Synthetic peptide-based vaccine and diagnostic system for effective control of FMD.

We have designed synthetic peptides corresponding to two different regions of the genome of foot-and-mouth disease virus (FMDV) that are effective as (a) a vaccine or (b) a diagnostic reagent which differentiates convalescent from vaccinated animals, respectively. The peptide vaccine is based on a sequence from the prominent G-H loop of VP1, one of the four capsid proteins. The sequence was optimized by the inclusion of a cyclic constraint and adjoining sequences, and broader immunogenicity was obtained by the incorporation of consensus residues at hypervariable positions. The peptide also included a promiscuous T-helper epitope for effective immunogenicity in outbred populations of large animals.The diagnostic reagent, a peptide based on non-structural (NS) protein 3B, is used in immuno-assays for the detection of antibodies. Antibodies to this NS protein are present in the sera of infected animals but not in the sera of vaccinated animals. The VP1 peptide can be used in complementary immuno-assays for confirmation of NS test results and to monitor for vaccination. This system for differential diagnosis is important to establish the disease-free status of a country.

Postexposure immunoprophylaxis of primary isolates by an antibody to HIV receptor complex.

mAb B4 is a monoclonal antibody directed against HIV receptor complex. The antibody had broad neutralizing activity against HIV and provided postexposure prophylaxis to hu-peripheral blood leukocyte (PBL)-severe combined immunodeficient mice and chimpanzees. B4 recognized a complex receptor site for HIV on the T cell surface that includes CD4 and also may be influenced by interaction with HIV coreceptors. mAb B4 preferentially neutralized primary HIV-1 isolates compared with T cell line-adapted strains, including syncytium-inducing and non-syncytium-inducing phenotypes, representatives from HIV-1 subtypes A-G, as well as HIV-2, simian immunodeficiency virus, and chimeric simian/human immunodeficiency virus (SHIV). Neutralization was demonstrated in both pre- and postinfection models. The administration of mAb B4 after infectious challenge totally interrupted the infection of hu-PBL-severe combined immunodeficient mice by PBL-grown HIV-1 and the infection of chimpanzees by chimp-adapted HIV-1. This mode of protection suggested that the anti-HIV receptor antibody is efficacious for prophylaxis after exposure to HIV and for prevention of maternal transmission and may be an effective antiretroviral agent for treatment.

Differentiation of convalescent animals from those vaccinated against foot-and-mouth disease by a peptide ELISA.

We have identified continuous antigenic determinants within the amino acid sequences of the conserved nonstructural region containing proteins 2C and 3ABC of foot-and-mouth disease virus which can distinguish between the sera from vaccinated and infected animals. An ELISA based on a 3B peptide gave a positive reaction with sera from cattle, pigs, sheep and guinea pigs infected with all seven serotypes of the virus, but not with sera from vaccinated animals. In experiments with cattle and pigs to determine the duration of the antibody response, positive reactions were obtained as late as one year after infection. The advantages of using peptides from the nonstructural viral proteins instead of recombinant proteins for differentiating vaccinees from infected animals include their exquisite specificity, nonreactivity with antibodies against host cell-derived proteins (e.g. E. coli and insect cell proteins), and their ease of preparation.

Active immunization against LHRH alone or combined with LHRH-analogue treatment impedes growth of androgen-dependent prostatic carcinoma.

PROBLEM: To determine whether active immunization against LHRH can serve as treatment for androgen-dependent prostatic carcinoma. METHOD: Male rats of Copenhagen X Fisher strain, implanted with Dunning R-3327 prostatic carcinoma cells were either immunized against LHRH, treated with LHRH-antagonist, or received a combined treatment of active immunization against LHRH and LHRH-antagonist. RESULTS: Testicular histology was consistent with infertility in all treatment groups. The rate of tumor growth was inhibited by all three treatment regimens. Tumor size increased by 3.8 +/- 1.4 cm2 in the LHRH-antagonist group, 3.2 +/- 1.1 cm2 in the immunized group, and 1.0 +/- 0.4 cm2 in the combined treatment group, as compared to 8.2 +/- 2.6 cm2 in non-treated control group. CONCLUSION: LHRH-antagonist administration combined with immunization against LHRH appeared to exert a synergistic effect. This may be due to the blockade of prostatic LHRH-like receptors by the antagonist, while androgen depletion was rapidly achieved by LHRH-antagonist, and maintained by continued gonadotropin suppression caused by active immunization against LHRH once antagonist treatment had been discontinued.

