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Vaccination of mice with the 42-kDa region of Plasmodium yoelii merozoite surface protein 1 (MSP1(42)) or its 19-kDa C-terminal processing product (MSP1(19)) can elicit protective antibody responses in mice. To investigate if the 33-kDa N-terminal fragment (MSP1(33)) of MSP1(42) also induces protection, the gene segment encoding MSP1(33) was expressed as a glutathione S-transferase (GST) fusion protein. C57BL/6 and BALB/c mice were immunized with GST-MSP1(33) and subsequently challenged with the lethal P. yoelii YM blood stage parasite. GST-MSP1(33) failed to induce protection, and all mice developed patent parasitemia at a level similar to that in naive or control (GST-immunized) mice; mice immunized with GST-MSP1(19) were protected, as has been shown previously. Specific prechallenge immunoglobulin G (IgG) antibody responses to MSP1 were analyzed by enzyme-linked immunosorbent assay and immunofluorescence. Despite being unprotected, several mice immunized with MSP1(33) had antibody titers (of all IgG subclasses) that were comparable to or higher than those in mice that were protected following immunization with MSP1(19). The finding that P. yoelii MSP1(33) elicits strong but nonprotective antibody responses may have implications for the design of vaccines for humans based on Plasmodium falciparum or Plasmodium vivax MSP1(42).

ABSTRACTVaccination of mice with the 42-kDa region of Plasmodium yoeliimerozoite surface protein 1 (MSP142) or its 19-kDa C-terminal processing product (MSP119) can elicit protective antibody responses in mice. To investigate if the 33-kDa N-terminal fragment (MSP133) of MSP142also induces protection, the gene segment encoding MSP133was expressed as a glutathione S-transferase (GST) fusion protein. C57BL/6 and BALB/c mice were immunized with GST-MSP133and subsequently challenged with the lethal P. yoeliiYM blood stage parasite. GST-MSP133failed to induce protection, and all mice developed patent parasitemia at a level similar to that in naive or control (GST-immunized) mice; mice immunized with GST-MSP119were protected, as has been shown previously. Specific prechallenge immunoglobulin G (IgG) antibody responses to MSP1 were analyzed by enzyme-linked immunosorbent assay and immunofluorescence. Despite being unprotected, several mice immunized with MSP133had antibody titers (of all IgG subclasses) that were comparable to or higher than those in mice that were protected following immunization with MSP119. The finding that P. yoeliiMSP133elicits strong but nonprotective antibody responses may have implications for the design of vaccines for humans based on Plasmodium falciparumor Plasmodium vivaxMSP142.

Evaluation of cytokine production in macaques has been hampered by a lack of availability of optimized and standardized immunoassays such as ELISA and enzyme-linked immune spot assay (ELISpot); only a limited number of macaque cytokines have been assessed by ELISpot. Using monoclonal antibodies (mAb) to human cytokines that cross-react with cynomolgus and rhesus macaque interferon-γ (IFN-γ), interleukin (IL)-2, IL-4, IL-5, IL-6, IL-12, IL-13 and granulocyte monocyte colony-stimulating factor, we measured macaque cytokine production by ELISA and ELISpot. Quantitation of spontaneous as well as phytohemagglutinin (PHA)-induced cytokine production in peripheral blood mononuclear cells (PBMC) from rhesus and cynomolgus macaques and humans were compared. The proportional distribution of the different cytokines, in terms of PBMC synthesizing different cytokines as well as the levels of the different cytokines produced, were similar in all species. Spontaneous- and PHA-induced cytokine productions thus appear to be similarly regulated in macaques and man. ELISpot and ELISA assays for macaque IFN-γ were further used to measure antigen-specific immune responses of PBMC from cynomolgus macaques exposed to, or vaccinated against, simian immunodeficiency virus (SIV). The establishment of reliable immunoassays for detection of macaque cytokines is of importance for future progress of research utilizing macaques as experimental animals.

ABSTRACTThe degree of protection against Plasmodium yoeliiasexual blood stages induced by immunization of mice with the 19-kDa region of merozoite surface protein 1 (MSP119) is H-2 dependent. As a strategy to improve the protection, mouse strains with disparate H-2 haplotypes were immunized with glutathioneS-transferase (GST)–MSP119proteins including either a universal T-cell epitope from tetanus toxin (P2) or an I-Ak-restricted T-cell epitope (P8) fromPlasmodium falciparumPf332. In H-2kmice which are poorly protected following immunization with GST-MSP119, GST-P2-MSP119significantly improved the protection. In mice partially (H-2k/b) or well protected by GST-MSP119(H-2dand H-2b), P2 did not further increase the protection. However, the protection of H-2k/bmice and to some extent H-2kmice was improved by immunization with GST-P8-MSP119. The magnitudes of immunoglobulin G1 (IgG1) and IgG2a responses in mice immunized with the GST-MSP119variants correlated with low peak parasitemia, indicating a protective capacity of these IgG subclasses. In H-2kmice immunized with GST-P2-MSP119, both IgG1 and IgG2a responses were significantly enhanced. The epitope P2 appeared to have a general ability to modulate the IgG subclass response since all four mouse strains displayed elevated IgG2a and/or IgG2b levels after immunization with GST-P2-MSP119. In contrast, GST-P8-MSP119induced a slight enhancement of IgG responses in H-2k/band H-2kmice without any major shift in IgG subclass patterns. The ability to improve the protective immunity elicited by P. yoeliiMSP119may have implications for improvement of human vaccines based on P. falciparumMSP119.

