Plasmodium yoelii: 17-kDa hepatic and erythrocytic stage protein is the target of an inhibitory monoclonal antibody.

Infected hepatocytes are important targets for malaria vaccines. To identify Plasmodium yoelii proteins expressed in infected hepatocytes, we immunized BALB/c ByJ mice with P. yoelii liver stage schizonts and produced a panel of monoclonal antibodies (Mabs). An IgG1 Mab, navy yoelii liver stage 3 (NYLS3), had the strongest reactivity against liver stage parasites and was selected for further characterization. The Mab does not recognize P. yoelii sporozoites, but recognizes liver stage parasites within 6 hr of invasion of mouse hepatocytes and throughout the hepatic and asexual erythrocytic stages of the parasite life cycle as determined by the immunofluorescent antibody test. This Mab is species-specific, and it reacts with liver stages of P. yoelii but does not react with liver stages of other Plasmodium species. The protein recognized by this Mab is present on the parasitophorous vacuole membrane of infected hepatocytes and erythrocytes as demonstrated by immunoelectron microscopy and has a relative molecular weight of 17 kDa as demonstrated by immunoblot of an extract of infected erythrocytes. It is therefore designated P. yoelii hepatic and erythrocytic stage protein, 17 kDa or PyHEP17. When added to primary cultures of mouse hepatocytes 24 hr after inoculation with P. yoelii sporozoites, when all sporozoites have invaded hepatocytes, NYLS3 eliminates up to 98% of liver-stage parasites. Intravenous injection of NYLS3 into mice delays the onset and reduces the density of blood-stage parasitemia after sporozoite or blood-stage challenge. The P. falciparum and P. vivax homologs of PyHEP17 may therefore be important targets for vaccines designed to attack the hepatic and erythrocytic stages of the parasite life cycle.

Effects of transmission-blocking immunity on Plasmodium vivax infections in Anopheles albimanus populations.

Two colonized populations of Anopheles albimanus isolated from the Suchiate region, Chiapas State, Mexico, were compared for their susceptibility to coindigenous Plasmodium vivax. Groups of mosquitoes were fed in vitro with either autologous donor blood or the same blood cells substituted with serum negative for anti-gametocyte antibody. Significant differences in susceptibility between the 2 colonies were encountered if the autologous blood from a patient was fed to mosquitoes: mean infection rates of AnA2-positive groups was double that in AnA1 mosquitoes. Consistent for both colonies, only 23.6% of samples positive from malaria-negative serum-substituted blood were infected with an autologous blood feed. Vector competence in these mosquito populations was partially linked to the human populations's immune response to the parasite.

Monoclonal antibodies of three different immunoglobulin G isotypes produced by immunization with a synthetic peptide or native protein protect mice against challenge with Plasmodium yoelii sporozoites.

Passive transfer of monoclonal antibodies (MAbs) against malaria circumsporozoite (CS) proteins protects animals against malaria. Active immunization with synthetic or recombinant peptides induces a level of polyclonal antibodies to sporozoites comparable to those found after passive immunization but does not provide comparable protection. In the Plasmodium yoelii system, synthetic or recombinant peptide-induced antibodies have never been shown to protect. The current studies were designed to determine whether immunogen structure (native protein versus synthetic peptide) or immunoglobulin G (IgG) subclass of antibodies was responsible for the absolute differences between protective, passively transferred MAbs and nonprotective, actively induced polyclonal antibodies. In this study we produced two MAbs, QGP-S1 (IgG1) and QGP-S2 (IgG2b), by immunization with a synthetic peptide based on the P. yoelii CS major repeat, (QGPGAP)4, conjugated to keyhole limpet hemocyanin. These MAbs were compared tp NYS1 (IgG3), an anti-CS protein MAb previously produced by immunization with irradiated P. yoelii sporozoites, which recognizes (QGP GAP)2. QGP-S1 and QGP-S2 passively transferred protection. However, when compared with NYS1, there was a hierarchy of protection, NYS1 > QGP-S1 > QGP-S2. There was no correlation between antibody level at challenge as determined by immunofluorescent antibody test against sporozoites or enzyme-linked immunosorbent assay against (QGPGAP)2 or apparent antibody avidity for (QGPGAP)2 by sodium thiocyanate elution assay. The data demonstrate that a synthetic peptide can induce protective antibodies and that a specific antibody subclass is not required for protection. Work to determine whether antibody affinity or fine specificity can explain the hierarchy of protection among the MAbs is under way.

Recombinant pseudorabies virus carrying a plasmodium gene: herpesvirus as a new live viral vector for inducing T- and B-cell immunity.

