RALP1 Is a Rhoptry Neck Erythrocyte-Binding Protein of Plasmodium falciparum Merozoites and a Potential Blood-Stage Vaccine Candidate Antigen

Erythrocyte invasion by merozoites is an obligatory stage of Plasmodium infection and is essential to disease progression. Proteins in the apical organelles of merozoites mediate the invasion of erythrocytes and are potential malaria vaccine candidates. Rhoptry-associated, leucine zipper-like protein 1 (RALP1) of Plasmodium falciparum was previously found to be specifically expressed in schizont stages and localized to the rhoptries of merozoites by immunofluorescence assay (IFA). Also, RALP1 has been refractory to gene knockout attempts, suggesting that it is essential for blood-stage parasite survival. These characteristics suggest that RALP1 can be a potential blood-stage vaccine candidate antigen, and here we assessed its potential in this regard. Antibodies were raised against recombinant RALP1 proteins synthesized by using the wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that RALP1 is a rhoptry neck protein of merozoites. Moreover, our IFA data showed that RALP1 translocates from the rhoptry neck to the moving junction during merozoite invasion. Growth and invasion inhibition assays revealed that anti-RALP1 antibodies inhibit the invasion of erythrocytes by merozoites. The findings that RALP1 possesses an erythrocyte-binding epitope in the C-terminal region and that anti-RALP1 antibodies disrupt tight-junction formation, are evidence that RALP1 plays an important role during merozoite invasion of erythrocytes. In addition, human sera collected from areas in Thailand and Mali where malaria is endemic recognized this protein. Overall, our findings indicate that RALP1 is a rhoptry neck erythrocyte-binding protein and that it qualifies as a potential blood-stage vaccine candidate.     

Enhancement by Ampicillin of Antibody Responses Induced by a Protein Antigen and a DNA Vaccine Carried by Live-Attenuated Salmonella enterica Serovar Typhi

Live-attenuated Salmonella species are effective carriers of microbial antigens and DNA vaccines. In a mouse model, the immunoglobulin M (IgM) and total antibody levels directed toward the lipopolysaccharide of Salmonella enterica serovar Typhi were significantly enhanced at day 21 after oral immunization with live-attenuated serovar Typhi (strain Ty21a) when ampicillin was concomitantly administered (P < 0.05 and P < 0.005, respectively). The heat-killed Ty21a-stimulated lymphocyte proliferation indices for the ampicillin group at day 21 were significantly higher than those for the normal saline (NS) group (P < 0.005, P < 0.001, and P < 0.01) for all three doses of antigen (10⁴, 10⁵, and 10⁶ heat-killed Ty21a per well, respectively). The 50% lethal doses for mice from the ampicillin and NS groups immunized with Ty21a with pBR322 after wild-type serovar Typhi challenge on day 24 were 3.4 × 10⁷ and 5.0 × 10⁶ CFU, respectively. The fecal bacterial counts for the ampicillin group at days 1, 3, and 5 were significantly lower than those for the NS group (P < 0.01, P < 0.01, and P < 0.05, respectively), and there was a trend toward recovery of Ty21a in a larger number of mice from the ampicillin group than from the NS group. Furthermore, the IgG2a levels directed toward tetanus toxoid were significantly enhanced at days 7 and 21 after oral immunization with Ty21a that carried the fragment c of tetanus toxoid when ampicillin was concomitantly administered (P < 0.05 and P < 0.005, respectively), and the IgM and total hepatitis B surface antibody levels were significantly enhanced at days 7 (P < 0.005 and P < 0.05, respectively) and 21 (P < 0.01 and P < 0.05, respectively) after oral immunization with Ty21a that carried the DNA vaccine that encodes hepatitis B surface antigen when ampicillin was concomitantly administered. The present observation may improve the efficacy of the protein antigens and DNA vaccines carried in live-attenuated bacteria, and further experiments should be carried out to determine the best antibiotics and dosage regimen to be used, as well as the best carrier system for individual protein antigens and DNA vaccines.     

Allelic Diversity and Naturally Acquired Allele-Specific Antibody Responses to Plasmodium falciparum Apical Membrane Antigen 1 in Kenya

