccinia virus Ankara

Malaria is a major global health problem for which an effective vaccine is required urgently. Prime-boost vaccination regimes involving plasmid DNA and recombinant modified vaccinia virus Ankara-encoding liver-stage malaria antigens have been shown to be powerfully immunogenic for T cells and capable of inducing partial protection against experimental malaria challenge in humans, manifested as a delay in time to patent parasitemia. Here, we report that substitution of plasmid DNA as the priming vector with a specific attenuated recombinant fowlpox virus, FP9, vaccine in such prime-boost regimes can elicit complete sterile protection that can last for 20 months. Protection at 20 months was associated with persisting memory but not effector T cell responses. The protective efficacy of various immunization regimes correlated with the magnitude of induced immune responses, supporting the strategy of maximizing durable T cell immunogenicity to develop more effective liver-stage vaccines against Plasmodium falciparum malaria.

Towards an RTS,S-based, multi-stage, multi-antigen vaccine against falciparum malaria: progress at the Walter Reed Army Institute of Research

The goal of the Malaria Vaccine Program at the Walter Reed Army Institute of Research (WRAIR) is to develop a licensed multi-antigen, multi-stage vaccine against Plasmodium falciparum able to prevent all symptomatic manifestations of malaria by preventing parasitemia. A secondary goal is to limit disease in vaccinees that do develop malaria. Malaria prevention will be achieved by inducing humoral and cellular immunity against the pre-erythrocytic circumsporozoite protein (CSP) and the liver stage antigen-1 (LSA-1). The strategy to limit disease will target immune responses against one or more blood stage antigens, merozoite surface protein-1 (MSP-1) and apical merozoite antigen-1 (AMA-1). The induction of T- and B-cell memory to achieve a sustained vaccine response may additionally require immunization with an adenovirus vector such as adenovirus serotype 35. RTS,S, a CSP-derived antigen developed by GlaxoSmithKline Biologicals in collaboration with the Walter Reed Army Institute of Research over the past 17 years, is the cornerstone of our program. RTS,S formulated in AS02A (a GSK proprietary formulation) is the only vaccine candidate shown in field trials to prevent malaria and, in one instance, to limit disease severity. Our vaccine development plan requires proof of an individual antigen's efficacy in a Phase 2 laboratory challenge or field trial prior to its integration into an RTS,S-based, multi-antigen vaccine. Progress has been accelerated through extensive partnerships with industrial, academic, governmental, and non-governmental organizations. Recent safety, immunogenicity, and efficacy trials in the US and Africa are presented, as well as plans for the development of a multi-antigen vaccine.

Gamma interferon responses to Plasmodium falciparum liver-stage antigen 1 and thrombospondin-related adhesive protein and their relationship to age, transmission intensity, and protection against malaria.

Gamma interferon (IFN-gamma) responses to the Plasmodium falciparum antigens liver-stage antigen 1 (LSA-1) and thrombospondin-related adhesive protein (TRAP) are thought to be important in protection against malaria. Optimal methods of testing and the effects of age and transmission intensity on these responses are unknown. IFN-gamma responses to LSA-1 and TRAP peptides were assessed by the enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA) in children and adults from areas of stable and unstable malaria transmission in Kenya. Adults in the areas of stable and unstable transmission had similar frequencies and levels of IFN-gamma responses to LSA-1 and TRAP as determined by ELISPOT and ELISA. In contrast, IFN-gamma responses to the LSA-1 T3 peptide (assessed by ELISPOT) and to any LSA-1 peptide (assessed by ELISA) were less frequent in children in the area of unstable transmission than in children in the area of stable transmission. IFN-gamma responses to LSA-1 were more frequently detected by ELISA than by ELISPOT in the stable-transmission area. IFN-gamma responses detected by ELISA and ELISPOT did not correlate with each other. In children in the stable-transmission area, IFN-gamma responses to LSA-1 peptides assessed by ELISA, but not by ELISPOT, were associated with protection against clinical malaria and anemia. IFN-gamma responses to LSA-1 appear to require repeated P. falciparum exposure and/or increased age and, as measured by ELISA, are associated with protection against clinical malaria and anemia.