In Balb/c mice, the sterile protective immunity induced by immunization with radiation-attenuated Plasmodium yoelii sporozoites is eliminated by in vivo depletion of CD8+ T lymphocytes, suggesting that cytotoxic T lymphocytes (CTL) against malaria antigens expressed on infected hepatocytes are required for mediating this protective immunity. To produce a vaccine that would induce CTL against the P. yoelii circumsporozoite protein (CS), we constructed an attenuated pseudorabies virus (PRV) containing a gene encoding this protein. Balb/c mice that received three doses of 10(7) plaque-forming units (p.f.u.) of this vaccine intravenously at 3 week intervals developed high levels of antibodies to sporozoites (indirect fluorescent antibody titre = 4096) and CTL against a 16 amino acid epitope (SYVPSAEQILEFVKQI, amino acids 281-296) from the P. yoelii CS protein designated PYCTL1. The cytotoxic activity of the CTL was antigen-specific, MHC-restricted, and dependent on CD8+ T cells. Furthermore, these CTL eliminated P. yoelii-infected hepatocytes from in vitro culture, indicating that they recognize this peptide on the surface of infected hepatocytes. However, all nine mice that were challenged with 200 sporozoites developed a blood-stage malaria infection. We attribute this lack of protection to the great difficulty of inducing sterile immunity against this highly infectious parasite P. yoelii. We conclude that recombinant pseudorabies virus (PRV) worked successfully as a live vaccine vector to induce both antibodies and CTL, albeit non-protective in vivo, and the herpesviruses should be considered as subunit vaccines where T- and B-cell immunity is required.

Antigenic analysis of Plasmodium yoelii liver stages by fluorescence antibody assays.

Little is known about the immune response against liver stage antigens which were first described for Plasmodium falciparum. In order to provide a basis for experimental studies, we analysed antigenically the liver stages of Plasmodium yoelii using sera of restricted specificity. Several distinct fluorescence patterns could be described in maturing liver forms. One pattern was identified as corresponding to antigens specific to the liver phase which are also species specific. Another pattern corresponds to sporozoite surface antigens which were predominant in liver trophozoites. Trophozoite-like liver forms were detected at least 7 days after the injection of irradiated sporozoites suggesting that parasites may persist and contribute to the immunity induced by this procedure.

Protection against malaria by vaccination with sporozoite surface protein 2 plus CS protein.

The circumsporozoite (CS) protein has been the target for development of malaria sporozoite vaccines for a decade. However, immunization with subunit vaccines based on the CS protein has never given the complete protection found after immunization with irradiated sporozoites. BALB/c mice immunized with irradiated Plasmodium yoelii sporozoites produced antibodies and cytotoxic T cells against a 140-kilodalton protein, sporozoite surface protein 2 (SSP2). Mice immunized with P815 cells that had been transfected with either SSP2 or CS genes were partially protected, and those immunized with a mixture of SSP2 and CS transfectants were completely protected against malaria. These studies emphasize the importance of vaccine delivery systems in achieving protection and define a multi-antigen sporozoite vaccine.

Inability of malaria vaccine to induce antibodies to a protective epitope within its sequence.

Saimiri monkeys immunized with a recombinant protein containing 20 copies of the nine amino acid repeat of the Plasmodium vivax circumsporozoite (CS) protein developed high concentrations of antibodies to the repeat sequence and to sporozoites, but were not protected against challenge. After intravenous injection of an immunoglobulin G3 monoclonal antibody (NVS3) against irradiated P. vivax sporozoites, four of six monkeys were protected against sporozoite-induced malaria, and the remaining two animals took significantly longer to become parasitemic. Epitope mapping demonstrated that NVS3 recognizes only four (AGDR) of the nine amino acids within the repeat region of the P. vivax CS protein. The monkeys immunized with (DRAADGQPAG)20 did not produce antibodies to the protective epitope AGDR. Thus, determination of the fine specificity of protective immune responses may be critical to the construction of successful subunit vaccines.

Monoclonal, but not polyclonal, antibodies protect against Plasmodium yoelii sporozoites.