Although Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate, extensive allelic diversity may compromise its vaccine potential. We have previously shown that naturally acquired antibodies to AMA1 were associated with protection from clinical malaria in this Kenyan population. To assess the impact of allelic diversity on naturally acquired immunity, we first sequenced the ectodomain-encoding region of P. falciparum ama1 from subjects with asymptomatic, mild, and severe malaria and measured allele frequency distributions. We then measured antibodies to three allelic AMA1 proteins (AMA1_3D7, AMA1_FVO, and AMA1_HB3) and used competition enzyme-linked immunosorbent assays (ELISAs) to analyze allele-specific antibodies. Seventy-eight unique haplotypes were identified from 129 alleles sampled. No clustering of allelic haplotypes with disease severity or year of sampling was observed. Differences in nucleotide frequencies in clinical (severe plus mild malaria) versus asymptomatic infections were observed at 16 polymorphic positions. Allele frequency distributions were indicative of balancing selection, with the strongest signature being identified in domain III (Tajima''s D = 2.51; P < 0.05). Antibody reactivities to each of the three allelic AMA1 proteins were highly correlated (P < 0.001 for all pairwise comparisons). Although antibodies to conserved epitopes were abundant, 48% of selected children with anti-AMA1 IgG (n = 106) had detectable reactivity to allele-specific epitopes as determined by a competition ELISA. Antibodies to both conserved and allele-specific epitopes in AMA1 may contribute to clinical protection.Many candidate antigens for subunit malaria vaccines are polymorphic in natural populations, posing challenges for vaccine development. It is important to know how many alleles of a particular candidate will need to be included in a vaccine to induce antibodies with specificity broad enough to recognize the existing antigenic diversity. Populations of Plasmodium falciparum in areas where the disease is highly endemic have high recombination rates (13, 37, 41) and can generate additional haplotypic diversity with every meiotic recombination (54). This is exemplified by apical membrane antigen 1 (AMA1), for which numerous distinct haplotypes are observed, particularly in areas with relatively high malaria transmission intensities (15, 20, 44, 45, 51). These haplotypes are comprised of single-nucleotide polymorphisms, which are distributed throughout the single-locus ama1 gene, but are especially numerous in the portion encoding its surface-accessible ectodomain. Independent studies provide strong evidence that balancing selection is acting to maintain these polymorphisms in the population (15, 20, 44, 45), reflecting the importance of AMA1 as a target of protective immunity. These polymorphisms may need to be incorporated into a vaccine based on AMA1. In animal models, immunization confers better protection against challenge with parasites bearing homologous rather than heterologous alleles of AMA1 (16, 29). Likewise, invasion inhibition is more efficient against parasites bearing homologous alleles (21, 27). Recent studies suggested that the allelic diversity in ama1 could be covered by vaccination with a combination of allelic types (27, 30). However, only a few allelic variants can realistically be included in a vaccine formulation, and it remains to be determined how effective this would be in populations where malaria is endemic, where individuals are repeatedly challenged with parasites bearing diverse ama1 alleles. For example, over 200 unique haplotypes of AMA1 were recently reported for a single geographical location in Mali (51).We have previously shown that naturally acquired antibodies to AMA1 were associated with protection from clinical malaria in a population in coastal Kenya (42). Here we explore the impact of the allelic diversity of ama1 on naturally acquired antibodies in this population. We compare the allelic diversities observed among parasite isolates obtained from children with asymptomatic infections and mild and severe clinical malaria. We test for signatures of balancing selection acting on the ama1 gene in this population, as reported previously for other populations, and describe antibody responses to proteins representing three allelic versions of AMA1 before, during, and after clinical infections.     

Comparative Immunization Study Using RNA and DNA Constructs Encoding a Part of the Plasmodium falciparum Antigen Pf332

Development of nucleic acid-based vaccines against parasitic diseases shows great promise, although certain concerns about safety aspects of conventional DNA vaccines have been raised. This study presents a comparison of antibody responses induced in mice by DNA and RNA-based immunization with vectors encoding a part of the P. falciparum antigen Pf332. Two types of plasmids were used, one conventional DNA plasmid containing a cytomegalovirus promoter and one suicidal DNA plasmid encoding the Semliki Forest virus (SFV) replicase. RNA, encoding the SFV replicase and the relevant antigen, was delivered either as naked RNA or packaged in SFV suicide particles. In general, the antibody responses induced by the DNA plasmids were low and peaking after three injections, the conventional plasmid giving the highest responses. Also the RNA delivered in SFV particles consistently induced antibody responses, although comparatively low. Analyses of the ratio of immunoglobulin (Ig)G1/IgG2a subclasses in the responses indicated that all plasmids resulted in a bias for a Th2-type of response, while the SFV-particles elicited a Th1 type of response. Importantly, all these immunogens induced an immunological memory, which could be efficiently activated by a booster injection with the corresponding protein, with unchanged patterns of IgG subclasses.     

Interleukin-10 Responses to Liver-Stage Antigen 1 Predict Human Resistance to Plasmodium falciparum

The design of an effective vaccine against Plasmodium falciparum, the most deadly malaria parasite of humans, requires a careful definition of the epitopes and the immune responses involved in protection. Liver-stage antigen 1 (LSA-1) is specifically expressed during the hepatic stage of P. falciparum and elicits cellular and humoral immune responses in naturally exposed individuals. We report here that interleukin-10 (IL-10) production in response to LSA-1 predicts resistance to P. falciparum after eradication therapy. Resistance was not related to gamma interferon or tumor necrosis factor alpha production. This is the first report that human IL-10 responses are associated with resistance after eradication therapy, and our findings support the inclusion of LSA-1 in a vaccine against malaria.     

