Immune responses of mice with different genetic backgrounds to improved multiepitope, multitarget malaria vaccine candidate antigen FALVAC-1A.

FALVAC-1A is a second-generation multitarget, multiepitope synthetic candidate vaccine against Plasmodium falciparum, incorporating elements designed to yield a stable and immunogenic molecule. Characteristics of the immunogenicity of FALVAC-1A were evaluated in congenic (H-2(b), H-2(k), and H-2(d)) and outbred strains of mice. The influences of four adjuvants (aluminum phosphate, QS-21, Montanide ISA-720, and copolymer CRL-1005) on different aspects of the immune response were also assessed. FALVAC-1A generated strong antibody responses in all mouse strains. The highest mean enzyme-linked immunosorbent assay (ELISA) antibody concentrations against FALVAC-1A were observed in the outbred ICR mice, followed by B10.BR, B10.D2, and C57BL/6 mice, though this order varied for the different adjuvants, with no statistical differences between mouse strains. In all mouse strains, the highest anti-FALVAC-1A antibody titers in ELISAs were induced by FALVAC-1A in copolymer and ISA-720 formulations, followed by QS-21 and AlPO4. These antibodies were of all four subclasses, though immunoglobulin G1 (IgG1) predominated, with the exception of FALVAC-1A with the QS-21 adjuvant, which induced predominantly IgG2c responses. Both sporozoites and blood stages of P. falciparum were recognized by anti-FALVAC-1A sera in the immunofluorescence assay. In addition to antibody, cellular immune responses were detected; these responses were studied by examining spleen cells producing gamma interferon and interleukin-4 in enzyme-linked immunospot assays. In summary, FALVAC-1A was found to be highly immunogenic and elicited functionally relevant antibodies that can recognize sporozoites and blood-stage parasites in diverse genetic backgrounds.

Predicted and observed alleles of Plasmodium falciparum merozoite surface protein-1 (MSP-1), a potential malaria vaccine antigen.

The 19-kDa antigenic domain of Plasmodium falciparum merozoite surface protein (MSP)-1 is a potential malaria vaccine candidate. Based on the amino acid substitution, four known alleles, E-TSR (PNG-MAD20 type), E-KNG (Uganda-PA type), Q-KNG (Wellcome type), and Q-TSR (Indo type) of this domain have been identified. Using single or double crossover recombinational events, we predicted the existence of additional alleles of this antigen. The presence of the predicted alleles was determined in parasite isolates from western Kenya, by undertaking a cross-sectional and a longitudinal study. Of the ten predicted alleles, we have revealed the presence of three new alleles: E-KSG-L (Kenya-1 type); E-KSR-L (Kenya-2 type); and E-KNG-F (Kenya-3 type). The results of this study suggest that it may be possible to predict the complexity of the genetic makeup of natural parasite populations.

Plasmodium vivax infections in chimpanzees for sporozoite challenge studies in monkeys.

The development and testing of vaccines directed against Plasmodium vivax has relied on Saimiri and Aotus monkeys as the animal test system and on chimpanzees to provide infective gametocytes to produce sporozoites for monkey challenge studies and vaccine development. One sporozoite-induced and 29 blood-induced infections with the Salvador I strain of P. vivax were studied in splenectomized chimpanzees. Eighteen primary infections with P. vivax resulted in maximum parasite counts ranging from 1,519 to 81,810/ microliters (median 29,100/microliters). Twelve infections induced in animals previously infected with the homologous or heterologous strains of P. vivax had maximum parasite counts ranging from 155 to 14,136/microliters (median 1,736/microliters). A total of 202 of 237 lots containing a total of 293,175 Anopheles freeborni, An. stephensi, An. gambiae, An. dirus, An. quadrimaculatus, and An. maculatus mosquitoes were infected by membrane feeding on gametocytes from chimpanzees. Despite lower levels of parasitemia during secondary (reinfection) parasitemia, 66 of 70 lots of mosquitoes (94.3%) were infected. Based on the mean number of oocysts per positive mosquito gut, An. freeborni was more heavily infected than An. stephensi; An. stephensi was more heavily infected than An. gambiae; there was no significant difference between An. stephensi and An. dirus. Sporozoites from An. stephensi, An. gambiae, An. dirus, and An. freeborni infected with the Salvador I strain of P. vivax produced in chimpanzees were used to infect 193 Saimiri and six Aotus monkeys as well as one chimpanzee.

The Nigerian I/CDC strain of Plasmodium ovale in chimpanzees.