Five isoforms of tachykinin‐related peptides (TRPs), designated LemTRP‐1–5, have been identified in the midgut of the cockroach Leucophaea maderae. These peptides have a conserved C‐terminus hexapeptide (GFX1GX2Ramide; X1and X2are variable residues) and variable N‐termini. Here, we address the question of whether these five isoforms are all colocalized in the two types of cells in the cockroach midgut, the endocrine cells and the neuronal processes. We also investigate whether the N‐terminally extended isoforms LemTRP‐2 and ‐3, which contain putative endoproteolytic cleavage sites, are expressed in intact form or are cleaved in the midgut cells. To this end, we used two approaches. (1) Extracts from portions of the midgut containing each of the cell types were subjected to reverse‐phase high performance liquid chromatography (HPLC) and the fractions monitored in a radioimmunoassay (RIA) with an antiserum to the conserved C‐terminus of insect TRPs. (2) Antisera were raised to the variable N‐termini of the extended LemTRP‐2 and ‐3 and used for immunocytochemistry. The HPLC‐RIA and immunocytochemical findings indicate that LemTRP‐1 and 4–5 are present in the neuronal processes and in endocrine cells of the midgut proper and of the gastric cecae. The two extended forms LemTRP‐2 and ‐3 display a differential distribution: LemTRP‐2 was found in endocrine cells of midgut and gastric cecae, but not in neuronal processes, whereas LemTRP‐3 was seen in neuronal processes and endocrine cells of the midgut proper, but not in the gastric cecae. LemTRP‐3 and ‐4 have not been identified in the brain, suggesting further cell‐ and tissue‐specific expression of LemTRPs. The mechanisms behind the cell‐specific expression of the LemTRPs are not yet understood, but the demonstration of differential distribution of the peptide isoforms provide a first indication that the isoforms may have different actions. J. Comp. Neurol. 406:15–28, 1999. © 1999 Wiley‐Liss, Inc.

Plasmodium falciparum-reactive antibodies can inhibit growth of parasite blood stagesin vitroby interfering at different stages of parasite development. Antibodies reactive with repeat sequences in Pf155/ring-infected surface antigen (Pf155/RESA) or in antigen Pf332 inhibit parasite growth as determined by a reduction of newly infected erythrocytes. Antibodies to Pf155/RESA have been implicated in merozoite invasion inhibition. Since it was considered unlikely that antibodies to the late stage antigen Pf332 act on merozoite invasion, we investigated at what developmental stage this growth inhibition takes place. Parasites were cultured in the presence of antibodies to repeat sequences of either Pf332 or Pf155/RESA and were examined with regard to the distribution of different stages and their morphology. As expected, antibodies to both antigens decreased the number of ring stages after reinfection. They did not affect the intraerythrocytic development of ring stages or trophozoites. However, antibodies to Pf332 induced high levels of schizonts displaying an abnormal morphology while antibodies to Pf155/RESA induced considerably lower levels of degenerated schizonts. Thus, Pf332-reactive antibodies seem to interfere with the schizont development by blocking the rupture of mature schizonts or, alternatively, by interfering intraerythrocytically with late stage parasites.

The presence of a signal sequence preceding the gene encoding a target antigen in a DNA vaccine should facilitate secretion of the in vivo translated antigen. The immune responses elicited upon injection with such a vector could differ from those induced by the same vector lacking a signal sequence. In the present study, the humoral responses elicited in mice immunized with two plasmids, either containing or lacking the human tissue plasminogen activator signal sequence, were compared. Both plasmids encode the chimeric antigen ZZN4, containing a malaria antigen Pf332‐derived sequence (N4) linked to a bacterial fusion partner (ZZ). In vitro transfection of COS cells with each plasmid and treatment of the transfectants with brefeldin A confirmed that secretion of ZZN4 via the endoplasmic reticulum and Golgi pathway only occurred in cells transfected with the signal peptide‐encoding plasmid. Repeated intramuscular injections of mice with either of the plasmids elicited comparable antibody responses to ZZN4 with regard to kinetics, specific IgG levels and persistence. These results indicate that in vivo transfection of muscle cells by either of these two plasmids generated comparable levels of antigen available for B‐cell recognition and for uptake by antigen‐presenting cells, despite the differential intracellular targeting of the encoded antigen. The relevance of these findings for the design of DNA vaccine vectors is discussed.