New Candidate Vaccines against Blood-Stage Plasmodium falciparum Malaria: Prime-Boost Immunization Regimens Incorporating Human and Simian Adenoviral Vectors and Poxviral Vectors Expressing an Optimized Antigen Based on Merozoite Surface Protein 1

Although merozoite surface protein 1 (MSP-1) is a leading candidate vaccine antigen for blood-stage malaria, its efficacy in clinical trials has been limited in part by antigenic polymorphism and potentially by the inability of protein-in-adjuvant vaccines to induce strong cellular immunity. Here we report the design of novel vectored Plasmodium falciparum vaccines capable of overcoming such limitations. We optimized an antigenic insert comprising the four conserved blocks of MSP-1 fused to tandemly arranged sequences that represent both allelic forms of the dimorphic 42-kDa C-terminal region. Inserts were expressed by adenoviral and poxviral vectors and employed in heterologous prime-boost regimens. Simian adenoviral vectors were used in an effort to circumvent preexisting immunity to human adenoviruses. In preclinical studies these vaccines induced potent cellular immune responses and high-titer antibodies directed against MSP-1. The antibodies induced were found to have growth-inhibitory activity against dimorphic allelic families of P. falciparum. These vectored vaccines should allow assessment in humans of the safety and efficacy of inducing strong cellular as well as cross-strain humoral immunity to P. falciparum MSP-1.Attempts to generate protective blood-stage immunity to Plasmodium falciparum by vaccination in humans have met with limited success to date (18). The focus for most vaccine candidates has been on the induction of antibodies against merozoite antigens and merozoite surface protein 1 (MSP-1) in particular (24). Antibodies against the blood stage of P. falciparum are known to contribute to protective immunity in humans (40). However, the induction of antibodies to the 42-kDa portion of MSP-1 (MSP-1₄₂) appeared to be insufficient to provide protective immunity in humans in one study (39). Evidence from both animal models and humans (detailed below) suggests that cell-mediated immune responses to MSP-1 could be additionally required to induce protective immune responses.During the process of merozoite invasion into erythrocytes, MSP-1 undergoes two proteolytic processing steps; following the first step, only MSP-1₄₂ remains membrane bound, and a second cleavage of MSP-1₄₂ into 33-kDa (MSP-1₃₃) and 19-kDa (MSP-1₁₉) portions is then required for erythrocyte invasion (4). MSP-1₁₉ is a major target of protective antibodies, and MSP-1₃₃ is a target of both CD8⁺ T cells and CD4⁺ helper T cells (11, 21, 25). Antibodies to MSP-1₁₉ are thought to act though the direct inhibition of merozoite invasion into the red blood cell and via cytophilic antibody-mediated antibody-dependent cellular inhibition (24, 33). CD4⁺ T cells specific to MSP-1₃₃ are able to partially protect nude mice from lethal Plasmodium chabaudi and Plasmodium yoelii infections (53, 57), while transferred antibodies to MSP-1₁₉ alone are unable to protect nude mice against P. yoelii (22). CD4⁺ T cells against MSP-1₃₃ play an important role in providing help for priming MSP-1₁₉-specific B cells in vaccine-induced protection against murine malaria (11), and depletion of CD4⁺ T cells has been shown to reduce protection against P. yoelii (23).Following the discovery that MSP-1 is also expressed late in the liver stage (49), CD8⁺ T cells directed against MSP-1₃₃ have been shown to protect against P. yoelii in the preerythrocytic stage (11, 27). In addition, immune responses induced by immunization with nonlethal blood-stage parasites of P. yoelii have been shown to protect against sporozoite challenge, through CD4⁺ and CD8⁺ T cell mechanisms and at least partly through release of gamma interferon (IFN-γ) (2). This discovery that CD8⁺ T cells mediate significant antiparasitic activity against the liver stage of P. yoelii provides an argument that similar mechanisms may occur in human P. falciparum malaria. Further suggestion of the role of cellular immunity in protection against P. falciparum comes from those studies in humans in which protective immunity has been associated with significant cellular immune responses to blood-stage parasites, in the absence of strong blood-stage antibody responses (42, 47). In the first study, the secretion of IFN-γ appeared to be associated with protection against blood-stage P. falciparum malaria (42), and in the second, the presence of polyfunctional T cells, secreting tumor necrosis factor alpha (TNF-α) and interleukin-2 (IL-2) in combination with IFN-γ when stimulated by blood-stage parasites, was shown to be associated with protection against P. falciparum (47). We therefore sought to develop a vaccine targeting MSP-1, which would induce strong cellular immune responses in conjunction with high antibody titers.While inhibitory antibodies prevent MSP-1₁₉ processing and erythrocyte invasion and appear to be beneficial to the human host, blocking antibodies act to inhibit the action of these beneficial antibodies (19). Enzyme-linked immunosorbent assays (ELISAs) and immunofluorescence assays (IFAs) are routinely used to quantify the responses to vaccination but give no functional information as to levels of invasion-inhibitory antibodies. Growth-inhibitory activity (GIA) assays measure the growth of P. falciparum in the presence of immune sera in vitro, and such assays may be of greater clinical relevance when assessing vaccines targeting regions of MSP-1. The definition of epitopes for inhibitory and blocking monoclonal antibodies has enabled the design of vaccines that aim to induce inhibitory, in preference to blocking, antibodies against MSP-1₁₉ (13, 16). We investigated the inclusion of this approach in the design of MSP-1 antigens in this study.Regions of MSP-1, such as MSP-1₃₃, show extensive polymorphism, and this divides those sequences into two sets of allelic families that demonstrate extensive diversification (55). Allelic variation has been shown to reduce antibody production by single-allele vaccine constructs to heterologous strains in humans (37) and to reduce protection against heterologous strain challenge in nonhuman primates (31). Moreover human T cell responses against some peptides representing these two major allelic types of MSP-1 have been found to be mutually inhibitory in in vitro assays, a phenomenon termed altered peptide ligand antagonism (28). Within the ∼190-kDa protein sequence of MSP-1, blocks have been defined on the basis of their extensive, more limited, and minimal genetic diversity (55). For example in block 8 there is only 10% homology at the amino acid level, while in block 17, encoding MSP-1₁₉, there is 90% homology (55). Blocks 16 (MSP-1₃₃) and 17 (MSP-1₁₉) together encode MSP-1₄₂.Virus-vectored vaccines are becoming established as relatively inexpensive, effective, and safe alternative vaccine platforms to conjugate protein-in-adjuvant vaccines (12, 20). We have previously demonstrated the induction of high-titer protective antibodies against P. yoelii rodent malaria by using a recombinant replication-incompetent form of human adenovirus serotype 5 (AdHu5) and modified vaccinia virus Ankara (MVA) as vaccine vectors in a heterologous prime-boost regimen (13). A heterologous human adenoviral regimen has recently been shown to induce a strong, polyfunctional, and protective T cell response against simian immunodeficiency virus (SIV) in rhesus macaques (30), and a homologous AdHu5 regimen expressing MSP-1₄₂ has been shown to induce GIA in rabbits (5). Experience in the HIV vaccine field has emphasized the importance of avoiding preexisting antivector immunity when developing vectored vaccines (6). The immunogenicity of virus-vectored vaccines is reduced in the presence of preexisting vector-neutralizing antibodies against AdHu5 (6-8), but such antibodies do not reduce the immunogenicity of simian adenoviral vectors in humans (K. Ewer et al. unpublished data). We hypothesized that a heterologous chimpanzee adenoviral vector regimen may induce strong immune responses and be more suitable for clinical use. The simian adenoviruses C6, C7, and C9 have structural similarity and sequences close to those of human adenovirus 4 in subgroup E. These vaccine vectors have shown promise in preclinical vaccines against infections such as rabies and HIV (45, 59). An alternative simian adenovirus, AdCh63, which is closely related to C6, C7, and C9, has recently been found to be safe, immunogenic, and efficacious for human use when used to express the well-studied preerythrocytic vaccine candidate antigen ME-TRAP (Ewer et al., submitted for publication). We therefore assessed our MSP-1 vaccine constructs in simian adenovirus-vectored vaccines.To develop an MSP-1 vaccine for clinical trials, we investigated the possibility of including in vectors (i) the conserved blocks of MSP-1, (ii) both allelic forms of MSP-1₄₂, and (iii) a modified MSP-1 sequence to improve antibody fine specificity and of using a variety of DNA-based virus-vectored vaccines, including several simian adenoviral serotypes. We show in animal models that both cellular and humoral immune responses can be generated by this approach. We propose that such responses may help to overcome some of the limitations of previous generations of vaccines against MSP-1.     