Liver stage antigen and malaria

Liver stage antigen and malaria

Antibodies That Inhibit Binding of Plasmodium falciparum -Infected Erythrocytes to Chondroitin Sulfate A and to the C Terminus of Merozoite Surface Protein 1 Correlate with Reduced Placental Malaria in Cameroonian Women

Plasmodium falciparum-infected erythrocytes often sequester in the placenta of pregnant women, producing placental malaria, a condition that can compromise the health of the developing fetus. Scientists are hopeful that a vaccine can be developed to prevent this condition. Immunological mechanisms responsible for eliminating parasites from the placenta remain unclear, but antibodies to the carboxyl-terminal 19-kDa segment of the merozoite surface protein 1 (MSP1-19), the ring-infected erythrocyte surface antigen (RESA), and an erythrocyte-surface ligand that binds chondroitin sulfate A (CSA-L) have been implicated. In addition, antibodies to sporozoite and liver-stage antigens could reduce initial parasite burdens. This study sought to determine if antibodies to the circumsporozoite protein (CSP), liver-stage antigen 1 (LSA1), RESA, MSP1-19, or CSA-L correlated with either the absence of placental parasites or low placental parasitemias. Using a frequency-matched case-control study design, we compared antibody levels in women (gravidity 1 to 11) with and without placental malaria. Results showed that women who were antibody negative for MSP1-19 were at a higher risk of having placental malaria than women with antibodies (P < 0.007). Furthermore, an association between high levels of antibodies that blocked the binding of infected erythrocytes to CSA and low placental parasitemias was observed (P = 0.02). On the other hand, women with high antibody levels at term to CSP, LSA1, and RESA were more likely to have placental malaria than antibody-negative women. Since antibodies to MSP1-19 and CSA-L were associated with reduced placental malaria, both antigens show promise for inclusion in a vaccine for women of child-bearing age.

Sporozoite and Liver Stage Antigen Plasmodium falciparum peptides bind specifically to human hepatocytes

Sporozoite and Liver Stage Antigen (SALSA) sequence synthetic peptides were used in HepG2 cell binding assays to identify regions involved in parasite invasion. SALSA 20608 ( 21 IWASEKKDEKEASEQGEESHY 40 ) and 20611 ( 64 KKDDGTDKVQEKVLEKSPKY 83 ) peptides were determined as having high binding activity in HepG2 cell assays, some of them were located in immunogenic regions. Immune-fluorescence antibody test with 24276 (20608 peptide analogue, CGIWS SM K M DEK M A AM QGEESHCG) showed sporozoite and merozoite reactivity. This data suggests SALSA high activity binding peptides’ (HABPs) possible role in hepatic cell invasion and merozoite invasion of erythrocytes.

Antibodies to the Plasmodium falciparum antigens circumsporozoite protein, thrombospondin-related adhesive protein, and liver-stage antigen 1 vary by ages of subjects and by season in a highland area of Kenya.

Immunoglobulin G (IgG) antibodies to three vaccine candidate preerythrocytic Plasmodium falciparum antigens were evaluated in children and adults in an epidemic-prone highland area of Kenya during rainy (high-transmission) and dry (low-transmission) seasons. The frequencies and median levels of IgG antibodies to circumsporozoite protein (CSP) and thrombospondin-related adhesive protein (TRAP) were compared to the frequencies and median levels of IgG antibodies to liver-stage antigen 1 (LSA-1) reported previously. The frequencies and median levels of IgG antibodies to CSP and TRAP were similar in children and adults in the rainy season, but they were lower in children than in adults in the dry season. The frequencies and median levels of antibodies to LSA-1 were lower in children than in adults in both the rainy and dry seasons. Antibodies to CSP and LSA-1 were primarily members of the IgG1 and IgG3 subclasses, while antibodies to TRAP were primarily members of the IgG3 and IgG4 subclasses. In a treatment-reinfection study following dry season testing, antibodies to TRAP were associated with a trend toward protection from infection in children (P = 0.051) but not in adults. Antibodies to LSA-1 and CSP did not correlate with protection in children or adults. In this highland area of Kenya with unstable transmission, IgG antibodies to preerythrocytic P. falciparum antigens vary in subjects by age and season, and the protective effects of these antibodies against infection may be different in adults and children.