Development of an antifertility vaccine for pets based on active immunization against luteinizing hormone-releasing hormone.

Male dogs and cats were immunized against LHRH in order to evaluate the feasibility of an immunological approach to pet contraception. In the first study, dogs were immunized with 100, 500, or 2500 micrograms of LHRH conjugated to tetanus toxoid. A significant decline in serum testosterone (T) levels was observed in all immunized dogs, reaching castration levels in some animals by Week 4 and remaining suppressed in all the immunized dogs through the course of the study. Testicular histology suggested arrest of spermatogenesis (infertility). The effects of "immunological castration" were reversible (study 2): steroidogenesis suppressed by "immunological castration" was restored as antibody titers declined. Effective antibodies were rapidly reinduced in dogs by a single injection of LHRH1-TT. In contrast, the level of antibodies induced in male cats (study 3) was not sufficient for "immunological castration." The conclusion was that active immunization against LHRH could provide a cost-effective, nonsurgical, reversible means to control the fertility of companion animals.

Long-term high-titer neutralizing activity induced by octameric synthetic HIV-1 antigen.

A titer for homologous viral neutralization activity (greater than 1:19,683) was observed after a 3.5-year immunization period with an octameric, branching peptide representing the principal neutralizing determinant (PND) of the human immunodeficiency virus-1IIIB envelope protein. Booster immunizations elicited persistent and potent antibodies in guinea pigs, exceeding responses produced by a conventional bovine serum albumin conjugate by 100-fold. Peptide length, central presentation of a conserved sequence, and inclusion of an upstream sequence contributed to immunogenicity. Titers (greater than 1:1,000) of heterotypic neutralizing antibodies also developed. Octameric PND peptides are a promising approach for an acquired immunodeficiency syndrome (AIDS) vaccine.

Primary structure of an oligomeric antigen of Treponema pallidum.

The properties and sequence of an oligomeric antigen of Treponema pallidum are presented. Antigen C1-5 assembles into oligomers of 140,000 and greater. The nucleotide sequence predicts an open reading frame for a protein monomer of 19,400, confirmed by amino-terminal sequencing of the recombinant antigen.

Structural relatedness between mosquitocidal endotoxins of Bacillus thuringiensis subsp. israelensis.

A mosquitocidal toxin gene, cloned from Bacillus thuringiensis subsp. israelensis, was introduced into mutant crystal-negative B. thuringiensis subsp. israelensis cells. Partial toxicity to mosquitos was restored. The 58-kilodalton cloned gene product is a minor protein component of B. thuringiensis subsp. israelensis crystals and is structurally related to a major, 135-kilodalton crystal toxin.

Structural similarity between the lepidoptera- and diptera-specific insecticidal endotoxin genes of Bacillus thuringiensis subsp. "kurstaki" and "israelensis".

A gene from Bacillus thuringiensis subsp. "israelensis" was cloned from the large plasmids of this subspecies and was shown to code for a mosquitocidal polypeptide. The gene could be expressed in either Escherichia coli, Bacillus subtilis, or B. thuringiensis subsp. "israelensis" to produce the larvicidal activity. Similarly, a Lepidoptera-specific toxin gene from B. thuringiensis subsp. "kurstaki" was also cloned and expressed in E. coli and B. subtilis. Both cloned genes were sequenced and subjected to computer analysis. A long open translational reading frame coded for the B. thuringiensis subsp. "kurstaki" gene product. However, the B. thuringiensis subsp. "israelensis" clone was composed of two adjacent open reading frames oriented as if they were in a transcriptional operon. The products of the cloned genes retained their specificity for either Lepidoptera or Diptera. The control regions immediately preceding the toxin genes of both B. thuringiensis subspecies showed considerable DNA homology, most likely because both toxins are expressed only during sporulation. In addition, the deduced amino acid sequences from the two contiguous B. thuringiensis subsp. "israelensis" genes bore a striking resemblance to the deduced amino acid sequence from the single larger B. thuringiensis subsp. "kurstaki" gene, as if these two arrangements were evolutionarily related.

Native surface association of a recombinant 38-kilodalton Treponema pallidum antigen isolated from the Escherichia coli outer membrane.

