Ubiquitin-fused and/or multiple early genes from cottontail rabbit papillomavirus as DNA vaccines

Human papillomavirus (HPV) vaccines have the potential to prevent cervical cancer by preventing HPV infection or treating premalignant disease. We previously showed that DNA vaccination with the cottontail rabbit papillomavirus (CRPV) E6 gene induced partial protection against CRPV challenge and that the vaccine's effects were greatly enhanced by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF). In the present study, two additional strategies for augmenting the clinical efficacy of CRPV E6 vaccination were evaluated. The first was to fuse a ubiquitin monomer to the CRPV E6 protein to enhance antigen processing and presentation through the major histocompatibility complex class I pathway. Rabbits vaccinated with the wild-type E6 gene plus GM-CSF or with the ubiquitin-fused E6 gene formed significantly fewer papillomas than the controls. The papillomas also required a longer time to appear and grew more slowly. Finally, a significant proportion of the papillomas subsequently regressed. The ubiquitin-fused E6 vaccine was significantly more effective than the wild-type E6 vaccine plus GM-CSF priming. The second strategy was to vaccinate with multiple CRPV early genes to increase the breadth of the CRPV-specific response. DNA vaccines encoding the wild-type CRPV E1-E2, E6, or E7 protein were tested alone and in all possible combinations. All vaccines and combinations suppressed papilloma formation, slowed papilloma growth, and stimulated subsequent papilloma regression. Finally, the two strategies were merged and a combination DNA vaccine containing ubiquitin-fused versions of the CRPV E1, E2, and E7 genes was tested. This last vaccine prevented papilloma formation at all challenge sites in all rabbits, demonstrating complete protection.     

Antibacterial property of the antipsychotic agent prochlorperazine, and its synergism with methdilazine

The antipsychotic drug prochlorperazine was screened in vitro for possible antimicrobial property against 157 strains of bacteria, belonging to gram positive and gram negative genera. The minimum inhibitory concentration (MIC) of prochlorperazine was determined by agar dilution method, which ranged from 25 to 200 microg/ml with respect to most of the strains. Based on such findings, a further study was undertaken to determine whether the efficacy of this drug could be enhanced in the presence of an antihistaminic agent methdilazine, reported to have remarkable antimicrobial action. Four bacterial strains, sensitive to prochlorperazine as well as to three antibacterial chemotherapeutics, viz., methdilazine, fluphenazine and thioridazine were chosen. Disc diffusion tests with prochlorperazine and methdilazine revealed marked synergism between the combination, compared to their individual effects. The synergism was found to be statistically significant (p<0.01). To assess the degree of synergism, the checkerboard analysis was performed. The FIC index of this combination turned out to be 0.37, which confirmed synergism. Therefore, this synergistic drug combination might open a new therapeutic approach to combat drug-resistance in bacterial infections.     

SAM domains can utilize similar surfaces for the formation of polymers and closed oligomers

The mitogen-activated protein kinase (MAPK) Byr2 and its activator Ste4 are involved in the mating pheromone response pathway of Schizosaccharomyces pombe and interact via their SAM domains. SAM domains can self-associate to form higher-order structures, including dimers, polymers and closed oligomers. Ste4-SAM is adjacent to a trimeric leucine zipper domain and we have shown previously that the two domains together (Ste4-LZ-SAM) bind to a monomeric Byr2-SAM with high affinity (Kd approximately 20 nM), forming a 3:1 complex. Here, we map the surfaces of Byr2-SAM and Ste4-SAM that is involved the interaction. A set of 38 mutants of Byr2-SAM and 33 mutants of Ste4-SAM were prepared, covering most of the protein surfaces. These mutants were purified and screened for binding, yielding a map of residues that are required for binding and a complementary map of residues that are not required. We find that the interface maps to regions of the SAM domains that are known to be important for the formation of SAM polymers. These results indicate that SAM domains can create a variety of oligomeric architectures utilizing common binding surfaces.     

Phospholipase B activity enhances adhesion of Cryptococcus neoformans to a human lung epithelial cell line