Peptides of the liver stage antigen-1 (LSA-1) of Plasmodium falciparum bind to human hepatocytes

Synthetic peptides from the liver stage antigen-1 (LSA-1) antigen sequence were used in HepG2 cell and erythrocyte binding assays to identify regions that could be involved in parasite invasion. LSA-1 protein peptides 20630 ( 21 INGKIIKNSEKDEIIKSNLRY 40 ), 20637 ( 157 KEKLQGQQSDSEQERRAY 173 ), 20638 ( 174 KEKLQEQQSDLEQERLAY 190 ) and 20639 ( 191 KEKLQEQQSDLEQERRAY 207 ) had high binding activity in HepG2 assays. Were located in immunogenic regions; peptide cell binding was saturable. Peptide 20630 bound specifically to 48 kDa HepG2 membrane surface protein. LSA-1 peptides 20630 ( 21 INGKIIKNSEKDEIIKSNLRY 40 ) and 20633 ( 81 DKELTMSNVKNVSQTNFKSLY 100 ) showed specific erythrocyte binding activity and inhibited merozoite invasion of erythrocytes in vitro. A monkey serum prepared against LSA-1 20630 peptide analog (CGINGKNIKNAEKPMIIKSNLRGC) inhibited merozoite invasion in vitro. The data suggest LSA-1 “High Activity Binding Peptides” could play a possible role in hepatic cell invasion as well as merozoite invasion of erythrocytes.

EFFECTS OF PERMETHRIN-TREATED BED NETS ON IMMUNITY TO MALARIA IN WESTERN KENYA II. ANTIBODY RESPONSES IN YOUNG CHILDREN IN AN AREA OF INTENSE MALARIA TRANSMISSION

As part of a large community-based trial on the impact of insecticide (permethrin)-treated bed nets (ITNs) on childhood morbidity and mortality in an area of intense perennial malaria transmission in western Kenya, we assessed the effects of ITNs on malaria-specific humoral responses in young children. The IgG responses to Plasmodium falciparum pre-erythrocytic antigens circumsporozoite protein (CSP) and liver stage antigen-1 (LSA-1) and the blood stage antigen merozoite surface protein-1 (MSP-1(19) kD) in children less than three years old were investigated during a series of cross-sectional surveys. At 14 and 22 months after the introduction of ITNs, the frequencies and levels of IgG to CSP and LSA-1 were significantly lower in children from ITN villages than in children from control villages (P < 0.001). In contrast, the prevalence of IgG to MSP-1 was significantly higher in children from ITN villages at 14 months (P = 0.0069), but not at 22 months. Our results show that decreased exposure by ITNs reduces IgG responses to pre-erythrocytic antigens, but there was no evidence that two years of ITN use compromises IgG responses to blood stage antigens in these young children in this malaria holoendemic area.

Analysis of intra-hepatic peptide-specific cell recruitment in mice immunised with Plasmodium falciparum antigens