One of the primary strategies for malaria vaccine development has been to design subunit vaccines that induce protective levels of antibodies against the circumsporozoite (CS) protein of malaria sporozoites. In the Plasmodium yoelii mouse model system such vaccines have been uniformly unsuccessful in protecting against sporozoite-induced malaria. To demonstrate that antibodies to P. yoelii CS protein could provide protection we established a passive transfer model. Passive transfer of Navy yoelii sporozoite 1 (NYS1), an IgG3 mAb against the P. yoelii CS protein, protected 100% of mice against challenge with 5000 P. yoelii sporozoites. Binding of NYS1 to sporozoites was inhibited by incubation with (QGPGAP)2, a synthetic peptide derived from the repeat region of the P. yoelii CS protein, indicating that the epitope on sporozoites recognized by this mAb was included within this peptide. The levels of antibodies to (QGPGAP)2 by ELISA, and to sporozoites by indirect fluorescent antibody test and CS precipitation reaction were similar in sera from mice that received NYS1 in passive transfer and were protected against challenge with 5000 sporozoites, and from mice that had been immunized with subunit vaccines containing (QGPGAP)2 but were not protected against challenge with 40-200 sporozoites. To determine if antibody avidity, not absolute concentration could explain the striking differences in protection, we established a thiocyanate elution assay. The results suggest that NYS1, the protective mAb, has a lower avidity for (QGPGAP)2 and for sporozoites than do the vaccine-induced antibodies. Although the results of the conventional antibody assays did not correlate with protection, sera from the protected animals inhibited sporozoite development in mouse hepatocyte cultures significantly more than did the sera from the unprotected, subunit vaccine-immunized animals, correlating with protection. The data clearly demonstrate that antibodies to the CS protein can protect against intense sporozoite infection. Improved understanding of the differences between protective mAb and nonprotective polyclonal antibodies will be important in the further development of malaria vaccines.

Evaluation of monoclonal antibodies against Plasmodium vivax sporozoites for ELISA development.

Nine monoclonal antibodies (MAbs) developed against Plasmodium vivax (Grassi & Feletti) salivary gland sporozoites were evaluated for use in an enzyme-linked immunosorbent assay (ELISA), using sporozoites developed in Anopheles dirus Peyton & Harrison An. gambiae Giles and An.maculatus Theobald. Four of the antibodies were unsuitable due to the low sensitivity of the resulting assays or the requirement for high concentrations of capture antibody. An additional two MAbs were rejected because they resulted in assays with high background absorbance, attributed to self-binding. Of the three remaining MAbs, the use of Navy vivax sporozoite (NVS) 3 resulted in an ELISA with the highest sensitivity and the lowest concentration requirement for capture antibody. Assay sensitivity varied with sporozoite strain indicating possible quantitative epitope heterogeneity. None of the MAbs cross-reacted with the heterologous sporozoites tested by immunofluorescence antibody assay (IFA). The IFA activity was not an indicator of ELISA sensitivity. The use of MAb NVS 3 in a standardized ELISA method resulted in an assay 10 times more sensitive than reported previously for P. vivax sporozoites, with a detection limit of fewer than 100 sporozoites per mosquito.

Plasmodium falciparum-infected Anopheles stephensi inconsistently transmit malaria to humans.

Malaria was transmitted to only 5 of 10 volunteers bitten by 1-2 Anopheles stephensi carrying sporozoites of the 3D7 clone of the NF54 strain of Plasmodium falciparum in their salivary glands. Parasites were detectable by culture in blood taken 7-10 days following exposure and by thick blood film 14-16.5 days after exposure. Infectivity did not correlate with the numbers of sporozoites in the salivary glands.

Evaluation of vaccines designed to induce protective cellular immunity against the Plasmodium yoelii circumsporozoite protein: vaccinia, pseudorabies, and Salmonella transformed with circumsporozoite gene.

In an attempt to induce a protective cytotoxic T-cell mediated immunity against sporozoites of Plasmodium yoelii, the gene encoding the P. yoelii circumsporozoite (CS) protein was engineered into three live vectors: vaccinia, attenuated pseudorabies, and attenuated Salmonella typhimurium. Balb/c mice were immunized with 1-4 doses of 10(8) pfu of the vaccinia construct (IP), 3 doses of 10(5), 10(6) or 10(7) pfu of pseudorabies construct (IV), and 3 doses of 10(9) salmonella transformants (orally). In the case of vaccinia and pseudorabies constructs, an excellent immune response was obtained as measured by antibodies to sporozoites. No protection or delay in prepatent period was seen in any of the experimental animals when challenged with 200 (vaccinia, pseudorabies) or 100 (salmonella) sporozoites, although mice immunized with irradiation-attenuated sporozoites were consistently protected against challenge with greater than 10(4) sporozoites. Since other vaccinia, pseudorabies, and salmonella CS constructs have been shown to induce cytotoxic T lymphocytes (CTL) against the CS protein, it is likely that CTL against the CS protein were induced during these studies. It is currently unclear if the vaccines did not induce the appropriate CTL or inadequate numbers of CTL, or if CTL against the P. yoelii CS protein are inadequate to protect against sporozoite challenge.

A malaria sporozoite surface antigen distinct from the circumsporozoite protein.