Potent Malaria Transmission-Blocking Antibody Responses Elicited by Plasmodium falciparum Pfs25 Expressed in Escherichia coli after Successful Protein Refolding

Production of Pfs25, a Plasmodium falciparum transmission-blocking vaccine target antigen, in functional conformation with the potential to elicit effective immunogenicity still remains a major challenge. In the current study, codon-harmonized recombinant Pfs25 (CHrPfs25) was expressed in Escherichia coli, and purified protein after simple oxidative refolding steps retained reduction-sensitive conformational epitopes of transmission-blocking monoclonal antibodies. CHrPfs25 formulated in several adjuvants elicited strong immunogenicity in preclinical studies in mice. Antibodies elicited after immunization recognized native Pfs25 on the surface of live gametes of P. falciparum and demonstrated complete malaria transmission-blocking activity. The transmission-blocking efficacy was 100% even after a 1:128 dilution of sera from immunized mice in the complete Freund''s adjuvant and Montanide ISA51 groups and after a 1:16 dilution of sera from mice in the alum group. The blocking was mediated by antibodies; purified IgG at concentrations as low as 31.25 μg/ml exhibited 100% transmission blocking in membrane feeding assays employing two different species of mosquitoes, Anopheles gambiae and Anopheles stephensi. This study provides the first evidence for successful expression of biologically functional rPfs25 in E. coli. The extremely potent malaria transmission-blocking activity of antibodies elicited by immunization with purified protein provides strong support for further evaluation of E. coli-derived CHrPfs25 as a malaria transmission-blocking vaccine in human clinical trials.     