The liver stage of Plasmodium spp. now appears as a relevant target of immune effectors triggered by the so-called “anti-sporozoite” vaccine. Since the monitoring of immune responses at the systemic level may not faithfully reflect the local protective mechanisms, the aim of the present work was to set up a model to study the local intra-hepatic cellular responses and to compare these with the peripheral immune responses. This was achieved by intra-portal delivery of epitopic peptides, i.e. peptides containing B and T cell epitopes, which were coated onto the surface of polystyrene microbeads. The peptide-coated beads presumably mimic the hepatic schizont, and when distinct peptides are administered separately, this method of delivery allows us to decipher the immune responses resulting in mice immunised with recombinant proteins spanning several such epitopes. Using the P. falciparum liver stage antigen-3 (LSA3) molecule, which can induce protection against a sporozoite challenge, our results show that 25-μm microbeads could easily access the liver parenchyma by intra-portal injection and were distributed evenly in the liver. Also, LSA3-derived synthetic peptides coated onto microbeads initiated specific cell recruitment within 6 h. Depending on the LSA3 peptide used, the infiltrates induced differed in size, with the strongest cell recruitment obtained using nonrepeat II peptide (NR2)-coated microbeads with a mean leukocyte number of 79 per granuloma. Immunohistological studies of liver sections revealed that, irrespective of the delivered peptide, cells infiltrating the liver towards microbeads were mainly CD3 + T lymphocytes, both CD4 + (70 to 80%) and CD8 + (20 to 30%) subtypes, macrophages and dendritic cells. Cells infiltrating the granuloma had features of activated cells, with evidence of VLA-4 cell-surface expression, and production of IFN-γ and IL-4. Analysis of the peripheral B and T-cell responses in the same animals revealed that, whereas the local responses were directed mainly towards NR2 and repeat peptides (RE), the peripheral T-cell response to these peptides was weak and infrequent, although antibody production was high.

Immunity to liver stage malaria: considerations for vaccine design.

Malaria is the world's deadliest parasitic disease and effective control measures are a public health priority. Most deaths in humans from malaria are caused by one species of the protozoa, Plasmodium falciparum. An efficacious and cost-effective vaccine against this parasite is considered a holy grail of modern molecular medicine. A vaccine that targets liver stage parasites would prevent infection from reaching the blood and causing clinical disease. Among known P. falciparum antigens, liver stage antigen-1 (LSA-1) is the only protein expressed exclusively by infected hepatocytes. Several independent studies in humans have consistently related immune responses to LSA-1 with resistance to infection or disease, providing a powerful rationale for the development of liver stage vaccines. Investigations by ourselves and others aim to dissect the mechanism of cellular immunity to LSA-1 and to evaluate in different delivery systems epitopes associated with protection as components of a multiantigen malaria vaccine. The first clinical trials are already being conducted, the results of which are eagerly awaited.

Interferon-gamma responses to Plasmodium falciparum liver-stage antigen-1 and merozoite-surface protein-1 increase with age in children in a malaria holoendemic area of western Kenya

BackgroundIn areas of high-level, year-round malaria transmission, morbidity and mortality due to malaria decrease after the first two to three years of life. This reduction may be related to the development of cellular immunity to specific antigens expressed in the different life-cycle stages of Plasmodium falciparum.MethodsA cross sectional study was conducted to evaluate T cell cytokine responses to the P. falciparum pre-erythrocytic antigen liver-stage antigen-1 (LSA-1) and the blood-stage antigen merozoite-surface protein-1 (MSP-1) in children under five years of age residing in a malaria holoendemic region of western Kenya. Interferon-γ (IFN-γ) and interleukin-10 (IL-10) responses to the LSA-1 T3 peptide (aa 1813–1835) and the MSP-1 aa20–39 peptide were tested in 48 children.ResultsThe proportion of children producing IFN-γ to LSA-1 and to MSP-1 increased with age: in the 0–12, 13–24, 25–36 and 37–48 month age groups, zero, 11.1, 36.4 and 40% of children had IFN-γ responses to LSA-1 (p = 0.019), and 10, 10, 27.7 and 40% of children had IFN-γ responses to MSP-1 (p = 0.07), respectively. In contrast, the proportion of children producing IL-10 to LSA-1 and MSP-1 was similar in all age groups.ConclusionThe data suggest that development of IFN-γ responses to LSA-1 and MSP-1 requires increased age and/or repeated exposure, whereas IL-10 responses to these antigens may occur at any age and with limited exposure. The data also demonstrate that by the age of 4 years, children in a malaria holoendemic area develop frequencies of IFN-γ responses to LSA-1 and MSP-1 similar to those seen in adults in the area.

