Human and murine CD4 T cell epitopes map to the same region of the malaria circumsporozoite protein: limited immunogenicity of sporozoites and circumsporozoite protein.

The circumsporozoite (CS) protein is a candidate vaccine antigen for the sporozoite stage in the life cycle of the malaria parasite. Using CS protein purified from recombinant baculovirus-infected cells and a panel of H-2 congenic mice, we are able to demonstrate that this protein is poorly immunogenic in terms of antibody production as a result of Ir gene control. The immune response to the protein is also restricted following immunization with a CS-recombinant vaccinia virus or with sporozoites. Using a panel of overlapping peptides spanning the entire protein, we are able to show that the high responder mice recognize helper T cell epitopes from the same region of the protein as do humans. This region, however, is the polymorphic segment of the protein, which has implications for vaccine development. However, the close overlap of human and murine T cell epitopes demonstrates that murine models may be very useful in epitope mapping and vaccine development for human pathogens. The T cell antigenic regions of this protein fulfil the predictive requirements for the amphipathic helicity algorithm.

Identification of a 43-kDa polypeptide associated with acetylcholine receptor-enriched membranes as MM creatine kinase.

Creatine kinase isoenzymes from Torpedo californica electric organ, skeletal muscle, and brain were purified and characterized. Torpedo electric organ and skeletal muscle creatine kinase have identical apparent Mr, electrophoretic mobility, and cyanogen bromide fragments. The electrophoretic mobility of the Torpedo creatine kinase was anodal as compared to mammalian MM creatine kinase. No creatine kinase isoenzyme with an electrophoretic mobility similar to mammalian BB creatine kinase was seen in any of the Torpedo tissues examined. Hybridization studies demonstrate the Torpedo electric organ creatine kinase to be composed of identical subunits and capable of producing an enzymatically active heterodimer when combined with canine BB creatine kinase. Creatine kinase from sucrose gradient-purified Torpedo electric organ acetylcholine receptor-rich membranes has an electrophoretic mobility identical with the cytoplasmic isoenzyme and an apparent Mr identical with mammalian MM creatine kinase. Western blot analysis showed Torpedo electric organ skeletal muscle creatine kinase and acetylcholine receptor-enriched membrane creatine kinase reacted with antiserum specific for canine MM creatine kinase. NH2-terminal amino acid sequence determinations show considerable sequence homology between human MM, Torpedo electric organ, chicken MM, and porcine MM creatine kinase. The acetylcholine receptor-associated creatine kinase is, therefore, identical with the cytoplasmic form from the electric organ and is composed of M-subunits.

Molecular mechanism for the production of multiple forms of MM creatine kinase.

Incubation of human, canine or rabbit MM creatine kinase with carboxypeptidase-N or B resulted in the production of 2 additional enzyme forms with increased anodal migration on polyacrylamide gels. The C-terminal amino acid of tissue MM creatine kinase from all 3 species was shown to be lysine, a specific substrate for carboxypeptidase-N and B.

Carboxypeptidase-catalyzed hydrolysis of C-terminal lysine: mechanism for in vivo production of multiple forms of creatine kinase in plasma.

Human myocardial creatine kinase isoenzyme MM is present as a single form in tissue, but upon its release into plasma two additional forms, with faster anodal migration, are apparent on polyacrylamide electrophoresis. We designate the three forms as MM3, MM2, and MM1 in increasing order of anodal mobility. When tissue creatine kinase isoenzyme MM (MM3) is incubated with either carboxypeptidase N or carboxypeptidase B it is converted into the two additional forms, MM2 and MM1. The carboxy terminal amino acid of human, canine, and rabbit tissue MM3 was determined to be lysine, a specific substrate for carboxypeptidases N and B. Evidently the mechanism for the production of multiple forms of creatine MM in human plasma is the hydrolysis of a positively charged C-terminal lysine residue from one M subunit (MM2), followed by hydrolysis of the C-terminal lysine from the other subunit (MM1).

Serological analysis of 17 baculoviruses from subgroups A and B using protein blot immunoassay.