A Longitudinal Study of Human Antibody Responses to Plasmodium falciparum Rhoptry-Associated Protein 1 in a Region of Seasonal and Unstable Malaria Transmission

Rhoptry-associated protein 1 (RAP1) of Plasmodium falciparum is a nonpolymorphic merozoite antigen that is considered a potential candidate for a malaria vaccine against asexual blood stages. In this longitudinal study, recombinant RAP1 (rRAP1) proteins with antigenicity similar to that of P. falciparum-derived RAP1 were used to analyze antibody responses to RAP1 over a period of 4 years (1991 to 1995) of 53 individuals naturally exposed to P. falciparum malaria. In any 1 year during the study, between 23 and 39% of individuals who had malaria developed immunoglobulin G (IgG) antibodies detectable with at least one rRAP1 protein. However, the anti-RAP1 antibody responses were detected only during or shortly after clinical malarial infections. RAP1 antibody levels declined rapidly (within 1 to 2 months) following drug treatment of the infections. No anti-RAP1 antibodies were usually detected a few months after the end of malaria transmission, during the dry season, or by the start of the next malaria season. Thus, RAP1 IgG responses were very short-lived. The short duration of RAP1 antibody response may explain the apparent lack of response in a surprisingly high proportion of individuals after clinical malarial infections. For some individuals who experienced more than one malarial infection, a higher anti-RAP1 antibody response to subsequent infections than to earlier infections was observed. This suggested secondary responses to RAP1 and thus the development of immunological memory for RAP1.     

Induction of Protective Immune Responses by Immunization with Linear Multiepitope Peptides Based on Conserved Sequences from Plasmodium falciparum Antigens

A cysteine-containing peptide motif, EWSPCSVTCG, is found highly conserved in the circumsporozoite protein (CSP) and the thrombospondin-related anonymous protein (TRAP) of all the Plasmodium species analyzed so far and has been shown to be crucially involved in the sporozoite invasion of hepatocytes. We have recently shown that peptide sequences containing this motif, and also the antibodies raised against the motif, inhibit the merozoite invasion of erythrocytes. However, during natural infection, and upon immunization with recombinant CSP, this motif represents a cryptic epitope. Here we present the results of immunization studies with two linear multiepitopic constructs, a 60-residue (P60) and a 32-residue (P32) peptide, containing the conserved motif sequence. Both the peptides per se generated high levels of specific antibodies in BALB/c mice. P32 was found to be genetically restricted to H-2^d and H-2^b haplotypes of mice, whereas P60 was found to be immunogenic in five different strains of mice. The antibody response was predominantly targeted to the otherwise cryptic, conserved motif sequence in P60. Anti-P60 antibodies specifically stained the asexual blood stages of Plasmodium falciparum and Plasmodium yoelii in an immunofluorescence assay, recognized a 60- to 65-kDa parasite protein in an immunoblot assay, and blocked P. falciparum merozoite invasion of erythrocytes in a dose-dependent manner. Immunization with P60 also induced significant levels of the cytokines interleukin-2 (IL-2), IL-4, and gamma interferon in BALB/c mice. Moreover, >60% of mice immunized with P60 survived a heterologous challenge infection with a lethal strain of P. yoelii. These results indicate that appropriate medium-sized synthetic peptides might prove useful in generating specific immune responses to an otherwise cryptic but critical and putatively protective epitope in an antigen and could form part of a multicomponent malaria vaccine.     

Antigenic Characterization of an Intrinsically Unstructured Protein,Plasmodium falciparum Merozoite Surface Protein 2

Merozoite surface protein 2 (MSP2) is an abundant glycosylphosphatidylinositol (GPI)-anchored protein of Plasmodium falciparum, which is a potential component of a malaria vaccine. As all forms of MSP2 can be categorized into two allelic families, a vaccine containing two representative forms of MSP2 may overcome the problem of diversity in this highly polymorphic protein. Monomeric recombinant MSP2 is an intrinsically unstructured protein, but its conformational properties on the merozoite surface are unknown. This question is addressed here by analyzing the 3D7 and FC27 forms of recombinant and parasite MSP2 using a panel of monoclonal antibodies raised against recombinant MSP2. The epitopes of all antibodies, mapped using both a peptide array and by nuclear magnetic resonance (NMR) spectroscopy on full-length recombinant MSP2, were shown to be linear. The antibodies revealed antigenic differences, which indicate that the conserved N- and C-terminal regions, but not the central variable region, are less accessible in the parasite antigen. This appears to be an intrinsic property of parasite MSP2 and is not dependent on interactions with other merozoite surface proteins as the loss of some conserved-region epitopes seen using the immunofluorescence assay (IFA) on parasite smears was also seen on Western blot analyses of parasite lysates. Further studies of the structural basis of these antigenic differences are required in order to optimize recombinant MSP2 constructs being evaluated as potential vaccine components.     

