Development of a novel recombinant LHRH fusion protein for therapy of androgen and estrogen dependent cancers.

LHRH based vaccines are promising candidates for therapy of androgen and estrogen dependent cancers. We report in this communication development of a novel recombinant protein vaccine candidate against LHRH. A synthetic gene was designed in which the codon sequence in the LHRH decapeptide was modified by substituting the codon for 6-glycine with that of l-leucine. Further the LHRH(6leu) gene was linked to heat-labile enterotoxin of E. coli (LTB) as carrier. This LHRH(6leu)-LTB gene was cloned into a prokaryotic expression vector under the control of inducible and strong bacteriophage T7 promoter to over-express LHRH(leu) fused to LTB as recombinant protein in E. coli. Recombinant LHRH(leu)-LTB protein of ∼14 kDa size, was purified from inclusion bodies using in-situ refolding on the column and Ni-NTA based immobilized affinity chromatography. Western blot confirmed the immunoreactivity of purified LHRH(leu)-LTB fusion protein with anti-LHRH monoclonal antibody. The vaccine protein was further characterized by mass spectroscopy, circular dichroism and fluorescence spectroscopy. This communication reports a recombinant LHRH fusion protein with potential for blocking of sex hormones production for eventual therapy of sex hormones dependent neoplasms.

Immunoreactivity with native zona pellucida of antibodies against a 19 amino acid synthetic peptide corresponding to human ZP3.

Immunization of female Syrian hamsters with a 19mer-synthetic peptide corresponding to amino acid sequence 323-341 of human ZP3 and coupled to diphtheria toxoid (DT) led to generation of antibodies against both the peptide as well as DT. Antisera showed positive reaction in ELISA with solubilized isolated zona pellucida (SIZP) from human oocytes. In an indirect immunofluorescence assay, the anti-peptide antibodies bound to zona pellucida of human and bonnet monkey but failed to recognize that of mouse, rabbit, hamster and dog. These studies will help in designing a synthetic peptide based ZP immunocontraceptive vaccine for human application.

A vaccine that prevents pregnancy in women.

We report here results of clinical trials on a birth control vaccine, consisting of a heterospecies dimer of the beta subunit of human chorionic gonadotropin (hCG) associated noncovalently with the alpha subunit of ovine luteinizing hormone and conjugated to tetanus and diphtheria toxoids as carriers, that induces antibodies of high avidity (K(a) approximately 10(10) M-1) against hCG. Fertile women exposed to conception over 1224 cycles recorded only one pregnancy at antibody titers of > 50 ng/ml (hCG bioneutralization capacity). The antibody response declines with time; fertility was regained when titers fell to < 35 ng/ml. This study presents evidence of the feasibility of a vaccine for control of human fertility.

Production and characterization of new murine monoclonal antibodies reactive to Mycobacterium tuberculosis.

Murine monoclonal antibodies were raised against Mycobacterium tuberculosis (H37Rv) employing conventional hybridoma procedure. The binding characteristics of the four selected monoclonal antibodies reactive to M. tuberculosis were assessed by enzyme-linked immunosorbent assay (ELISA) using sonic extracts. The immunofluorescence test (IFT) was done using intact M. tuberculosis, 16 other mycobacterial species, and 10 bacteria of other genera. Monoclonal antibody A30 reacted strongly to M. tuberculosis both in ELISA and IFT. Monoclonal antibody A25 showed restricted cross-reactivity with mycobacterial species whereas two other monoclonal antibodies B6 and H2 appeared broadly cross-reactive. In Western blot analysis, A30 reacted with the 30-kD protein antigen, A25 with the 18- and 28-kD protein bands, H2 with the 18-kD antigen alone, and B6 with three bands of 17-19, 22, and 28 kD of M. tuberculosis H37Rv. A30 exhibited high reactivity with virulent M. tuberculosis H37Rv and a clinical South Indian Strain and minimal reactivity with avirulent M. tuberculosis H37Ra strain.

Effects of a tryptophan supplemented diet and U.V. radiation on the rat lens.

Rats maintained on a tryptophan supplemented diet and exposed to U.V. radiation showed decreased concentration of ascorbic acid in serum. In the lens, a small increase in the urea-mercaptoethanol soluble fraction was observed suggesting some oxidation of P-SH groups. The decreased concentrations of lens glutathione and ascorbic acid were accompanied with increased concentration of malondialdehyde suggesting increased oxidative stress. The activities of glutathione peroxidase decreased by about 40%. Though the activity of glutathione reductase decreased by about 58%, addition of FAD in the enzyme assay system showed restoration of lost activity. Additive effect of raised serum tryptophan concentration and ultraviolet radiation in causing damage to the eye lens is suggested.