Marinko Sremac

Antigen presentation using novel particulate organelles from halophilic archaea

A presentation vehicle was developed based on particulate gas vesicles produced by halophilic archaea. Gas vesicle epitope displays were prepared using standard coupling methods or recombinant DNA technology. When presented in the context of gas vesicle preparations, either the hapten, TNP, or a model six amino acid recombinant insert in the outer gas vesicle protein, GvpC was rendered immunogenic. Assays to quantify humoral responses indicated that each preparation elicited strong antibody responses in the absence of exogenous adjuvant. Thus, each preparation elicited a humoral response when injected into mice and this response was long lived and exhibited immunologic memory. Recombinant gas vesicle preparations therefore constitute a new, self-adjuvanting carrier/display vehicle for presentation of an array of peptidyl epitopes.

Survey for Winter Moth (Lepidoptera: Geometridae) in Northeastern North America with Pheromone-Baited Traps and Hybridization with the Native Bruce Spanworm (Lepidoptera: Geometridae)

ABSTRACT We used pheromone-baited traps to survey the distribution of winter moth, Operophtera brumata (L.) (Lepidoptera: Geometridae), a new invasive defoliator from Europe in eastern New England. The traps also attracted Bruce spanworm, Operophtera bruceata (Hulst) (Lepidoptera: Geometridae), native to North America. We distinguished between the two species by examining male genitalia and sequencing the mitochondrial cytochrome oxidase subunit 1 (COI) gene, the DNA barcoding region. In 2005, we recovered winter moths at sites stretching from eastern Long Island, southeastern Connecticut, all of Rhode Island, eastern Massachusetts, coastal New Hampshire, and southern coastal Maine. At sites further west and north we captured only Bruce spanworm. In 2006, we confirmed that both winter moth and Bruce spanworm are present in Nova Scotia and in coastal Maine, but only Bruce spanworm was recovered in coastal New Brunswick, Canada; Pennsylvania; Vermont; or Quebec City, Canada. In 2007, we collected Bruce spanworm, but no winter moths, in New Brunswick and the interior areas of Maine, New Hampshire, and New York. Winter moth and Brace spanworm differed in the COI sequence by 7.45% of their nucleotides. The prevalence of intermediate genitalia in the zone of overlap suggested that hybridization between the two species may be occurring. To confirm the presence of hybrids, we sequenced the nuclear gene, glucose-6phosphate dehydrogenase (G6PD). We identified six nucleotides that routinely distinguished winter moth and Bruce spanworm, of which three were always diagnostic. We showed that eggs produced by hybridizing the two species in the laboratory contained copies of both species at these six sites. We found that most of the moths collected in the field with intermediate genitalia had winter moth CO1 and G6PD sequences and thus were not hybrids (or at least F1 hybrids). We found three hybrids out of 158 moths with intermediate genitalia in the region where both species were caught. We conclude that hybrids occur in nature, but are not as common as previously reported. Introgression of genes between the two species may still be significant.

Recombinant gas vesicles from Halobacterium sp . displaying SIV peptides demonstrate biotechnology potential as a pathogen peptide delivery vehicle

BackgroundPrevious studies indicated that recombinant gas vesicles (r-GV) from a mutant strain of Halobacterium sp. NRC-1 could express a cassette containing test sequences of SIVmac gag derived DNA, and function as an antigen display/delivery system. Tests using mice indicated that the humoral immune response to the gag encoded sequences evoked immunologic memory in the absence of an exogenous adjuvant.ResultsThe goal of this research was to extend this demonstration to diverse gene sequences by testing recombinant gas vesicles displaying peptides encoded by different SIV genes (SIVtat, rev or nef). Verification that different peptides can be successfully incorporated into the GvpC surface protein of gas vesicle would support a more general biotechnology application of this potential display/delivery system. Selected SIVsm-GvpC fusion peptides were generated by creating and expressing fusion genes, then assessing the resulting recombinant gas vesicles for SIV peptide specific antigenic and immunogenic capabilities. Results from these analyses support three conclusions: (i) Different recombinant gvpC-SIV genes will support the biosynthesis of chimeric, GvpC fusion proteins which are incorporated into the gas vesicles and generate functional organelles. (ii) Monkey antibody elicited by in vivo infection with SHIV recognizes these expressed SIV sequences in the fusion proteins encoded by the gvpC-SIV fusion genes as SIV peptides. (iii) Test of antiserum elicited by immunizing mice with recombinant gas vesicles demonstrated notable and long term antibody titers. The observed level of humoral responses, and the maintenance of elevated responses to, Tat, Rev and Nef1 encoded peptides carried by the respective r-GV, are consistent with the suggestion that in vivo there may be a natural and slow release of epitope over time.ConclusionThe findings therefore suggest that in addition to providing information about these specific inserts, r-GV displaying peptide inserts from other relevant pathogens could have significant biotechnological potential for display and delivery, or serve as a cost effective initial screen of pathogen derived peptides naturally expressed during infections in vivo.