Resistance to Coccidioides immitis in Mice after Immunization with Recombinant Protein or a DNA Vaccine of a Proline-Rich Antigen

Two inbred strains of mice (BALB/c and C57BL/6) were vaccinated with either recombinant expression protein of a Coccidioides immitis spherule-derived proline-rich antigen (rPRA) in monophosphoryl lipid A-oil emulsion adjuvant or a DNA vaccine based on the same antigen. Four weeks after vaccination, mice were infected intraperitoneally with arthroconidia. By 2 weeks, groups of mice receiving saline or plasmids with no PRA insert exhibited significant weight loss, and quantitative CFUs in the lungs ranged from 5.9 to 6.4 log10. In contrast, groups of mice immunized with either rPRA or DNA vaccine had significantly smaller pulmonary fungal burdens, ranging from 3.0 to 4.5 log10 fewer CFUs. In vitro immunologic markers of lymphocyte proliferation and gamma interferon (IFN-gamma) release after splenocytes were stimulated with rPRA correlated with protection. Also, plasma concentrations of rPRA-specific total immunoglobulin G (IgG), IgG1, and IgG2a showed increases in vaccinated mice. These studies expand earlier work by demonstrating protection in mice which differ in H-2 background, by using an adjuvant that is potentially applicable to human use, and by achieving comparable protections with a DNA-based vaccine. Our in vitro results substantiate a Th1 response as evidenced by IFN-gamma release and increased IgG2a. However, IgG1 was also stimulated, suggesting some Th2 response as well. PRA is a promising vaccine candidate for prevention of coccidioidomycosis and warrants further investigation.     

Partial Protection against Plasmodium vivax Blood-Stage Infection in Saimiri Monkeys by Immunization with a Recombinant C-Terminal Fragment of Merozoite Surface Protein 1 in Block Copolymer Adjuvant

Merozoite surface protein 1 is a candidate for blood-stage vaccines against malaria parasites. We report here an immunization study of Saimiri monkeys with a yeast-expressed recombinant protein containing the C terminus of Plasmodium vivax merozoite surface protein 1 and two T-helper epitopes of tetanus toxin (yP₂P₃₀Pv200₁₉), formulated in aluminum hydroxide (alum) and block copolymer P1005. Monkeys immunized three times with yP₂P₃₀Pv200₁₉ in block copolymer P1005 had significantly higher prechallenge titers of immunoglobulin G (IgG) antibodies against the immunogen and asexual blood-stage parasites than those immunized with yP₂P₃₀Pv200₁₉ in alum, antigen alone, or phosphate-buffered saline (PBS) (P < 0.05). Their peripheral blood mononuclear cell proliferative responses to immunogen stimulation 4 weeks after the second immunization were also significantly higher than those from the PBS control group (P < 0.05). Upon challenge with 100,000 asexual blood-stage parasites 5 weeks after the last immunization, monkeys immunized with yP₂P₃₀Pv200₁₉ in block copolymer P1005 had prepatent periods longer than those for the control alone group (P > 0.05). Three of the five animals in this group also had low parasitemia (peak parasitemia, ≤20 parasites/μl of blood). Partially protected monkeys had significantly higher levels of prechallenge antibodies against the immunogen than those unprotected (P < 0.05). There was also a positive correlation between the prepatent period and titers of IgG antibodies against the immunogen and asexual blood-stage parasites and a negative correlation between accumulated parasitemia and titers of IgG antibodies against the immunogen (P < 0.05). These results indicate that when combined with block copolymer and potent T-helper epitopes, the yeast-expressed P₂P₃₀Pv200₁₉ recombinant protein may offer some protection against malaria.     

Immunization of Mice with Live-Attenuated Late Liver Stage-Arresting Plasmodium yoelii Parasites Generates Protective Antibody Responses to Preerythrocytic Stages of Malaria