The chimpanzee is the only animal host currently available that can support the development of the human malaria parasite Plasmodium ovale. Thirty-one infections with the Nigerian I/CDC strain were induced in splenectomized chimpanzees. Maximum parasite counts ranged from 1,240 to 127,224/microliters. Infections were transient and unpredictable. Anopheles stephensi, Anopheles gambiae, Anopheles freeborni, and Anopheles dirus mosquitoes were infected by feeding through parafilm membranes on heparinized blood containing gametocytes; each species supported development to sporozoites in the salivary glands. Mean oocyst counts per infected mosquito ranged from 1 to 85.1; 21.7% of infected lots of mosquitoes averaged > 20 oocysts per positive mosquito gut. One infection was induced via the bites of infected An. gambiae. The prepatent period was 16 days.

Blood and sporozoite stage-specific small subunit ribosomal RNA-encoding genes of the human malaria parasite Plasmodium vivax.

Malaria parasites, unlike other eukaryotes, have developmentally controlled distinct small subunit ribosomal RNA (SSUrRNA)-encoding genes (SSUrDNA), sporozoite stage-specific C and blood stage-specific A genes. This report describes characterization of the C and A forms of SSUrDNA from the human malaria parasite Plasmodium vivax. We have aligned and compared these sequences with the reported SSUrDNA sequences of other human malaria parasites to identify the regions with potential for diagnostic probes. The comparison revealed the presence of seven conserved regions (> or = 90% similarity), four highly variable regions (< 60% similarity) and three semiconserved regions. The analysis also revealed that the A and C genes of P. vivax share more similarity with each other, as compared to the A and C genes of P. falciparum. Comparison of the SSUrDNA of human, monkey and rodent malaria parasites revealed that the A genes share more similarity with each other than the C genes share with each other.

Inhibition of malaria parasite development in mosquitoes by anti-mosquito-midgut antibodies.

The mosquito midgut plays a central role in the development and subsequent transmission of malaria parasites. Using a rodent malaria parasite, Plasmodium berghei, and the mosquito vector Anopheles stephensi, we investigated the effect of anti-mosquito-midgut antibodies on the development of malaria parasites in the mosquito. In agreement with previous studies, we found that mosquitoes that ingested antimidgut antibodies along with infectious parasites had significantly fewer oocysts than mosquitoes in the control group. We also found that the antimidgut antibodies inhibit the development and/or translocation of the sporozoites. Together, these observations open an avenue for research toward the development of a vector-based malaria parasite transmission-blocking vaccine.

Identification of Plasmodium vivax-like human malaria parasite.

There are four species of human malarial parasite and several monkey ones, and in evolutionary terms the human and non-human primate plasmodia may be related. The tools of molecular biology have lately pointed to the existence of two types of Plasmodium vivax. Using specific oligonucleotides we have identified a human malaria parasite resembling P vivax under the microscope but with circumsporozoite (CS) protein differing from those of P vivax types 1 and 2. The CS protein of this "P vivax-like" malaria parasite is identical to that of P simiovale, a monkey parasite resembling P ovale, a human one, morphologically. Polyclonal serum raised against a partial repeat sequence of the P vivax-like malaria parasite specifically reacted with P simiovale sporozoites but not with those of P vivax types 1 and 2, P ovale, or P simium. Sera collected from people living in malaria endemic regions of Papua New Guinea and Brazil contained antibodies that specifically reacted with the synthetic peptides representing the repeat sequences of CS protein of this P vivax-like parasite. A comparison of the CS protein gene sequences of P simiovale and the P vivax-like malaria parasites with those of other primate parasites, data on serological cross-reactivity, and 18S ribosomal DNA analyses suggest that the new human malaria parasite described here is distinct from P falciparum, P malariae, P vivax, and P ovale, the four known species of human parasite.

Polymorphism in the circumsporozoite protein of the human malaria parasite Plasmodium vivax.

The circumsporozoite (CS) protein that covers the surface of infectious sporozoites is a candidate antigen in malaria vaccine development. To determine the extent of B- and T-epitope polymorphism and to understand the mechanisms of antigenic variability, we have characterized the CS protein gene of Plasmodium vivax from field isolates representing geographically distant regions of Papua New Guinea (PNG) and Brazil. In the central repeat region of the CS protein, in addition to variation in the number of repeats, an array of mutations was observed which suggests that point mutations have led to the emergence of the variant CS repeat sequence ANGA(G/D)(N/D)QPG from GDRA(D/A)GQPA. Outside the repeat region of the protein, the nonsilent nucleotide substitutions of independent origin are localized in three domains of the protein that either harbor known T-cell determinants or are analogous to the Plasmodium falciparum immunodominant determinants, Th2R and Th3R. We have found that, with the exception of one CS clone sequence that was shared by one P. vivax isolate each from PNG and Brazil, the P. vivax CS protein types can be grouped into Papuan and Brazilian types. These results suggest that an in-depth study of parasite population dynamics is required before field trials for vaccine formulation based on polymorphic immunodominant determinants are conducted.

