Immunization of Aotus Monkeys with Recombinant Plasmodium falciparum Hybrid Proteins Does Not Reproducibly Result in Protection from Malaria Infection

Plasmodium falciparum antigens SERP, HRPII, MSAI, and 41-3 have shown promise as vaccine components. This study aimed at reproducing and extending previous results using three hybrid molecules. Antibody responses were reproduced in Aotus monkeys, but solid protection from a P. falciparum blood-stage challenge that showed an unintendedly enhanced pathogenicity was not observed.The increasing drug resistance of Plasmodium falciparum, the most pathogenic human malaria parasite, underlines the need for an effective malaria vaccine. Identification, testing, and optimization of candidate molecules originating from all developmental stages of the parasite are under way. Previously, a successful trial in Aotus monkeys employed the Escherichia coli-expressed hybrid proteins MS2/SERP/HRPII and SERP/MSAI/HRPII (11). Both hybrid proteins contain a region of the serine repeat protein SERP (1, 9), including two putative T-cell epitopes (13) and previously shown to induce a partial protective response in Aotus monkeys (5), and the C-terminal half of the histidine-rich protein HRPII (14), which has also been shown to induce a partially protective response (5, 8). SERP/MSAI/HRPII contains in addition a conserved N-terminal region of the merozoite surface antigen MSAI (7) that includes at least four T-cell epitopes (3, 6). Here we report on further analysis of three hybrid proteins of this type in a vaccination trial with Aotus monkeys. Two of the proteins, SERP/HRPII and SERP/MSAI/HRPII, are improved versions of the hybrid proteins mentioned above, obtained by deleting nonmalaria protein regions and changing an internal restart residue (methionine-729 of SERP) into alanine. Thus, the SERP/HRPII hybrid protein comprises residues 630 to 893 of SERP fused to the 189 C-terminal residues of HRPII, and SERP/MSAI/ HRPII comprises residues 630 to 764 of SERP fused to residues 146 to 259 of MSAI, which is fused to the 189 C-terminal residues of HRPII. SERP/41-3/HRPII contains the same components as SERP/HRPII, and additionally includes residues 77 to 188 of antigen 41-3 (10), which was previously shown to confer protection against a P. falciparum challenge (5). The internal restart residue (methionine-100) was also mutagenized into alanine and another residue, arginine-319, was changed into leucine to prevent proteolytic degradation. SERP/MSAI/HRPII was partially purified to a final purity of about 30%, as described previously (8), in order to match the quality of the proteins used in the successful previous trials (5, 11). The other two hybrid proteins were purified from bacterial lysates to over 90% purity by size exclusion chromatography (SERP/41-3/HRPII) or by sequential cation and anion exchange and then size exclusion chromatography (SERP/ HRPII) (data not shown). The final products were dialyzed against phosphate-buffered saline–3 M urea and adjusted to 100 μg of protein per ml. Efficacy was tested following an experimental protocol identical to the one used in the previous successful trial (11).Fifteen laboratory-raised Aotus azarae boliviensis karyotype VI monkeys were randomly assigned to one of four experimental groups (three groups of four and one group of three monkeys) and immunized with 1 ml of antigen or with the diluent alone (control group), both mixed with 100 μl of polyalphaolefin (4) as an adjuvant, on days 0, 21, and 42. Each vaccine dose was administered subcutaneously at two separate sites in the right and left flank and was well tolerated. The seroconversion results, as measured by enzyme-linked immunosorbent assay with SERP/HRPII as the solid-phase antigen and peroxidase-labelled rabbit anti-human immunoglobulin G (1:10,000 dilution; Pierce) as the secondary antibody, are shown in Fig. ​Fig.1.1. All experimental monkeys developed comparable antibody responses to SERP/HRPII, irrespective of the immunogen. Control monkey sera did not react significantly (not shown). A boosting effect is obvious after the second injection in all three groups (Fig. ​(Fig.1),1), as well as after the third SERP/41-3/HRPII injection (Fig. ​(Fig.1C).1C). This is similar to the seroconversion pattern observed previously (11). Prechallenge sera were also tested by immunofluorescence (IFA) for reactivity with P. falciparum schizonts. All preimmune sera and control group immune sera were negative (1:100 dilution). IFA titers from the experimental animals were all 1:1,600, except for animals A381 and A462 (titer, 1:800) and A452 and A292 (titer, 1:3,200). Thus, antibodies specific for native parasite determinants were induced. The relatively low IFA titers were comparable to those obtained in previous successful trials (5, 11). Open in a separate windowFIG. 1Development of antibody responses in Aotus monkeys during the immunization period as determined by enzyme-linked immunosorbent assay. Monkeys in different immunization groups were immunized at weeks 0, 3, and 6 (indicated by arrows) and challenged at week 8 (indicated by an asterisk). All sera were tested for reactivity with SERP/HRPII in a 1:100 dilution. The hybrid antigens used for immunization were SERP/MSAI/HRPII (A), SERP/HRPII (B), and SERP/41-3/HRPII (C). Sera of the three control monkeys remained negative in this assay (not shown). OD, optical density.At week 7 all monkeys were splenectomized, and at week 8 they received intravenously 2 × 10⁶ parasitized erythrocytes, which had been isolated from an Aotus monkey infected with an in vivo-passaged FUP-Cayenne isolate of P. falciparum (a kind gift of W. E. Collins) (Fig. ​(Fig.1).1). Monkey A293 appeared to have no spleen, although there was no prior history of splenectomy. The immunoglobulin G response of monkey A293 was nevertheless comparable to that of the other animals (Fig. ​(Fig.1B).1B). Figure ​Figure22 shows the course of parasitemia after challenge. Two of three control animals rapidly developed a parasitemia which required mefloquine therapy (20 mg/kg of body weight orally) when parasitemia reached 10% (day 8 for A371 and day 10 for A320). In A432, parasitemia developed to 8.6% (day 10) and then fluctuated until rapidly reaching 19% (day 21), at which point mefloquine was administered (Fig. ​(Fig.2A).2A). None of the three immunized groups showed a solid protective response (Fig. ​(Fig.2B2B to D). A340 (SERP/HRPII group) (Fig. ​(Fig.2C)2C) and A292 (SERP/41-3/HRPII group) (Fig. ​(Fig.2D)2D) showed low fluctuating parasitemias with a peak around 2.5% at the end of the observation period (day 25). Otherwise, parasitemias of the experimental animals did not significantly differ from those of the controls. No obvious correlation between prechallenge antibody levels and protection was evident. Open in a separate windowFIG. 2Course of infection with the FUP-Cayenne isolate of P. falciparum in control Aotus monkeys (A) and in Aotus monkeys immunized with SERP/MSAI/HRPII (B), SERP/HRPII (C), and SERP/41-3/HRPII (D). Parasitemias of ≥10% were cured with mefloquine.It seems unlikely that small conservative changes designed to improve SERP/HRPII and SERP/MSAI/HRPII expression in E. coli and to remove nonrelevant sequences adversely affected immune response development. After challenge, parasitemia developed markedly faster than in the previous trial, which had shown protection. Challenge with 2 × 10⁶ parasitized erythrocytes now resulted in high parasitemias on days 7 to 9 in 2 of the 3 controls and in 6 of the 12 experimental animals, whereas previously controls were untreated until day 14 (11). Also, one control and three experimental animals suffered recrudescence, which was not seen previously (11) or with later infections with the same parasite stock (5). It is remarkable that this apparent enhanced pathogenicity developed after a single passage in A. nancymai just before the present trial started. It is likely that this unintended pathogenicity influenced the experimental outcome. The protection of two monkeys in the SERP/HRPII and SERP/41-3/HRPII group may, however, reveal some protective effect of these vaccine candidates.For demonstration of the protective potential of antigens in the primate model the pathogenicity of the challenge strain in the respective primate (sub)species, i.e., the equilibrium of immune response and pathogenicity, seems to be crucial (2, 12). The disturbance of this equilibrium may explain the discrepancy between previous successful trials (5, 11) and the present study. Recombinant proteins shown to be protective in the Aotus model (5, 11) failed to protect Saimiri monkeys, in which the course of parasitemia is quite different from that observed in Aotus monkeys. Similarly, no protection could be demonstrated in A. nancymai against the same challenge strain as was used in the successful trials with A. azarae boliviensis and A. lemurinus griseimembra (7a). The poor standardization of these models due to the scarcity of monkeys susceptible to human malaria remains an obstacle for the evaluation of human malaria vaccine candidates.     