We used the protein blot radioimmunoassay technique (Smith and Summers, 1981) to evaluate the immunoreactivity of antisera made against SDS-disrupted baculovirus structural proteins. Eight antisera (six for NPVs and two for GVs) were tested against eleven nuclear polyhedrosis (NPV) and six granulosis viruses (GV). Homologous reactions revealed that SDS-disrupted baculovirus antigens elicited antibodies which reacted with several of the virus proteins. Heterologous reactions of each viral antiserum were determined with a total of 17 baculoviruses. Using these data and that of Smith and Summers (1981), we were able to detect a number of shared antigenic determinants which revealed some specific serological relationships among some of the 17 viruses tested. The MNPVs showing distinct serological relatedness included those of Autographa californica, Rachiplusia ou, Anticarsia gemmatalis, Choristoneura fumiferana, and Orgyia pseudotsugata. Heliothis armiger MNPV and Heliothis zea SNPV shared cross-reacting antigenic determinants as did Trichoplusia ni SNPV, Pseudoplusia includens SNPV, and Heliothis zea SNPV. The GVs. of T. ni, H. armiger, and Spodoptera frugiperda were also serologically related. Also reactions were detected which indicated the presence of common antigenic determinants among different baculovirus subgroups in addition to those originally detected in our earlier study (Smith and Summers, 1981). Because of the large number of structural proteins in baculoviruses and the lack of information concerning their structural or functional roles in the virus group specific antigenic determinants could not be identified. However, protein blot RIA allows specific identification of baculoviruses and the number and molecular weight of homologous and heterologous proteins sharing antigenic determinants.

Investigation of genetic heterogeneity in wild isolates of Spodoptera frugiperda nuclear polyhedrosis virus by restriction endonuclease analysis of plaque-purified variants.

Spodoptera frugiperda nuclear polyhedrosis virus (SfMNPV was obtained from four different sources and each isolate was cultured in IPLB-SF21 cells. Viral DNA from each isolate was analyzed with EcoR1, Xho1, and BamH1 restriction endonucleases. All four isolates could be distinguished on the basis of minor differences in EcoR1 patterns. Submolar DNA restriction fragments were seen in all isolates indicating that each isolate consisted of a mixture of different genotypes. To investigate this, two of the isolates were plaque purified. Of the total of 13 clones isolated, the DNA from 5 of these had distinctly different EcoR1 restriction endonuclease patterns. The five cloned variants were further analyzed with Xho1, BamH1, and Sac1. Three of the five clones had fragments which comigrated with submolar fragments seen in the wild isolates. Thus, the submolar fragments observed in digests of the DNA from two wild isolates of SfMNPV were due to a mixture of genetic variants.

Physical Maps of Autographa californica and Rachiplusia ou Nuclear Polyhedrosis Virus Recombinants.

TN-368 cells were infected simultaneously with the closely related Autographa california (AcMNPV) and Rachiplusia ou (RoMNPV) nuclear polyhedrosis viruses. Progeny viral isolates were plaque purified, and their DNAs were analyzed with restriction endonucleases. Of 100 randomly cloned plaques, 7 were AcMNPV and RoMNPV recombinants, 5 were RoMNPV, and 88 were AcMNPV. The recombinants contained DNA sequences derived from both parental genomes. By comparing the restriction cleavage patterns of parental and recombinant DNAs, the crossover sites were mapped. A single double crossover was detected in each of the seven recombinant genomes. In addition, six of the seven recombinants revealed a crossover site mapping between 78 and 89% of the genome. The structural polypeptides of the seven recombinants and two parental viruses were analyzed by polyacrylamide gel electrophoresis, and their polyhedrins were identified by tryptic peptide mapping. An analysis of the segregation of three enveloped nucleocapsid proteins and of the polyhedrins among the recombinants located the DNA sequences coding for AcMNPV structural polypeptides with molecular weights of 37,000 (a capsid polypeptide), 56,000, and 90,000 and the RoMNPV structural polypeptides with molecular weights of 36,000 (a capsid polypeptide), 56,000, and 91,000. The AcMNPV and RoMNPV polypeptides of molecular weights 37,000 and 36,000, respectively, mapped within 78 to 89% or 1 to 29%, the polypeptides of molecular weights 55,000 and 56,000 mapped within 78 to 29%, and the polypeptides of molecular weights 90,000 and 91,000 mapped within 19 to 56% of the genome. The region of the parental DNAs that codes for polyhedrin was located within 70 to 89% of the genome.