Use of glass beads and CF 11 cellulose for removal of leukocytes from malaria-infected human blood in field settings.

Passage of malaria-infected blood through a two-layered column composed of acid-washed glass beads and CF 11 cellulose removes white cells from parasitized blood. However, because use of glass beads and CF 11 cellulose requires filtration of infected blood separately through these two resins and the addition of ADP, the procedure is time-consuming and may be inappropriate for use in the field, especially when large numbers of blood samples are to be treated. Our modification of this process yields parasitized cells free of contaminating leukocytes, and because of its operational simplicity, large numbers of blood samples can be processed. Our procedure also compares well with those using expensive commercial Sepacell resins in its ability to separate leukocytes from whole blood. As a test of usefulness in molecular biologic investigations, the parasites obtained from the blood of malaria-infected patients using the modified procedure yield genomic DNA whose single copy gene, the circumsporozoite gene, efficiently amplifies by polymerase chain reaction.

Wide distribution of the variant form of the human malaria parasite Plasmodium vivax.

We have found polymorphism in the repetitive and nonrepetitive regions of the sporozoite vaccine antigen, the circumsporozoite (CS) protein, in Plasmodium vivax malaria parasites from two geographically distant malaria endemic regions of the world. Like the recently described variant repeat sequence of P. vivax from Thailand, the CS protein repeat sequence of the variant P. vivax parasites from Papua New Guinea and Brazil is ANGA(G/D)(N/D)QPG, which differs from the previously identified CS repeat sequence, GDRA(D/A)GQPA, of P. vivax parasites from South America, Central America, and North Korea. Comparison of the P. vivax CS protein outside the repeat region revealed restricted polymorphism in regions that have exhibited T-cell immune function and sequence heterogeneity in the CS protein of Plasmodium falciparum. Our results show that P. vivax malaria parasites with the variant CS repeat sequences are widespread in nature and that the polymorphism in the CS protein of P. vivax is also present in the nonrepeat region.

Circumsporozoite protein gene from Plasmodium reichenowi, a chimpanzee malaria parasite evolutionarily related to the human malaria parasite Plasmodium falciparum.

We have cloned and sequenced the gene encoding the circumsporozoite (CS) protein of Plasmodium reichenowi a Plasmodium falciparum-like malaria parasite of chimpanzees. Comparison of the two CS proteins reveals both similarities and differences in these two evolutionarily related parasites that have adapted to different hosts. The P. reichenowi CS protein has a new repeat sequence, NVNP, in addition to the P. falciparum-like NANP and NVDP repeats. In the immunodominant TH2R and TH3R regions of the CS protein, the amino acid sequences are similar in both parasite proteins. The differences in the two proteins exist in domains around the conserved regions, Region I and Region II, which are otherwise conserved in the CS proteins of P. falciparum analyzed to date. Studies of parasite protein genes of evolutionarily related malaria parasites, together with other immunologic and biologic characteristics, will help better understand the evolution and host parasite relationship of malaria parasites and may provide a tool for identifying protein determinants for malaria vaccine development.

Characterization of the DA26 gene in a hypervariable region of the Autographa californica nuclear polyhedrosis virus genome.

A region of the baculovirus Autographa californica nuclear polyhedrosis virus genome that is frequently found to be altered after serial passage of the virus in cell culture was characterized. Sequence analysis of this region of the genome in wild-type and mutant viruses revealed that some of the mutations affected a 675 bp open reading frame, designated DA26. The DA26 gene was disrupted both by deletion and by insertion of sequences that resembled transposable elements. Northern blot analysis of DA26 showed that it was expressed very early after infection. DA26-specific transcripts could be detected after the 1 h viral adsorption period upon infection of cultured Trichoplusia ni cells. These transcripts were mapped by nuclease protection assays. A recombinant virus was constructed in which DA26 was disrupted by insertion of the Escherichia coli lacZ gene. This virus was viable in both T. ni and Spodoptera frugiperda cells and analysis of the kinetics of protein synthesis revealed no differences between wild-type and recombinant viruses. The disruption of DA26 also did not interfere with the ability of the virus to infect T. ni or S. frugiperda larvae.