Pharmacological control of leukotriene and prostaglandin production from mouse peritoneal macrophages

Leukotriene and prostaglandin production by mouse peritoneal macrophages was investigated. It could be shown that the tumour promoter 12-O-tetradecanoylphorbol-13-acetate initiated the release of prostaglandin E₂ but had little effect on the release of leukotriene C₄-like immunoreactivity. The divalent cation ionophore A 23187 at concentrations between 10^–6 and 10^–8 mol/l initiated prostaglandin as well as leukotriene release. This prostaglandin and leukotriene release could be modulated by drugs. Non-steroidal anti-inflammatory drugs including benoxaprofen inhibited prostaglandin release but simultaneously enhanced leukotriene production. The analgesics paracetamol and 4-methylaminoantipyrine had similar effects at high concentrations. The experimental compound BW 755 c inhibited prostaglandin and leukotriene production while the antithrombotic compound nafazatrom inhibited the production of leukotriene C₄-like immunoreactivity but enhanced prostaglandin E₂ production. Nordihydroguaiaretic acid inhibited prostaglandin and leukotriene production. The results show that the metabolism of arachidonic acid in macrophages via the cyclooxygenase or the lipoxygenase pathway is dependent on the stimulus applied. Both pathways can be inhibited conjointly or selectively by drugs. Our results do not provide evidence that differences in anti-inflammatory activity claimed for some of the drugs tested can be explained by differential inhibition of either pathway. The experimental system described may be used for assessing the potency of drugs to inhibit the lipoxygenase and the cyclooxygenase pathway of arachidonic acid metabolism.     