Monoclonal antibody NYS4 recognizes a single 140 kDa antigen on the surface of Plasmodium yoelii sporozoites, an antigen which is distinct from the extensively characterized circumsporozoite (CS) protein. To more thoroughly characterize this additional surface component, a genomic expression library was screened with NYS4 and an immunoreactive clone (M4) was obtained which expressed part of the antigen gene. The deduced amino acid sequence of the M4 peptide included two unique repetitive sequences of amino acids and a conserved sequence motif which is found in several proteins including the CS protein (region II). The cloned DNA hybridized to a PCR (polymerase chain reaction) amplified sporozoite mRNA demonstrating the sporozoite-stage expression of this gene. A synthetic peptide of one of the repeats, (Asn-Pro-Asn-Glu-Pro-Ser), was recognized by NYS4 and mice immunized with (Asn-Pro-Asn-Glu-Pro-Ser)3 conjugated to KLH (keyhole limpet haemocyanin) produced high levels of antibodies that reacted with the surface of sporozoites and specifically to the 140 kDa antigen. Thus, at least two different proteins are on the surface of the P. yoelii sporozoite indicating that the immunoreactive exterior of the infective stage of malaria parasites is more antigenically complex than previously thought.

Active and passive immunization against Plasmodium yoelii sporozoites.

Three subunit vaccines based on the major repeat, (QGPGAP)n, and flanking regions of the Plasmodium yoelii circumsporozoite protein were designed, produced, and tested. All were immunogenic, but none gave consistent protection against a 40-200 sporozoite challenge. To demonstrate that antibodies to P. yoelii CS protein could provide protection we established a passive transfer model. Passive transfer of NYS1, an IgG3 MAb against the P. yoelii CS protein, protected 100% of mice against challenge with 5000 P. yoelii sporozoites. Binding of NYS1 to sporozoites was inhibited by incubation with (QGPGAP)2, indicating that the epitope on sporozoites recognized by this MAb was included within this peptide. The levels of antibodies to (QGPGAP)2 by ELISA, and to sporozoites by IFAT and CS precipitation reaction were similar in sera from mice that received NYS1 in passive transfer and were protected against challenge with 5000 sporozoites, and from mice that had been immunized with subunit vaccines containing QGPGAP but were not protected against challenge with 40-200 sporozoites. To determine if antibody avidity, not the absolute concentration, could explain the striking differences in protection, we established a thiocyanate elution assay. The results suggest that NYS1, the protective MAb, has a lower avidity for (QGPGAP)2 and for sporozoites than do the vaccine-induced antibodies. The data clearly demonstrate that antibodies to the CS protein can protect against intense sporozoite infection. Improved understanding of the differences between protective MAbs and non-protective polyclonal antibodies will be important in the further development of malaria vaccines.

Freeze-fracture studies on the sporoblast and sporozoite development in the early oocyst.

Freeze-fracturing has been used to study the formation of the triple layer pellicular complex of budding sporozoites of Plasmodium falciparum in the early oocyst. Sporozoites are formed from sporoblasts within the oocyst. The outer membrane of the sporozoites is derived from the single plasma membrane of the sporoblast while the inner 2 membranes are formed anew at the base of the differentiating sporozoites. A dense collar of intramembranous particles located on the P face of the outer membrane encircles the base of each budding sporozoite. The fact that this collar of intramembranous particles is located in the same region where the inner membranes of the sporozoites first make their appearance strongly suggests that the 2 are related, and that the collar may be related to either membrane synthesis or to membrane organization and assembly.

Dual action of anti-sporozoite antibodies in vitro.

With the use of a double staining technique that permits localization of the sporozoite during the process of entering a host cell, we studied the biologic effects of three mAb directed against determinants contained in the circumsporozoite of Plasmodium yoelii. These mAb, which included one IgM and two IgG3, were studied in primary cultures of rodent hepatocytes inoculated with sporozoites of P. yoelii. These results confirm previous reports of the extended action of antibodies on Plasmodium falciparum after entering hepatocytes by producing a strong intrahepatocyte inhibitory effect in addition to the inhibitory effect on sporozoite entry. As with P. falciparum the intracellular effects on P. yoelii liver stages are only observed when the antibodies are present at the time the sporozoite enters the cell. While carrying out experiments on this phenomenon, it was discovered that, at lowered antibody concentrations, an increase in number of maturing liver schizonts occurs, with the increase or enhancement of infection reaching up to 150% of that of controls. It was also observed that there was an inverse relationship between the antibody concentration that was inhibitory and that which enhanced parasite infectivity.

Cloning of Plasmodium yoelii genes expressing three different sporozoite-specific antigens.

Genomic DNA isolated from Plasmodium yoelii (strain #17XNL) was prepared by partial digestion and cloned in Escherichia coli TB-1 with pUC18 plasmid. Antigen-producing recombinants were detected by a battery of monoclonal antibodies against antigens of the sporozoite stages. Four clones producing stage-specific sporozoite antigens were identified. One produced P. yoelii circumsporozoite protein, and three produced other P. yoelii sporozoite antigens.