Lymphocyte proliferation and antibody responses to Plasmodium falciparum liver-stage antigen-1 in a highland area of Kenya with seasonal variation in malaria transmission.

Lymphocyte proliferation and antibody responses to five peptides corresponding to the N- and C-terminal non-repeat and central repeat regions of Plasmodium falciparum liver-stage antigen-1 (LSA-1) were examined in residents of a highland area of Kenya where malaria transmission is episodic and varies with rainfall. The frequency of lymphocyte proliferation responses (stimulation index > 2) by children (persons > or = 6 years old) and adults (persons > or = 18 years old) was similar and did not differ significantly across seasons. In contrast, the proportion of individuals with IgG antibodies to LSA-1 peptides was higher in the rainy than dry season, and the frequency of these responses was greater for adults than children (39.4% versus 18.7% during the period of high transmission; P = 0.009). Antibodies to LSA-1 were primarily of the IgG1 and IgG3 subclasses, and these also varied with season (30.1% and 32.5% of individuals had IgG1 and IgG3 in the rainy season versus none and 10.9% in the dry season). There was no significant difference in the time to re-infection between groups of persons with or without IgG antibody or lymphocyte proliferation responses to LSA-1 peptides. These data indicate that age and transmission intensity independently affect IgG antibody responses to LSA-1 but do not influence lymphocyte proliferation in this highland area where malaria transmission is highly variable.

Antibody responses to repetitive epitopes of the circumsporozoite protein, liver stage antigen-1, and merozoite surface protein-2 in infants residing in a Plasmodium falciparum-hyperendemic area of western Kenya. XIII. Asembo Bay Cohort Project.

The present study was initiated to characterize antibody responses to repetitive epitopes of the circumsporozoite protein (CSP), liver stage antigen-1 (LSA-1), and merozoite surface protein-2 (MSP-2) of Plasmodium falciparum in infants residing in a P. falciparum-hyperendemic area of western Kenya. In this study, development and maintenance of these antibody responses in 28 infants were studied longitudinally by use of monthly serum samples collected from birth to age 1 year. Mother plasma and infant umbilical cord plasma were also tested to assess the transplacental transfer of maternal antibodies. Results showed that antibodies passively transferred from mothers were detectable for CSP, LSA-1, and MSP-2 repeat epitopes. Infants were able to mount and maintain a strong antibody response against LSA-1 in their first year of life. Infants often responded to CSP repeats, but with a much lower antibody titer. Antibody responses in infants against Fc27 and 3D7 repeats of MSP-2 were low throughout their first year. In addition, 51 infants whose first detected infection occurred at > 4 months of age were selected to determine antibody responses to the antigens tested upon their first and second detected infections. Antibody responses to LSA-1 and, to a lesser degree, CSP increased in positivity rates and titer upon second infection. Antibody responses to Fc27-type and 3D7-type repeats of MSP-2 were low upon both infections. There was no association between maternally transferred anti-LSA-1, anti-CSP, or anti-MSP-2 antibodies and an infant's first detected infection. No significant correlation was found between an infant's antibody responses to the 4 antigen repetitive epitopes and protection against malarial parasitemia during the first year of life.

Influence of age and HLA type on interferon-gamma (IFN-gamma) responses to a naturally occurring polymorphic epitope of Plasmodium falciparum liver stage antigen-1 (LSA-1).