Dielectric properties of a combined main-chain/side-chain liquid-crystalline polymer

The dielectric relaxation properties of a combined main-chain/side-chain liquid-crystalline polymer were investigated. It was found that the rotation of the side chain about the main chain (δ-process) is not as strongly restricted as in side-chain liquid-crystalline polymers. This is attributed to the facts that the side chain is attached to the flexible spacer within the chain backbone and that the concentration of the side chains is comparatively small. Two low-temperature relaxation processes were observed to occur in the glassy smectic and the crystalline state. They are attributed to intramolecular motions with in the mesogenic groups.     

Microproteins in amniotic fluid as an index of changes in fetal renal function during development

Protein content and protein composition were studied in amniotic fluid obtained from 171 healthy pregnant women between the 16th and 38th week of gestation, using microgradient gel electrophoresis to separate proteins according to their molecular size into albumin (68 KD), proteins of low molecular weight (LMW proteins, <68 KD), and proteins of high molecular weight (HMW proteins, >68 KD). Additionally -1-microglobulin (-1-MG, 33 KD) and -2-microglobulin (-2-MG, 11,8 KD) were analysed as micromolecular marker proteins. Concentrations of LMW proteins were 0.15–0.22 g/l, of -1-MG 28.4–34.5 mg/l, and of -2-MG 7.2–11.6 mg/l during the second trimester of gestation, and thereafter decreased progressively to 0.03 g/l, 14.1 mg/l and 2.4 mg/l respectively near term. The same developmental trends were confirmed by calculating the protein/creatinine ratios in amniotic fluid. The concentrations of LMW proteins found in the first postnatal urine of 73 healthy infants born prematurely or at term were similar to those in amniotic fluid of corresponding fetal age. Concentrations of albumin and HMW proteins in postnatal urine were about 5% and 15% respectively when compared with amniotic fluid concentrations. No strong correlation existed between gestational age and either of the analysed proteins which would allow accurate assessment of fetal maturation by protein analysis in amniotic fluid. It is concluded that fetal urinary excretion is the major determinant of the microprotein content of amniotic fluid. Microproteins seem to reflect an increasing tubular reabsorption capacity, which accelerates rapidly after the second trimester of gestation.     

Cerebral blood flow in autogenic training and hypnosis

In 12 healthy volunteers with at least an experience of six months in autogenic training (AT), the cerebral blood flow (CBF) was measured at rest, in AT and in hypnosis (H). The results were correlated with individual test profiles. The cortical flow pattern at rest of our AT trained volunteers did not show the hyperfrontality which is described in the literature. This may be interpreted as an effect of better and habitualized relaxation in long trained AT practitioners. This flow pattern corresponds to the low grades of neuroticism and aggressivity found in the tests. Furthermore an activation in central cortical areas and a deactivation in regions which are associated with acoustic and autonomous functions occur.Possible explanations for these phenomena as well as for the relatively low perfusion of the left hemisphere at rest and activation in AT are discussed. The global rise of CBF in H may be an activation effect caused by resistance against the hypnotizer: the deeper the trance, the smaller the CBF increase in the motor cortical area needed for maintaining catalepsy of the right arm and in temporal cortical fields processing acoustic inputs.     

Pharmacological characterization of A1 adenosine receptors in isolated rat ventricular myocytes