Understanding protective immunity to malaria is essential for the design of an effective vaccine to prevent the large number of infections and deaths caused by this parasitic disease. To date, whole-parasite immunization with attenuated parasites is the most effective method to confer sterile protection against malaria infection in clinical trials. Mouse model studies have highlighted the essential role that CD8⁺ T cells play in protection against preerythrocytic stages of malaria; however, there is mounting evidence that antibodies are also important in these stages. Here, we show that experimental immunization of mice with Plasmodium yoelii fabb/f⁻ (Pyfabb/f⁻), a genetically attenuated rodent malaria parasite that arrests late in the liver stage, induced functional antibodies that inhibited hepatocyte invasion in vitro and reduced liver-stage burden in vivo. These antibodies were sufficient to induce sterile protection from challenge by P. yoelii sporozoites in the absence of T cells in 50% of mice when sporozoites were administered by mosquito bite but not when they were administered by intravenous injection. Moreover, among mice challenged by mosquito bite, a higher proportion of BALB/c mice than C57BL/6 mice developed sterile protection (62.5% and 37.5%, respectively). Analysis of the antibody isotypes induced by immunization with Pyfabb/f⁻ showed that, overall, BALB/c mice developed an IgG1-biased response, whereas C57BL/6 mice developed an IgG2b/c-biased response. Our data demonstrate for the first time that antibodies induced by experimental immunization of mice with a genetically attenuated rodent parasite play a protective role during the preerythrocytic stages of malaria. Furthermore, they highlight the importance of considering both the route of challenge and the genetic background of the mouse strains used when interpreting vaccine efficacy studies in animal models of malaria infection.     

Immunization of Saimiri sciureus Monkeys with a Recombinant Hybrid Protein Derived from the Plasmodium falciparum Antigen Glutamate-Rich Protein and Merozoite Surface Protein 3 Can Induce Partial Protection with Freund and Montanide ISA720 Adjuvants

The immunogenicity and efficacy of a hybrid recombinant protein derived from the N-terminal end of the glutamate-rich protein (GLURP) and the C-terminal portion of the merozoite surface protein 3 (MSP3) of Plasmodium falciparum was evaluated in Saimiri sciureus monkeys. The GLURP/MSP3 hybrid protein, expressed in Lactococcus lactis, was administered in association with alum, Montanide ISA720, or complete or incomplete Freund adjuvant (CFA/IFA) in groups of five animals each. The three formulations were shown to be immunogenic, but the one with alum was shown to be weak compared to the other two, particularly CFA/IFA, which provided very high antibody titers (enzyme-linked immunosorbent assay titers of >3,000,000 and immunofluorescence antibody test titers of 6,400). After a challenge infection with P. falciparum FUP strain, all five monkeys from the GLURP/MSP3-alum group showed a rapid increase in parasitemia, reaching 10% and were treated early. The two monkeys with the highest antibody titers in group GLURP/MSP3-Montanide ISA720 had a delay in the course of parasitemia and were treated late due to a low hematocrit. In the GLURP/MSP3-CFA/IFA group, parasitemia remained below this threshold in four of the five animals and, after it reached a peak, parasitemia started to decrease and monkeys were treated late. When all animals were grouped according to the outcome, a statistically significant association between high antibody titers and partial protection was observed. The challenge infection boosted the antibody titers, and the importance of this event for vaccine efficacy in areas where this parasite is endemic is discussed. In conclusion, these data suggest that GLURP and MSP3 can induce protection against malaria infection if antibodies are induced at properly high titers.     

Simultaneous Induction of Multiple Antigen-Specific Cytotoxic T Lymphocytes in Nonhuman Primates by Immunization with a Mixture of Four Plasmodium falciparum DNA Plasmids

CD8⁺ T cells have been implicated as critical effector cells in protective immunity against malaria parasites developing within hepatocytes. A vaccine that protects against malaria by inducing CD8⁺ T cells will probably have to include multiple epitopes on the same protein or different proteins, because of parasite polymorphism and genetic restriction of T-cell responses. To determine if CD8⁺ T-cell responses against multiple P. falciparum proteins can be induced in primates by immunization with plasmid DNA, rhesus monkeys were immunized intramuscularly with a mixture of DNA plasmids encoding four P. falciparum proteins or with individual plasmids. All six monkeys immunized with PfCSP DNA, seven of nine immunized with PfSSP2 DNA, and five of six immunized with PfExp-1 or PfLSA-1 DNA had detectable antigen-specific cytotoxic T lymphocytes (CTL) after in vitro restimulation of peripheral blood mononuclear cells. CTL activity was genetically restricted and dependent on CD8⁺ T cells. By providing the first evidence for primates that immunization with a mixture of DNA plasmids induces CD8⁺ T-cell responses against all the components of the mixture, these studies provide the foundation for multigene immunization of humans.     

