Juan A. Cooper

A rhoptry antigen of Plasmodium falciparum contains conserved and variable epitopes recognized by inhibitory monoclonal antibodies.

Four monoclonal antibodies produced against Plasmodium falciparum recognize an antigen in merozoites that is localized in rhoptries, as judged by a punctate, double dot fluorescence pattern. All four antibodies bound to the same affinity purified antigen in a two site immunoradiometric assay. Immunoprecipitation of antigen by monoclonal antibody followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis yielded protein bands of 80, 66 and 42 kDa. Western blotting gave bands of 80 and 66 kDa only with three of the antibodies: the fourth did not blot. Based on protease inhibitor data the 66 kDa band is considered to be a cleavage product of the 80 kDa band, but the 42 kDa band does not appear to derive from the latter and may be a coprecipitation product. This group of antigens labels with both [35S]methionine and [3H]histidine. Two of the monoclonal antibodies inhibited merozoite invasion of erythrocytes. One of these inhibitors recognizes a variable epitope, whereas the second recognizes a highly conserved epitope present in all 106 primary isolates of P. falciparum tested from Brazil, Thailand and Papua New Guinea.

An epitope recognised by inhibitory monoclonal antibodies that react with a 51 kilodalton merozoite surface antigen in Plasmodium falciparum

Monoclonal antibodies designated 8G10/48 and 9E3/48 raised against mature asexual blood stages of Plasmodium falciparum inhibit parasite growth in vitro. Both antibodies bind to an epitope which includes the linear sequence Ser Thr Asn Ser and which is present in a cDNA clone from a P. falciparum expression library. These antibodies recognise a glycosylated antigen of approximately 51 kDa which is located on the merozoite surface membrane.

Plasmodium falciparum: two antigens of similar size are located in different compartments of the rhoptry.

Two previously described antigens, AMA-1 and QF3, which are located in the rhoptries of Plasmodium falciparum merozoites have polypeptides of similar relative molecular masses. On immunoblots, antibodies to both antigens recognized polypeptides of relative molecular mass 80,000 and 62,000 in all isolates tested. Two-dimensional electrophoresis showed that the isoelectric points of the two antigens were different. QF3 being more basic than AMA-1. AMA-1 was soluble in Triton X-114 whereas QF3 partitioned into the aqueous phase after temperature-dependent phase separation. In immunoelectron microscopic studies. QF3 was found in the body of the rhoptry whereas AMA-1 was consistently found in the neck of the rhoptry. Both antigens gave a punctate double-dot pattern in mature schizonts and merozoites when visualized by fluorescence microscopy, but AMA-1 antibodies also appeared to label the merozoite surface. QF3 was also detected in ring-infected erythrocytes whereas AMA-1 was not. Synthesis of both antigens was first observed in mature trophozoites and immature schizonts. Pulse-chase experiments showed that the Mr 80,000 polypeptide of the AMA-1 gene was subject to immediate processing to the Mr 62,000 product. This cleavage pattern was not stage specific. The Mr 80,000 polypeptide of QF3 was derived from a short-lived Mr 84,000 precursor polypeptide. Processing of the Mr 80,000 polypeptide to an Mr 62,000 polypeptide was restricted to the period of merozoite maturation and reinvasion. Hence AMA-1 and QF3 are different antigens with polypeptides of similar size but located in different compartments of the merozoite rhoptries.

Opsonic human antibodies from an endemic population specific for a conserved epitope on the M protein of group A streptococci

This study demonstrates the presence of epitope‐specific opsonic human antibodies in a population living in an area endemic for group A streptococci (GAS) infection. Antibodies recognizing a conserved C‐terminal region epitope (p145, sequence in single letter amino acids: LRRDLDASREAKKQVEKALE) of the M protein of GAS were isolated from human patients by affinity chromatography and were shown to be of the immunoglobulin G1 (IgG1) and IgG3 subclasses. These antibodies could reduce the number of colonies of serotype 5 GAS in an in vitro opsonization assay by 71–92%, compared with an equal amount of IgG from control adult donors living in non‐endemic areas and without antibodies to p145. Addition of the peptide, p145, completely inhibited this opsonization. Indirect immunofluorescence showed that p145‐specific antibodies were capable of binding to the surface of M5 GAS whereas control IgG did not. Using chimeric peptides, which contain overlapping segments of p145, each 12 amino acids in length, inserted into a known helical peptide derived from the DNA binding protein of yeast, GCN4, we have been able to further define two minimal regions within p145, referred to as pJ2 and pJ7. These peptides, pJ2 and pJ7, were able to inhibit opsonization by p145 specific antibodies. Finally, we have observed an association between the age‐related development of immunity to GAS and the acquisition of antibodies to the conserved epitope, p145, raising the possibility of using this epitope as a target in a prophylactic vaccine administered during early childhood.

Mapping of the region predominantly recognized by antibodies to the Plasmodium falciparum merozoite surface antigen MSA 1

The locations of the epitopes of a panel of mouse monoclonal antibodies directed against the Plasmodium falciparum merozoite surface antigen MSA 1 were mapped by using naturally occurring processed fragments, by chemical cleavage of the protein and by comparison of the isolate-specificity of binding with known sequence variation. By these criteria, the most antigenic region occurs in the cysteine-rich, invariant 19-kDa carboxyl terminal domain with 12/19 monoclonal antibodies (mAbs) binding to this region. One of these mAbs recognized an epitope near the C-terminal putative glycosylphosphatidylinositol anchor site. This was the only mAb which significantly inhibited parasite growth in vitro. The other mAbs recognized conformational epitopes involving the cysteine residues located throughout this fragment. This study has identified further naturally occurring processing sites and a consensus processing site sequence is now emerging.