A recombinant plasmid designated pAW305, containing a 6-kilobase insert of Treponema pallidum DNA, directed the expression of a 38-kilodalton (kDa) treponemal antigen in Escherichia coli. The 38-kDa antigen copurified with the outer membrane fraction of the E. coli cell envelope after treatment with nonionic detergents or sucrose density gradient centrifugation. Rabbits immunized with the recombinant 38-kDa antigen developed antibodies which reacted specifically with a 38-kDa T. pallidum antigen on immunoblots, and 38-kDa antisera specifically immobilized T. pallidum in a complement-dependent manner in the T. pallidum immobilization test. Antisera to the 38-kDa recombinant antigen were also used to demonstrate its native surface association on T. pallidum by immunoelectron microscopy.

Purification and characterization of a cloned protease-resistant Treponema pallidum-specific antigen.

A cloned Treponema pallidum antigen, designated 4D, was purified from Escherichia coli predominantly as a 190-kilodalton (kd) polypeptide, although higher oligomeric forms exist. Extensive proteolysis of 4D created a limit digestion product of 90 kd which retained antigenicity with sera from patients with primary, secondary, early latent, late latent, and tertiary syphilis. A molecule indistinguishable from 90-kd 4D in size, isoelectric point, and antigenicity was isolated from T. pallidum after proteolysis. The 190- and 90-kd forms of 4D were stable at 68 degrees C but converted to 19- and 14-kd species, respectively, after boiling in sodium dodecyl sulfate. The low-molecular-weight species did not react with syphilitic sera. Rabbits immunized with the purified 4D antigen developed antibodies which immobilized virulent T. pallidum in a complement-dependent assay system, suggesting that the antigen has a native surface location.

Expression of Treponema pallidum antigens in Escherichia coli.

Treponema pallidum DNA was cloned in a bacteriophage. Clones were screened for expression of Treponema pallidum antigens by an in situ radioimmunoassay on nitrocellulose, with the use of subsequent reactions with syphilitic serum and radioiodinated Staphylococcus aureus protein A. One clone, which gave a strong signal, codes for at least seven antigens that react specifically with human antibodies to Treponema pallidum.

Misalignment of V and J gene segments resulting in a nonfunctional immunoglobulin gene.

The myeloma variant NS-1n has lost the functional immunoglobulin kappa gene which is present in its parent, myeloma MOPC-21. The variant retains a nonfunctional rearranged gene, M.21N, which undergoes RNA transcription and processing to yield a mature size kmRNA. This kRNA, however, is not translated into kappa polypeptide chains. The nonfunctional gene was cloned into Charon 4A to determine the basis for its inactivity. Nucleotide sequence analysis of a DNA fragment overlapping the V-J recombination site in the M.21N gene indicated that a misalignment had taken place during somatic recombination. This misalignment results in a deletion of four nucleotides at the 3' end of the V gene and, thus, a translational reading frame shift. In other respects the M.21n V gene, which corresponds to a different VK subgroup than the functional gene of MOPC-21, appears normal.

Rearranged and germline immunoglobulin kappa genes: different states of DNase I sensitivity of constant kappa genes in immunocompetent and nonimmune cells.

The rearrangement of a variable (V) and a constant (C) gene appears to be a necessary prerequisite for immunoglobulin gene expression. Multiple different rearranged kappa genes were found in several mouse myelomas, although these cells produce only one type of kappa chain [Wilson, R., Miller, J., & Storb, U. (1979) Biochemistry 18, 5013--5021]. It is therefore of interest to understand how only one allele within a lymphoid cell becomes expressed, while the other allele remains nonfunctional ("allelic exclusion"). We have studied the chromatin conformation of kappa genes by making use of the preferential digestion of potentially active genes by DNase I described, for example, for globin genes [Weintraub, H., & Groudine, M. (1976) Science (Washington, D.C.) 193, 848--856]. The DNase I sensitivity of kappa genes in myeloma tumors, in a B cell lymphoma, and in liver was determined by hybridization with DNA on Southern blots. It was found that rearranged C kappa genes are DNase I sensitive in myelomas in which several kappa genes are rearranged, regardless of whether the rearranged genes code for the kappa chains synthesized by the cell. Furthermore, the C kappa gene in germline configuration is also DNase I sensitive in a B cell lymphoma; i.e., it is in the same chromatin state as the rearranged C kappa gene which probably codes for the kappa chains produced by the cell. The altered chromatin state appears to be localized: V kappa genes in germline context are not DNase I sensitive in myeloma or B lymphoma cells while C kappa genes present in a kappa gene cluster on the same chromosomes are sensitive. When rearranged, however, the V kappa genes are as sensitive to DNase I as are rearranged C kappa genes. V lambda and C lambda genes are not DNase I sensitive in kappa myelomas. Thus, commitment to kappa gene expression is apparently correlated with a chromatin conformation which confers increased DNase I sensitivity to the DNA in the vicinity of all C kappa genes in the cell. "Allelic exclusion" does not operate on the level of chromatin conformation which can be detected by altered DNase I sensitivity.

