Antonio Silvio Verdini

Age-related prevalence of antibody response against three different, defined Plasmodium falciparum antigens in children from the Haut-Ogooué province in Gabon

The kinetics of the humoral response to defined Plasmodium falciparum antigens was studied in 543 children, 1 month to 15 years old, living in an area endemic for malaria. The antigens used for enzyme-linked immunosorbent assay were (i) the synthetic peptide (NANP)40 representing the immunodominant repeated region of the circumsporozoite protein, and (ii) the fusion peptide 31.1, representing the N-terminal portion of the 83 kDa polypeptide expressed at the surface of merozoites which is a processed product of the 190-200 kDa glycoprotein. In addition, glutaraldehyde-fixed infected red blood cells (RBC) were used to detect ring-infected erythrocyte surface antigen (RESA) and unfixed infected RBC to detect intra-erythrocytic asexual form (IEF) antigens by immunofluorescence. In the 1 to 2 months age group, 50%, 26% and 21% of the children had antibodies for IEF, (NANP)40 and 31.1 respectively, but none had anti-RESA antibodies. The proportions of positive subjects decreased until 3 to 6 months and then increased progressively for the 4 antigens, approaching, but not reaching, adult values by the age of 15 years. Antibodies against specific antigens were acquired concomitantly. Children born from (NANP)40-positive mothers showed enhanced anti-(NANP)40 IgG responses.

Sporozoite antibodies and malaria in children in a rural area of The Gambia.

Sporozoite antibody levels were measured in a group of children aged one to nine years resident in a rural area of The Gambia, using an ELISA to the repeat peptide (NANP)40. The prevalence and titre of antibodies varied with age but not with sex or ethnic group. Significant variations in prevalence were recorded within a group of adjacent villages. Children who were seropositive at the beginning of the dry season had higher spleen and parasite rates both at this time and at the end of the subsequent rainy season than did seronegative children, suggesting that they were exposed more frequently to infection. However, seropositive children had fewer episodes of fever accompanied by high levels of parasitaemia than did seronegative children, suggesting that they had a greater degree of clinical immunity. No differences were found in seroprevalence rates or in mean antibody titres between children who slept under conventional or Permethrin treated bed nets and those who did not, even though bed nets significantly reduced the number of bites by vector mosquitoes.

Antibodies to Plasmodium falciparum sporozoites following a malarial outbreak in a non-endemic area of Sri Lanka

An enzyme-linked immunosorbent assay (ELISA) based on the synthetic peptide (NANP)40 was used to characterize the sporozoite antibodies in an unusual Plasmodium falciparum outbreak in a non-malarious area in Sri Lanka. A positive antibody response was seen in 62% of patients with their first P. falciparum illness. There was no correlation between sporozoite antibodies and the antibody against blood stages, determined by immunofluorescence assay. The majority (91%) of the patients lost the antibodies to circumsporozoite (CS) protein within one year (in the absence of re-exposure). Three patients had high levels of CS antibodies even after one year, and this persistence was related to the level of the initial antibody response. In the area of the outbreak 10% of schoolchildren had antibodies to the (NANP)40 peptide. 21% of the 42 children with present or past overt malaria were antibody positive. Of the children with no such background, 8% were antibody positive. The corresponding seropositivity rates for asexual blood stages were 31% and 1% for the 2 groups respectively. It is concluded that (NANP)40 ELISA is potentially a valuable tool in sero-epidemiology, particularly in situations of seasonal transmission and recurrences due to drug resistance.

Preparative purification of Plasmodium falciparum circumsporozoite protein synthetic polypeptides by displacement chromatography

Displacement chromatography was used for the preparative purification of a synthetic polypeptide that is a promising malaria vaccine. It was prepared by solid-phase synthesis and contains two important epitopes of circumsporozoite (CS) protein of Plasmodium falciparum sporozoite. With apparatus typically employed in analytical high-performance liquid chromatography (HPLC) and on a 250 x 4.6 mm I.D. reversed-phase column, up to 50 mg of crude polypeptide were purified in a single run and with a yield higher than 95%. The results demonstrate that displacement chromatography is suitable for the isolation of several milligrams of a pure polypeptide from a complex mixture that is difficult to separate even by analytical HPLC. In such a preparative application, displacement appears to be superior to elution chromatography as used traditionally.