Antigenic polymorphism and HLA restriction may limit the immunogenicity of a subunit vaccine against liver-stage Plasmodium falciparum. We examined 59 clinical isolates and five laboratory clones of P. falciparum for polymorphism in the N- and C-terminal regions of LSA-1, evaluated binding of the corresponding peptides to selected HLA class I alleles, and measured IFN-gamma responses in residents of a malaria-endemic area of Papua New Guinea where HLA-A*1101, -24, -B13, and -B40 are the most common class I alleles. LSA-1 polymorphism was limited to a single non-synonymous mutation encoding serine (S), proline (P), or threonine (T) at amino acid 85. Nine-mer 84-92 peptides with S, T, or P at the primary anchor position bound differentially to HLA-A11, -A2, and -B7. IFN-gamma ELISPOT responses increased with age in malaria-exposed subjects: 14-16% and 30-36% of 2-5- and 6-54-year-olds, respectively, had > or =10 IFN-gamma-secreting cells/106 peripheral blood mononuclear cells when stimulated with at least one peptide variant (P<0.05). IFN-gamma responses to all three peptides were also greater for older than younger individuals. No children < 3 years old had lymphocytes that responded to all three 84-92 peptides, whereas 45% of adults (mean age 48 years) had aggregated IFN-gamma responses. These data support the notion that age-related cumulative exposure to P. falciparum increases the frequency of IFN-gamma responses to polymorphic epitopes of liver-stage antigens such as LSA-1.

Laser capture microdissection and molecular analysis of Plasmodium yoelii liver-stage parasites

Understanding the liver stage development of Plasmodial parasites, and by extension the proteins expressed during that phase of the life cycle, has become increasingly more important as investigators have identified the pre-erythrocytic stage of the parasite as a target for effector T lymphocytes and their products. It has been clearly demonstrated that immunization with radiation-attenuated sporozoites can confer long lasting sterile protective immunity and that the immunity is stage-specific [1–3]. This form of immunization is effective against a sporozoite challenge, but not a blood stage challenge. Therefore, the protective immune response must be directed against the sporozoite and/or liver stage antigens. Experimental evidence, in mice, has clearly demonstrated that this protection requires the cellular immune system [4,5] and specifically CD8 T lymphocytes [6–8]. Malarial antigens expressed during the liver stage are processed and presented, in the context of class I MHC molecules, on the surface of infected hepatocytes [9,10]. Identifying these antigens, that may act as targets for cell mediated immunity, would significantly enhance the understanding of how the irradiated sporozoite vaccine model confers protection and ultimately lead to the identification of candidates for a subunit vaccine. Recent advances in tissue microdissection have provided a methodology to allow the isolation of a pure population of liver stage schizonts from heterogeneous liver tissue. In the past, microdissection was done manually or with the aid of a micromanipulator. The area of interest was scraped off of a tissue section using a needle and subsequent processing was performed with the tissue that was recovered. This was a slow, laborious and unreliable practice that frequently yielded an impure cell population. The development of Laser-capture microdissection (LCM) has turned microdissection into a fast, reliable and precise technique. LCM utilizes an infra red laser and a transparent thermoplastic film to selectively isolate cells or regions of interest [11,12]. Briefly, a slide containing a section of infected liver is placed on the microscope stage. A transport arm, holding the thermoplastic film, is then moved over the region of interest. The cell(s) to be isolated are moved to the center of the optical field and the laser is activated. The laser melts the film directly onto the tissue containing the targeted cell. The film rapidly cools and solidifies such that when the film is moved by the transport arm the film/tissue combination shears away from the surrounding tissue. The process can be repeated many times, utilizing areas of the film that have not been melted with the laser. The film, which is held in a plastic ring, the same dimensions as a miAbbre iations: AMA-1, apical membrane antigen 1; CS, circumsporozoite; LCM, laser capture microdissection; MSP1, merrozoite surface protein 1; NYLS3, navy yoelii liver stage 3; RT-PCR, reverse transcriptase-polymerase chain reaction. * Corresponding author. E-mail address: jsacc001@umaryland.edu (J.B. Sacci, Jr). 1 Present address: Celera Genomics, 45 West Gude Dr., Rockville, MD 20852, USA.

Immunogenicity of well-characterized synthetic Plasmodium falciparum multiple antigen peptide conjugates.