Comparison of different rearranged immunoglobulin kappa genes of a myeloma by electronmicroscopy and restriction mapping of cloned DNA: implications for "allelic exclusion".

We have studied the organization and function of different rearranged kappa genes in a myeloma, MOPC-21. Two kappa genes were cloned into Charon 4A and compared with each other and with a cloned germline CK gene by restriction mapping and electron microscopy. One MOPC-21 clone corresponds to the gene coding for the MOPC-21 kappa chain polypeptide; it has the V21 gene joined with the CK gene at the J2 sequence. The other MOPC-21 clone corresponds to a nonfunctional rearranged MOPC-21 kappa gene, except for a lkb deletion, 3' of J4. A similar deletion is also found in a "new" kappa gene present in NS-1, a cellular subclone of MOPC-21. The clone of the "nonfunctional" kappa gene has a V gene which is distinct from V21 which is joined to CK in the vicinity of J2. The undeleted form of this gene codes for a KRNA having the size of mature KmRNA which, however, is not translated into kappa chains. Thus the defect of the "nonfunctional" gene manifests itself at a late step of gene expression. The basis for "allelic exclusion" of antibody genes may simply be the complexity of the processes between genes and gene products, resulting in the expression of only one gene.

Immunoglobulin genes in DNA restriction fragments.

We have investigated the organization of immunoglobulin genes in mice. High molecular weight DNA from myelomas and Krebs ascites cells was cleaved with EcoRI restriction endonuclease and fractionated using preparative agarose gel electrophoresis. Each fraction was then hybridized to an immunoglobulin mRNA or a cDNA transcribed from the mRNA. In two series of experiments, one with a kappa chain probe (MOPC 41 mRNA), the other with a lambda chain probe (SAPC 178 mRNA), we analyzed a variety of myeloma DNAs and Krebs DNA. In contrast to previously reported findings (Tonegawa, S., et al. (1976) Cold Spring Harbor Symp. Quant. Biol. 41, 877), we did not observe any unique restriction map pattern in the DNA from cells which exress a given immunoglobulin gene. We also found that restriction fragments containing c region genes do not appear to transpose, while DNA sequences corresponding to other portions of the kappa and lambda mRNAs do in some cases.

Turnover-synthesis of chloroplast DNA in developing chloroplasts.

The mechanism for the turnover-synthesis of chloroplast DNA in the absence of net synthesis during the chloroplast maturation in Euglena gracilis was determined. DNA synthesis was measured by incorporation of32Pi into chloroplast DNA. The density label, 15N, was incorporated to examine the mechanism of turnover-synthesis. The newly synthesized segments represent a replacement of segments in the DNA containing 1.5 X 10(3) to 6.1 X 10(3) nucleotides. Twenty-three fragments of chloroplast DNA, generated by digestion with the restriction endonuclease EcoRI, became labeled with 32Pi. Turnover-synthesis, therefore, replaces segments throughout the molecule of chloroplast DNA.

Light-stimulated synthesis of chloroplast DNA.

Light-stimulated chloroplast DNA synthesis was studied during chloroplast development in the absence of cell division and nuclear DNA synthesis. Incorporation of 32Pi was stimulated 10-15 fold, however, the ratio of chloroplast DNA to nuclear DNA remained constant. Isotope dilution experiments suggested that stimulated labeling of chloroplast DNA represented more efficient utilization of exogenously supplied Pi rather than stimulated turnover of chloroplast DNA. The low level of DNA synthesis and chloroplast development were resistant to nalidixic acid which inhibits semiconservative replication of chloroplast DNA.