The 140/130/105 kilodalton protein complex in the rhoptries of Plasmodium falciparum consists of discrete polypeptides

Four monoclonal antibodies (MAbs) recognise an antigen localised in the rhoptries of Plasmodium falciparum merozoites using both indirect immunofluorescence assay and immunoelectron microscopy with immunogold labeling. All MAbs immunoprecipitated bands at 140, 130 and 105 kDa from [35S]methionine-labeled parasites; however, one MAb immunoblotted only the 130 kDa protein and another MAb immunoblotted the 105 kDa protein. The affinity purified antigen complex consisted of proteins of 140, 130, 105 and 98 kDa. The individual proteins were subjected to peptide mapping with Staphylococcus aureus V8 protease; the 98 kDa protein was a degradation product of the 105 kDa protein and the 140, 130, and 105 kDa proteins were found to be unrelated. The antigen complex was synthesised at the mid trophozoite stage and was considered to be soluble as judged by release from mature schizonts by freeze/thaw lysis. One of the MAbs inhibited parasite growth and/or merozoite invasion of erythrocytes, in vitro, to a small but significant extent.

An antigenic complex in the rhoptries of Plasmodium falciparum.

A previously identified putative rhoptry antigen of Plasmodium falciparum is composed of two major components, one of 80 kDa and a doublet at 42/40 kDa. An inhibitory monoclonal antibody immunoprecipitated both the 80 kDa protein and the 42/40 kDa doublet, but immunoblotted only the 80 kDa component. A second monoclonal antibody, raised against the affinity purified complex, immunoblotted only the 42 kDa band under non-reducing conditions. Electron microscopic examination of thin sections of parasites immunolabeled with these monoclonal antibodies and colloidal gold anti-mouse conjugate has confirmed that this antigen is localised in the rhoptry organelles of mature schizonts and free merozoites. The antigen is associated with apparent membranous structures released from free merozoites. Immunoblotting and immunoprecipitation with two different monoclonal antibodies, and protease digestion experiments, have clearly demonstrated that this antigen is a complex composed of two separate and distinct proteins, and does not represent a monomer/dimer pair. The 80 kDa protein is synthesised as an 84 kDa precursor.

The 42-kilodalton rhoptry-associated protein of Plasmodium falciparum

The gene coding for a 42-kDa rhoptry protein of Plasmodium falciparum has been cloned. On the basis of prior monkey vaccination studies, this protein is regarded as an important vaccine candidate, but its identity has been the subject of considerable uncertainty. Analysis of the cloned sequence shows that it is a basic, hydrophobic protein, without repetitive elements, unrelated to any of the previously postulated gene products and shows minimal sequence diversity. The availability of the corresponding recombinant protein will enable studies of its efficacy in human vaccine trials to be undertaken.

Location of neutralizing epitopes on the G protein of bovine ephemeral fever rhabdovirus

The surface glycoprotein G is the major neutralizing and protective antigen of bovine ephemeral fever rhabdovirus (BEFV). Twelve neutralizing MAbs against BEFV strain BB7721 were used to select 33 neutralization escape mutants. The mutants had been classified previously into three major antigenic sites (G1-G3) based on their cross-neutralization patterns. The nucleotide sequence of the entire extracellular domain of the G protein gene was determined for all mutants. Each contained a single nucleotide change leading to a single amino acid substitution. The 16 mutants assigned to the linear antigenic site G1 mapped to aa 487-503 of the 623 aa G protein. Results of antibody binding to several overlapping octapeptides covering this region mapped the sequence of two common minimal B cell epitopes recognized by the five G1 MAbs to (488)EEDE(491) and (499)NPHE(502). Site G2 mutations mapped either at aa 169 or 187. The 12 mutants representing antigenic site G3 (G3a and G3b) mapped to aa 49, 57, 218, 229 and 265, indicating that this site is likely to combine complex discontinuous epitopes. Comparison of the deduced amino acid sequence from five BEFV field isolates and BB7721 identified aa 218 to be critical for the site G3a neutralization. Alignment of the glycoproteins of rabies virus, vesicular stomatitis Indiana virus, vesicular stomatitis New Jersey virus, infectious haematopoietic necrosis virus and BEFV revealed similarities in the location of the neutralizing epitopes and extensive conservation of cysteine residues, suggesting that basic elements of the folded structure of these glycoproteins are preserved.

Definition of T cell epitopes within the 19 kDa carboxylterminal fragment of Plasmodium yoelii merozoite surface protein 1 (MSP119) and their role in immunity to malaria

MSP119 is one of the leading malaria vaccine candidates. However, the mechanism of protection is not clear. To determine whether MSP119‐specific effector T cells can control parasitaemia, we analysed the specificity of T cells induced following immunization with recombinant forms of P. yoelii MSP119 and asked whether they could protect mice. There was no evidence that effector T cells were capable of protecting since: (1) immunization of mice with yMSP119, but not defined epitopes, was able to induce protection; and (2) long term MSP119‐specific CD4+ T cell lines were incapable of adoptively transferring protection. In contrast, priming mice with the T cell epitopes resulted in a rapid anamnestic antibody response to MSP119 after either challenge with MSP119 or parasite. Thus, MSP119 contains multiple T cell epitopes but such epitopes are the targets of helper T cells for antibody response but not of identified effector T cells capable of controlling parasitaemia.