Secreted phospholipase B (PLB1), which contains three enzyme activities in the one protein, is necessary for the initiation of pulmonary infection by Cryptococcus neoformans and for dissemination from the lung via the lymphatics and blood. Adhesion to lung epithelium is the first step in this process, therefore we investigated the role of PLB1 in adhesion to a human lung epithelial cell line, A549, using C. neoformans var. grubii wild-type strain H99, a PLB1 deletion mutant (deltaplb1), and a reconstituted strain (deltaplb1rec). Adhesion of H99 and deltaplb1rec was approximately 69% greater than deltaplb1 at 4 h. Adhesion of deltaplb1 significantly increased after killing by chemicals or heat, and Fourier-transformed analysis by FTIR spectroscopy indicated this was due to changes in capsular and/or cell wall polysaccharides and proteins. Inhibition by specific PLB1 antibodies, or inhibitors of phospholipase B (PLB), but not lysophospholipase (LPL) or lysophospholipase transacylase (LPTA) activities decreased the adhesion of H99 and deltaplb1rec by 33-58%. Growth under conditions of osmotic stress and high glucose concentration increased both PLB secretion and subsequent cryptococcal adhesion. Dose-dependent increases (to 67%) in adhesion of live deltaplb1 were observed in the presence of 0.1-2 mM palmitic acid. We conclude that PLB1 plays a role in the binding of C. neoformans to host lung epithelial cells, possibly due to production of fatty acids from plasma membranes and/or surfactant by PLB activity.     

Sequential evolution and escape from neutralization of simian immunodeficiency virus SIVsmE660 clones in rhesus macaques

Simian immunodeficiency virus (SIV) infection of rhesus macaques has become an important surrogate model for evaluating HIV vaccine strategies. The extreme resistance to neutralizing antibody (NAb) of many commonly used strains, such as SIVmac251/239 and SIVsmE543-3, limits their potential relevance for evaluating the role of NAb in vaccine protection. In contrast, SIVsmE660 is an uncloned virus that appears to be more sensitive to neutralizing antibody. To evaluate the role of NAb in this model, we generated full-length neutralization-sensitive molecular clones of SIVsmE660 and evaluated two of these by intravenous inoculation of rhesus macaques. All animals became infected and maintained persistent viremia that was accompanied by a decline in memory CD4(+) T cells in blood and bronchoalveolar lavage fluid. High titers of autologous NAb developed by 4 weeks postinoculation but were not associated with control of viremia, and neutralization escape variants were detected concurrently with the generation of NAb. Neutralization escape was associated with substitutions and insertion/deletion polymorphisms in the V1 and V4 domains of envelope. Analysis of representative variants revealed that escape variants also induced NAbs within a few weeks of their appearance in plasma, in a pattern that is reminiscent of the escape of human immunodeficiency virus type 1 (HIV-1) isolates in humans. Although early variants maintained a neutralization-sensitive phenotype, viruses obtained later in infection were significantly less sensitive to neutralization than the parental viruses. These results indicate that NAbs exert selective pressure that drives the evolution of the SIV envelope and that this model will be useful for evaluating the role of NAb in vaccine-mediated protection.     

Solid state fluorescence of lyophilized proteins

Fluorescence spectroscopy has been used to measure changes in the tertiary structure of proteins in the solution state. The sensitivity of fluorescence to the protein tryptophan environment has made it a useful tool for studying protein conformation and stability. Using fluorescence spectroscopy to probe structural alterations in lyophilized proteins has been limited due to technical challenges and overwhelming background light scattering. We have investigated the possibility of analyzing lyophilized proteins using the Cary-Eclipse spectrofluorometer by monitoring the fluorescence of the protein therapeutic after subjecting the lyophilized cake to heat-induced accelerated degradation. We have been able to obtain reproducible fluorescence spectra, detecting possible structural changes under these conditions. Fluorescence and circular dichroism spectroscopic analyses of the reconstituted proteins indicated that changes in fluorescence intensities observed in the solid state could be correlated to that in solution and to possible tertiary structural changes. Size exclusion chromatography analysis of protein Y subject to accelerated degradation showed a correlation between decreasing fluorescence intensity and increasing protein Y tetramer in solution, consistent with long-term stability. This suggests that solid state, intrinsic protein fluorescence measurements using the Cary-Eclipse holder may be feasible for long-term stability studies and formulation development.     

Mutation of the ptsG Gene Results in Increased Production of Succinate in Fermentation of Glucose by Escherichia coli

Escherichia coli NZN111 is blocked in the ability to grow fermentatively on glucose but gave rise spontaneously to a mutant that had this ability. The mutant carries out a balanced fermentation of glucose to give approximately 1 mol of succinate, 0.5 mol of acetate, and 0.5 mol of ethanol per mol of glucose. The causative mutation was mapped to the ptsG gene, which encodes the membrane-bound, glucose-specific permease of the phosphotransferase system, protein EIICB^glc. Replacement of the chromosomal ptsG gene with an insertionally inactivated form also restored growth on glucose and resulted in the same distribution of fermentation products. The physiological characteristics of the spontaneous and null mutants were consistent with loss of function of the ptsG gene product; the mutants possessed greatly reduced glucose phosphotransferase activity and lacked normal glucose repression. Introduction of the null mutant into strains not blocked in the ability to ferment glucose also increased succinate production in those strains. This phenomenon was widespread, occurring in different lineages of E. coli, including E. coli B.