Synthesis, resolution, and assignment of configuration of potent hypotensive retro-inverso bradykinin potentiating peptide 5a(BPP5a) analogues

The effect on the biological activity of an inverted amide group as an isosteric replacement for the Phe-Ala scissile bond of the BPP5a analogue [Phe3]BPP5a has been studied. The synthesis of the retroinverso pentapeptide [gPhe3,(R,S)-mAla4]BPP5a has been carried out in solution by coupling the pseudopeptide epimer HO-(R,S)-mAla-Pro-OBut to Glp-Lys(Boc)-gPhe-H·HCl, separation and identification of the resulting diastereoisomers [Lys2(Boc),gPhe3,(S)-mAla4,Pro5-OBut]BPP5a and [Lys2(Boc),gPhe3,(R)-mAla4,Pro5-OBut]BPP5a, and finally acidolytic cleavage. [gPhe3,(S)-mAla4]BPP5a, a moderate inhibitor of angiotensin converting enzyme (ACE)in vitro(I50= 14 × 10–5M), was obtained and proved to be more potent than BPP5a as a hypotensive in normotensive rats without manifesting the bradykinin potentiating action of the natural peptide.

A Rapid Inhibition Micro ELISA for Detecting Antibodies to Plasmodium Falciparum Sporozoites in Human Blood

A sensitive and specific micro ELISA, named MONOPLATE ELISA, for the detection of antibodies against P. falciparum sporozoites was developed. It can be applied to many kinds of samples including serum, plasma, whole blood, eluted bloodspot and mosquito bloodmeal as well. The method makes use of a single microtiter plate and the chemically synthesized (Asn-Ala-Asn-Pro)20 (NANP20) antigen both as coating material and as competitive (binding) inhibitor in the samples. The specific value of each sample is obtained as the absorbance difference between the uninhibited and the fully inhibited sample. Using appropriate conditions, the results can be evaluated by simple visual inspection of the plate, without any instrument. A rapid procedure, where the incubation times for sample and conjugate are just 15 minutes, is also described. When unknown samples from a P. falciparum endemic area were tested, a close correlation was found between our results and those obtained with the only commercial ELISA kit now available (Sclavo S.p.A). For screening purposes, as many as 48 samples per plate can be tested by this method.

Solid phase synthesis of retro-inverso peptide analogues. Synthesis of a partially modified retro-inverso analogue of substance P, [Glp6, gPhe8, mGly9] SP6–11[gPhe =–NHCH(CH2Ph)NH–,mGly =–OCCH2CO–]

The complete solid phase synthesis of a partially modified retro-inverso analogue of Substance P, [Glp6, gPhe8, mGly9]SP 6–11, [gPhe =–NHCH(CH2Ph)NH–, mGly =–OCCH2CO–] has been performed using a polydimethylacrylamide-based solid support and [bis(trifluoroacetoxy)iodo]benzene.

Reduced antibody response to the repetitive sequence of the Plasmodium falciparum circumsporozoite protein in mice infected with Plasmodium yoelii blood forms

The immunogenicity of the carrier-free synthetic peptide, (NANP)40, from the repetitive region of the Plasmodium falciparum circumsporozoite (CS) protein was investigated in genetically responder mice (C57BL/6, H-2b) acutely infected with blood forms of the non-lethal murine malaria parasite, P. yoelii. As compared to non-infected mice, P. yoelii-infected C57BL/6 mice produced significantly lower titers of anti-(NANP)40 IgG antibodies. This decrease in the anti-(NANP)40 antibody response peaked with the peak of parasitemia, and involved all the IgG subclasses. Interestingly, this P. yoelii-mediated effect was evident both on the development of the antibody response to the (NANP)40 peptide, and on an already established anti-(NANP)40 antibody titer, as seen in mice immunized with the peptide 1 month before the infection. Since (NANP)n-based constructs are strongly envisaged as potential vaccines against falciparum malaria, these results might be important in the evaluation of the efficacy of these vaccine candidates, when they will be used in individuals living in endemic areas.

Synthesis of a toxic octapeptide from the larvae of sawfly

The synthetic octapeptide lophyrotomin (PhCO-D-Ala-D-Phe-L-Val-L-lle-D-Asp-L-Asp-D-Glu-L-Gln), the toxic factor of the larvae of an Australian species of sawfly, Lophyrotoma interrupta, has been prepared by solid-phase synthesis and proved to be identical with the natural material.

Heterogeneity and Specificity of Murine Cell Response to Synthetic Peptide (NANP)n of P. Falciparum Circumsporozoite Protein

The major antigenic protein of the P. falciparum circumsporozoite (CS) is comprised of about 400 amino acids (1,2). The main feature of this protein is the presence of repeats consisting of four amino acids, ASN-ALA-ASN-PRO (NANP; Figure 1).