Given the emerging difficulties with malaria drug resistance and vector control, as well as the persistent lack of an effective vaccine, new malaria vaccine development strategies are needed. We used a novel methodology to synthesize and fully characterize multiple antigen peptide (MAP) conjugates containing protective epitopes from Plasmodium falciparum and evaluated their immunogenicity in four different strains of mice. A di-epitope MAP (T3-T1) containing two T-cell epitopes of liver stage antigen-1 (LSA-1), a di-epitope MAP containing T-cell epitopes from LSA-1 and from merozoite surface protein-1, and a tri-epitope MAP (T3-CS-T1) containing T3-T1 and a potent B-cell epitope from the circumsporozoite protein central repeat region were tested in this study. Mice of all four strains produced peptide-specific antibodies; however, the magnitude of the humoral response indicated strong genetic restriction between the different strains of mice. Anti-MAP antibodies recognized stage-specific proteins on the malaria parasites in an immunofluorescence assay. In addition, serum from hybrid BALB/cJ x A/J CAF1 mice that had been immunized with the tri-epitope MAP T3-CS-T1 successfully inhibited the malaria sporozoite invasion of hepatoma cells in vitro. Spleen cells from immunized mice also showed a genetically restricted cellular immune response when stimulated with the immunogen in vitro. This study indicates that well-characterized MAPs combining solid-phase synthesis and conjugation chemistries are potent immunogens and that this approach can be utilized for the development of subunit vaccines.

Long synthetic peptides encompassing the Plasmodium falciparum LSA3 are the target of human B and T cells and are potent inducers of B helper, T helper and cytolytic T cell responses in mice.

We synthesized 17 long synthetic peptides (LSP) spanning the whole 200-kDa Plasmodium falciparum liver stage antigen-3 (LSA3), an antigen that induces protection in chimpanzee, and analyzed their immunogenicity in BALB/c mice and their antigenicity in individuals living in a hyper-endemic malaria area. Our findings show that both specific antibodies and T cell proliferation against most LSA3-LSP develop in malaria-exposed adults. All individuals studied had detectable antibodies against a minimum of 6 and a maximum of 15 polypeptides. It is noteworthy that antibody prevalence and titers were as high against non-repeat as repeat regions. Although the extent of T cell reactivity was lower than that observed for B cells, most of the sequences contained at least one T helper epitope, indicating that the majority of LSA3-LSP contain both B and T cell epitopes within the same sequence. Injection of LSA3-LSP with SBSA2 adjuvant in mice, showed strong immunogenicity for most of them, eliciting both T cell responses and specific antibody production. While all the peptides were immunogenic for B cells, different patterns of T cell responses were induced. These peptides were thus classified in three sets according to the levels of the T cell proliferative and of the IFN-gamma-specific responses. Importantly, antibodies and T cells against some of the LSP were able to recognize LSA3 native protein on P. falciparum sporozoites. Additionally, some LSP (44-119, 1026-1095, 1601-1712) also contained epitopes recognized by H-2(d) class I-restricted T cells. These results led to the identification of numerous domains that are highly antigenic and immunogenic within the LSA3 protein, and underline the value of the LSP approach for vaccine development.

Pre-erythrocytic immunity to Plasmodium falciparum: the case for an LSA-1 vaccine.

A vaccine is urgently needed to stem the global resurgence of Plasmodium falciparum malaria. Vaccines targeting the erythrocytic stage are often viewed as an anti-disease strategy. By contrast, infection might be completely averted by a vaccine against the liver stage, a pre-erythrocytic stage during which the parasite multiplies 10000-fold within hepatocytes. Sterilizing immunity can be conferred by inoculating humans with irradiated pre-erythrocytic parasites, and a recombinant pre-erythrocytic vaccine partially protects humans from infection. Liver-stage antigen-1, one of a few proteins known to be expressed by liver-stage parasites, holds particular promise as a vaccine. Studies of naturally exposed populations have consistently related immune responses against this antigen to protection.