Mucosal Immunization of Lactating Female Rhesus Monkeys with a Transmitted/Founder HIV-1 Envelope Induces Strong Env-Specific IgA Antibody Responses in Breast Milk

We previously demonstrated that vaccination of lactating rhesus monkeys with a DNA prime/vector boost strategy induces strong T-cell responses but limited envelope (Env)-specific humoral responses in breast milk. To improve vaccine-elicited antibody responses in milk, hormone-induced lactating rhesus monkeys were vaccinated with a transmitted/founder (T/F) HIV Env immunogen in a prime-boost strategy modeled after the moderately protective RV144 HIV vaccine. Lactating rhesus monkeys were intramuscularly primed with either recombinant DNA (n = 4) or modified vaccinia virus Ankara (MVA) poxvirus vector (n = 4) expressing the T/F HIV Env C.1086 and then boosted twice intramuscularly with C.1086 gp120 and the adjuvant MF59. The vaccines induced Env-binding IgG and IgA as well as neutralizing and antibody-dependent cellular cytotoxicity (ADCC) responses in plasma and milk of most vaccinated animals. Importantly, plasma neutralization titers against clade C HIV variants MW965 (P = 0.03) and CAP45 (P = 0.04) were significantly higher in MVA-primed than in DNA-primed animals. The superior systemic prime-boost regimen was then compared to a mucosal-boost regimen, in which animals were boosted twice intranasally with C.1086 gp120 and the TLR 7/8 agonist R848 following the same systemic prime. While the systemic and mucosal vaccine regimens elicited comparable levels of Env-binding IgG antibodies, mucosal immunization induced significantly stronger Env-binding IgA responses in milk (P = 0.03). However, the mucosal regimen was not as potent at inducing functional IgG responses. This study shows that systemic MVA prime followed by either intranasal or systemic protein boosts can elicit strong humoral responses in breast milk and may be a useful strategy to interrupt postnatal HIV-1 transmission.     

Gene Conversion Transfers the GAF-A Domain of Phosphodiesterase TbrPDEB1 to One Allele of TbrPDEB2 of Trypanosoma brucei

Background

Chromosome 9 of Trypanosoma brucei contains two closely spaced, very similar open reading frames for cyclic nucleotide specific phosphodiesterases TbrPDEB1 and TbrPDEB2. They are separated by 2379 bp, and both code for phosphodiesterases with two GAF domains in their N-terminal moieties and a catalytic domain at the C-terminus.

Methods and Findings

The current study reveals that in the Lister427 strain of T. brucei, these two genes have undergone gene conversion, replacing the coding region for the GAF-A domain of TbrPDEB2 by the corresponding region of the upstream gene TbrPDEB1. As a consequence, these strains express two slightly different versions of TbrPDEB2. TbrPDEB2a represents the wild-type phosphodiesterase, while TbrPDEB2b represents the product of the converted gene. Earlier work on the subcellular localization of TbrPDEB1 and TbrPDEB2 had demonstrated that TbrPDEB1 is predominantly located in the flagellum, whereas TbrPDEB2 partially locates to the flagellum but largely remains in the cell body. The current findings raised the question of whether this dual localization of TbrPDEB2 may reflect the two alleles. To resolve this, TbrPDEB2 of strain STIB247 that is homozygous for TbrPDEB2a was tagged in situ, and its intracellular localization was analyzed.

Conclusions

The results obtained were very similar to those found earlier with Lister427, indicating that the dual localization of TbrPDEB2 reflects its true function and is not simply due to the presence of the two different alleles. Notably, the gene conversion event is unique for the Lister427 strain and all its derivatives. Based on this finding, a convenient PCR test has been developed that allows the stringent discrimination between Lister-derived strains that are common in many laboratories and other isolates. The technique is likely very useful to resolve questions about potential mix-ups of precious field isolates with the ubiquitous Lister strain.     

Spectral properties of fluorescent flavoproteins of isolated rat liver mitochondria

The different flavoproteins contributing to flavin fluorescence of isolated rat liver mitochondria have distinct excitation and emission spectra. The NAD-linked flavin component was identified as alpha-lipoamide dehydrogenase, while the non-NAD-linked component was found to be electron transfer flavoprotein. The differences in excitation and emission properties of the mitochondrial flavoproteins permit selective recording of their redox state changes in isolated mitochondria.     

TbPDE1, a novel class I phosphodiesterase of Trypanosoma brucei.

Cyclic nucleotide specific phosphodiesterases (PDEs) are important components of all cAMP signalling networks. In humans, 11 different PDE families have been identified to date, all of which belong to the class I PDEs. Pharmacologically, they have become of great interest as targets for the development of drugs for a large variety of clinical conditions. PDEs in parasitic protozoa have not yet been extensively investigated, despite their potential as antiparasitic drug targets. The current study presents the identification and characterization of a novel class I PDE from the parasitic protozoon Trypanosoma brucei, the causative agent of human sleeping sickness. This enzyme, TbPDE1, is encoded by a single-copy gene located on chromosome 10, and it functionally complements PDE-deficient strains of Saccharomyces cerevisiae. Its C-terminal catalytic domain shares about 30% amino acid identity, including all functionally important residues, with the catalytic domains of human PDEs. A fragment of TbPDE1 containing the catalytic domain could be expressed in active form in Escherichia coli. The recombinant enzyme is specific for cAMP, but exhibits a remarkably high Km of > 600 microm for this substrate.     

Sub-Cellular Element Analysis of a Cyanobacterium (Nostoc sp.) in Symbiosis with Gunnera manicata by ESI and EELS

Element analysis using electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) was performed in a symbiotic Nostoc sp. strain found in the upper stem tissue of Gunnera manicata, and in Nostoc PCC 9229, a free-living heterocyst-forming cyanobacterium able to enter into symbiosis with the angiosperm Gunnera in reconstitution experiments. ESI and EELS unequivocally identified the four elements nitrogen (N), sulphur (S), phosphorus (P) and oxygen (O) in different inclusion bodies of these biological specimens. High amounts of nitrogen were solely detected in huge cyanophycin granules in vegetative cells of the symbiotic Nostoc strain, whereas large polyphosphate bodies, containing high amounts of phosphorus, sulphur and oxygen, could be seen in the free-living Nostoc PCC 9229. The latter were usually not present or, when found, very small in vegetative cells of the cyanobiont.     

Fruit Bats (Chiroptera: Pteropodidae) as Seed Dispersers and Pollinators in a Lowland Malaysian Rain Forest1

The aims of this study were to (1) characterize the food resources exploited by fruit bats (Pteropodidae) within an old‐growth Malaysian dipterocarp forest, (2) test the viability of the seeds they disperse, and (3) provide an estimate of the proportion of trees that are to some degree dependent upon bats for seed dispersal and/or pollination. Fruit species exploited by bats could be distinguished from those eaten by birds largely on the basis of color (as perceived by human beings). Bat‐dispersed fruits were typically inconspicuous shades of green–yellow or dull red–brown, whereas fruits eaten by birds were generally bright orange to red. Dietary overlap between bats and nonflying mammals was relatively high. In contrast to primates and squirrels, which were major seed predators for several of the plant species under investigation, fruit bats had no negative impact on seed viability. A botanical survey in 1 ha of old‐growth forest revealed that 13.7 percent of trees (?15 cm girth at breast height) were at least partially dependent upon fruit bats for pollination and/or seed dispersal.