SIVsm Tat, Rev, and Nef1: functional characteristics of r-GV internalization on isotypes, cytokines, and intracellular degradation

BackgroundRecombinant gas vesicles (r-GV) from Halobacterium sp. strain SD109 expressing cassettes with different SIVsm inserts, have potential utility as an effective antigen display system for immunogen testing in vivo and for initial epitope assessments in vitro. Previous mouse model studies demonstrated immunization with r-GV expressing selected exogenous sequences elicited a prolonged immune response. Here we tested segments from three SIVsm genes (tat, rev, and nef) each surface displayed by r-GV. As with HIV, for SIVsm the proteins encoded by tat, rev and nef respectively serve critical and diverse functions: effects on efficient viral RNA polymerase II transcription, regulation of viral gene expression and effects on specific signaling functions through the assembly of multiprotein complexes. Humoral responses to r-GVTat, Rev or Nef1 elicited in vivo, associated changes in selected cell cytokine production following r-GV internalization, and the capacity of J774A.1 macrophage cells to degrade these internalized display/delivery particles in vitro were examined.ResultsThe in vivo studies involving r-GV immunizations and in vitro studies of r-GV uptake by J774A.1 macrophages demonstrated: (i) tests for antibody isotypes in immunized mice sera showed activation and re-stimulation of memory B cells, (ii) during long term immune response to the epitopes, primarily the IgG1 isotype was produced, (iii) in vitro, macrophage degradation of r-GV containing different SIVsm inserts occurred over a period of days resulting in an inherent slow breakdown and degradation of the SIVsm peptide inserts, (iv) vesicle specific GvpC, a larger protein, degraded more slowly than the recombinant peptide inserts and (v) in vitro uptake and degradation of the r-GV populations tested was associated with SIVsm insert specific patterns for cytokines IL-10, IL-12 and IL-18.ConclusionsTogether these findings provide new information underscoring r-GV potential. They can clearly: display various exogenous peptides, be intracellularly degraded in vitro over a period of days, affect cell cytokine levels, and retain their self-adjuvanting capacity irrespective of the specific peptide expressed within the GvpC protein. These features support the cost effective generation of vaccine components, and provide a simple, self-adjuvanting system for assessing immune visibility of and specific responses to individual pathogen peptides.

Phylogeographic Diversity of the Winter Moths Operophtera brumata and O. bruceata (Lepidoptera: Geometridae) in Europe and North America

ABSTRACT The European winter moth, Operophtera brumata (L.), an invasive forest defoliator, is undergoing a rapid range expansion in northeastern North America. The source of this invasion, and phylogeographic diversity throughout its native range, has not been explored. To do this, we used samples from a pheromone-baited trap survey of O. brumata collected across its native range in Europe, and invasive range in North America. Traps in North America also attract a congeneric species, the Bruce spanworm O. bruceata (Hulst), and the western Bruce spanworm O. b. occidentalis (Hulst). From this sampling, we sequenced two regions of the cytochrome c oxidase subunit I mitochondrial gene; one region corresponds to the DNA ‘barcode’ region, the other is a nonoverlapping section. We used these sequences, in combination with sequence data from a recent survey of the Geometridae in western North America, for phylogenetic and phylogeographic analyses to characterize genetic divergence and variation for O. brumata in North America and Europe, and O. bruceata and O. b. occidentalis in North America. We found O. brumata mtDNA diversity to be dominated by a single widespread, and common haplotype. In contrast, O. bruceata shows high haplotype diversity that is evenly distributed throughout North America. Phylogeographic patterns indicate an introduction of O. brumata in British Columbia likely originated from Germany, and suggest the invasive population in northeastern North America may have its origins in the United Kingdom, and/or Germany. We found uncorrected pairwise sequence divergence between Operophtera species to be ≈7%. O. b. occidentalis is ≈ 5% divergent from O. bruceata, has a restricted range in the Pacific Northwest, and has unique morphological characters. Together these lines of evidence suggest O. b. occidentalis may be deserving of species status. Additionally, a single morphologically unique Operophtera specimen, similar to O. bruceata, was collected in southern Arizona, far outside the known range of O. bruceata. This suggests that North America may contain further, unsampled, Operophtera diversity.