Naturally Acquired Antibody Responses to the C-Terminal Region of Merozoite Surface Protein 1 of Plasmodium vivax in Korea

We expressed a protein in Saccharomyces cerevisiae in order to evaluate the humoral immune responses to the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax. This protein (Pv200₁₈) had a molecular mass of 18 kDa and was reactive with the sera of individuals with patent vivax malaria on immunoblotting analysis. The levels of immunoglobulin M (IgM) and IgG antibodies against Pv200₁₈ were measured in 421 patients with vivax malaria (patient group), 528 healthy individuals from areas of nonendemicity (control group 1), and 470 healthy individuals from areas of endemicity (control group 2), using the indirect enzyme-linked immunosorbent assay (ELISA) method. To study the longevity of the antibodies, 20 subjects from the patient group were also tested for the antibody levels once a month for 1 year. When the cutoff values for seropositivity were determined as the mean + 3 × standard deviation of the antibody levels in control group 1, both IgG and IgM antibody levels were negative in 98.5% (465 of 472) of control group 2. The IgG and IgM antibodies were positive in 88.1% (371 of 421) and 94.5% (398 of 421) of the patient group, respectively. The IgM antibody became negative 2 to 4 months after the onset of symptoms, whereas the IgG antibody usually remained positive for more than 5 months. In conclusion, indirect ELISA using Pv200₁₈ expressed in S. cerevisiae may be a useful diagnostic method for vivax malaria.     

Relationship of Binding of Immunoglobulin G to Plasmodium falciparum-Infected Erythrocytes with Parasite Endemicity and Antibody Responses to Conserved Antigen in Immune Individuals

To investigate the potential for use of a well-established strain of Plasmodium falciparum as a reference strain for infected red blood cell (IRBC) surface reactivity, we monitored the binding of specific immunoglobulin G (IgG) from immune individuals to the reference Knob-positive FCR3 strain by flow cytometry. To permit interassay comparison for 162 plasma samples drawn after the rainy season, a labeling index (LI) was defined as the percentage of labeled parasites multiplied by the mean peak intensity. An LI ratio (LIR) was then calculated as the LI of the sample divided by the LI of the control. LIRs were calculated for individuals living in Dielmo and Ndiop, two Senegalese villages where P. falciparum is transmitted holoendemically and mesoendemically, respectively. The incidence (persons with an LIR of >3) observed in Dielmo was lower than that observed in Ndiop. Significantly higher LIRs were observed (i) for samples from Ndiop than for samples from Dielmo (P < 0.01) and (ii) in Ndiop, in subjects with hemoglobin AS (HbAS) than in those with hemoglobin AA (P = 0.03). No correlation with the cumulative age-associated immune status of the villagers was evidenced, contrary to antibody (Ab) responses against conserved IRBC-associated antigen (Ag) measured by enzyme-linked immunosorbent assay. These results are consistent with the notions that protection in HbAS individuals may relate to an increased IgG response to IRBC membrane Ags and that cell surface reactivity parallels IgG responses even though it is in itself a distinct indicator of the anti-P. falciparum Ab response. Measures of IgG binding to live IRBC are thus relevant for the functional screening of conserved IRBC-associated Ags that contribute to parasite destruction in vivo, as these Ags might be included in a multitarget vaccine.     

Protective Immunization with a Novel Membrane Protein of Plasmodium yoelii-Infected Erythrocytes

Immunization with a particulate fraction of blood-stage antigens was shown previously to protect mice against Plasmodium yoelii malaria. To identify antigens inducing the protective response, sera from immunized mice were used to screen a P. yoelii cDNA expression library. Sequence analysis of one 2.6-kb cDNA clone indicated that the identified gene, pypag-1, encoded a novel plasmodial antigen. Two nonoverlapping regions of pypag-1 were expressed in Escherichia coli. The first recombinant antigen, pAg-1N, contained the N-terminal 337 residues, which included a putative transmembrane domain and a region relatively rich in tryptophan residues. The second recombinant antigen, pAg-1C, contained the remaining C-terminal 211 residues, which included 31 copies of a 5-amino-acid degenerative repeat. Immunoblot studies using rabbit antiserum raised against recombinant pAg-1N showed that the native pypAg-1 protein migrated at approximately 98 kDa, considerably slower than its predicted molecular mass of 66 kDa. Immunofluorescence studies localized the expression of the native pypAg-1 protein both to the cytoplasm and at the surface of P. yoelii-infected erythrocytes. Immunization with either pAg-1N or pAg-1C induced a four- to sevenfold reduction in P. yoelii blood-stage parasitemia. As such, pypAg-1 appears to contain at least two distinct protective epitopes. To our knowledge, this is the first characterization of a protective antigen of P. yoelii that is associated with the erythrocyte membrane.     

Characterization of a 225 kilodalton rhoptry protein of Plasmodium falciparum

A monoclonal antibody (24C6 4F12) raised against Plasmodium falciparum culture supernatant antigens gave a multiple dot picture on schizonts when assayed by immunofluorescence on P. falciparum erythrocytic stages. The corresponding antigen was localized in the peduncle of rhoptries by immunoelectronmicroscopy. On Western blots of P. falciparum schizonts, a major antigen of 225 kDa and a minor one of 240 kDa were recognized by this McAb. Pulse chase analysis of [35S]methionine biosynthetic labeling of P. falciparum culture demonstrated that the 240 kDa molecule was the precursor of the 225 kDa and that its processing occurred between 0 and 4 h after synthesis. Biosynthesis of the 240-225 kDa antigen occurred only during schizogony.