Summary The aim of the present study was the characterization of adenosine receptors in isolated rat ventricular myocytes. The CAMP-levels of rat ventricular myocytes in the presence of 1 mol/l isoprenaline were reduced by up to 48% by adenosine analogues; the rank order of potency was: R-N⁶-phenylisopropyladenosine (IC₅₀ 60 nmol/1), 5-N-ethylcarboxamidoadenosine (IC₅₀ 360 nmol/l) and S-N⁶-phenylisopropyladenosine (IC₅₀ 16 ol/l). The adenosine receptor antagonist XAC (xanthine amine congener) antagonized the effect of R-N⁶-phenylisopropyladenosine in a concentration-dependent manner with a K_i-value of 20 nmol/l. The A₁ receptor-selective radioligand R-N⁶-¹²⁵I-p-hydroxyphenylisopropyladenosine bound to membranes prepared from rat ventricular myocytes in a saturable manner with a B _max of 17.7 fmol/mg protein and a K _D-value of 1.1 nmol/l. Adenosine analogues competed for the binding with the same rank order of potency as for the inhibition of the isoprenaline-induced cAMP-increase. GTP inhibited radioligand binding with an IC₅₀-value of 73 ol/l. These results suggest the presence of A₁ adenosine receptors on rat ventricular myocytes, which mediate an inhibition of adenylate cyclase. The receptors may be responsible for the effects of adenosine and its analogues on the heart.Abbreviations ¹²⁵I-HPIA R-N⁶-¹²⁵I-p-hydroxyphenylisopropyladenosine - PIA N⁶-phenylisopropyladenosine - NECA 5-N-ethyl-carboxamidoadenosine - XAC 8-4-[([(2-aminoethyl)aminocarbonyl]methyl)oxy]phenyl-1,3-dipropylxanthine (xanthine amine congener) - Ro 20-1724 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone - ScAMPTME 2-O-monosuccinyladenosine-3,5-cyclic monophosphate tyrosyl methyl ester - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - GTP guanosine-5-tri-phosphate Send offprint requests to D. Martens     

Omega-3 Fatty Acids and Their Role in Pediatric Cancer

Background: Malnutrition is common in children with cancer and is associated with adverse clinical outcomes. The need for supportive care is becoming ever more evident and the role of nutrition in oncology is still not sufficiently understood. In particular, the consequences of macro- and micronutrient deficiencies require further research. As epidemiological data suggest anti-tumoral properties of omega-3 (n-3) polyunsaturated fatty acids (PUFAs), we reviewed the role of nutrition and n-3 supplementation in pediatric oncology. Methods: A comprehensive literature search was conducted on PubMed through 5 February 2021 to select meta-analyses, systematic reviews, observational studies, and individual randomized controlled trials (RCTs) on macro- and micronutrient supplementation in pediatric oncology. The search strategy included the following medical subject headings (MeSH) and keywords: “childhood cancer”, “pediatric oncology”, “nutritional status”, “malnutrition”, and “omega-3-fatty-acids”. The reference lists of all relevant articles were screened to include potentially pertinent studies. Results: We summarize evidence about the importance of adequate nutrition in childhood cancer and the role of n-3 PUFAs and critically interpret findings. Possible effects of supplementation on the nutritional status and benefits during chemotherapy are discussed as well as strategies for primary and secondary prevention. Conclusion: We here describe the obvious benefits of omega-3 supplementation in childhood cancer. Further large scale clinical trials are required to verify potential anti-cancer effects of n-3 fatty acids.     

Emergence of SARS-CoV-2 B.1.1.7 Lineage at Outpatient Testing Site,Berlin, Germany,January–March 2021

Within 5 weeks in 2021, B.1.1.7 became the dominant severe acute respiratory syndrome coronavirus 2 lineage at an outpatient testing site in Berlin, Germany. Compared with outpatients with wild-type virus infection, patients with B.1.1.7 had similar cycle threshold values, more frequent sore throat and travel history, and less frequent anosmia/ageusia.     

The Functional Status of the ACL in Varus OA of the Knee: The Association With Varus Deformity and Coronal Tibiofemoral Subluxation

BackgroundThe present article analyzes the association of the functional anterior cruciate ligament (ACL) status and the overall varus deformity and coronal tibiofemoral subluxation (CTFS) in varus OA of the knee.MethodsOne hundred consecutive knees with varus OA in 84 patients were prospectively included. Knees were divided into two groups, in accordance with the ACL status (functionally sufficient or insufficient). All included patients were potential candidates for unicompartmental knee arthroplasty with predominantly medial compartment OA. Knees with Kellgren/Lawrence ≥ grade 3 in the lateral compartment were excluded leaving 79 knees to be included in this study. Mechanical varus deformity and CTFS were evaluated on AP radiographs and valgus stress radiographs, and compared between the two groups.ResultsKnees with a functionally insufficient ACL had significantly more varus deformity on hip-to-ankle AP standing radiographs (P = .001) and on valgus stress radiographs (P = .017). CTFS on AP standing radiographs was significantly higher (P = .045) in knees with a functionally insufficient ACL. Seventy-three percent (8/11) of the ACL-insufficient knees had a varus deformity of ≥10° and 64% (7/11) of ACL-insufficient knees had CTFS ≥ 6mm. By contrast, only one patient (2%, 1/41) with an insufficient ACL had< 10° varus deformity and a CTFS of < 6mm.ConclusionFunctional ACL insufficiency in osteoarthritic varus knees is associated with greater varus deformity and more advanced CTFS. Seventy-three percent of ACL-insufficient knees had a varus deformity of ≥10° and 64% of ACL-insufficient knees a CTFS of ≥ 6mm. In the work-up for medial unicompartmental knee arthroplasty, functional ACL insufficiency is likely in knees with varus deformity of ≥10° and CTFS of ≥ 6mm.