In vivo molecular imaging analysis of a nasal vaccine that induces protective immunity against botulism in nonhuman primates

Nasal administration is an effective route for a needle-free vaccine. However, nasally administered Ags have the potential to reach the CNS directly from the nasal cavity, thus raising safety concerns. In this study, we performed real-time quantitative tracking of a nasal vaccine candidate for botulism, which is a nontoxic subunit fragment of Clostridium botulinum type A neurotoxin (BoHc/A) effective in the induction of the toxin-neutralizing immune response, by using (18)F-labeled BoHc/A-positron-emission tomography, an in vivo molecular imaging method. This method provides results that are consistent with direct counting of [(18)F] radioactivity or the traditional [(111)In]-radiolabel method in dissected tissues of mice and nonhuman primates. We found no deposition of BoHc/A in the cerebrum or olfactory bulb after nasal administration of (18)F-labeled BoHc/A in both animals. We also established a real-time quantitative profile of elimination of this nasal vaccine candidate and demonstrated that it induces highly protective immunity against botulism in nonhuman primates. Our findings demonstrate the efficiency and safety of a nasal vaccine candidate against botulism in mice and nonhuman primates using in vivo molecular imaging.     

Photoreaction cycle of the light, oxygen, and voltage domain in FKF1 determined by low-temperature absorption spectroscopy

Flavin-binding Kelch repeat F-box (FKF1) protein plays important roles in the photoregulation of flowering in Arabidopsis. FKF1 has a light, oxygen, and voltage (LOV) sensing domain binding a flavin mononucleotide (FMN) as a chromophore noncovalently. Photoreaction of the FKF1-LOV polypeptide was studied by low-temperature absorption spectroscopy. Upon blue light irradiation, a ground state, D(450), is converted to S(390) known as a cysteinyl-flavin adduct intermediate in the photoreaction of phototropin. Below 150 K, bleaching of D(450) was much reduced and a new photoproduct, Z(370), appeared as well as S(390) formation. The calculated absorption spectrum for Z(370) is very similar to those of flavoproteins in an anion radical state. On the basis of the results that S(390) formation proceeds to Z(370) formation and that Z(370) formed at low temperatures reverts to D(450) upon temperature increase, Z(370) is concluded to be not an intermediate from D(450) to S(390). Z(370) is suggested to be formed from the biradical triplet-excited state after relaxing to the ground state with the FMN anion radical trapped at the low temperature, in which the SH of the cysteine is in the wrong position that is able to produce a radical pair but unable to form the cysteinyl-flavin adduct. The counter SH in the cationic radical state may revert to the ground state by extracting an electron from the unidentified amino acid residue. Interestingly, S(390) that has been thought to be irreversible to D(450) was revealed to revert to D(450) very slowly with a half-life time of 62.5 h in solution at 298 K. The photoreaction mechanism is discussed in reference to the calculated activation energy of the reaction processes.     

An N-glycan structure correlates with pulmonary metastatic ability of cancer cells

N-Glycan structures on the surface of cancer cells have diverse structures and play significant roles in metastatic process. However, little is known about their roles in organ-selective metastasis. Our study revealed that an alpha1,6-fucosylated biantennary N-glycan structure designated A2G2F is characteristic of lungs, with far more abundant expression in normal human and murine lungs than in other organs. In this study, we further examined the role of A2G2F in pulmonary metastasis. We stained metastatic cancers by alpha1,6-fucose-specific Lens culinaris agglutinin lectin and revealed that pulmonary metastatic nodules more abundantly expressed alpha1,6-fucosylated N-glycans than hepatic metastatic nodules from common primary cancers. The most specific alpha1,6-fucosylated N-glycan structure in pulmonary metastatic cancer was identified to be A2G2F. Using a B16 melanoma cell metastasis model, we showed that A2G2F-rich B16 cells formed more pulmonary metastatic nodules than A2G2F-poor cells. Our results suggest that A2G2F plays a critical role in pulmonary metastasis.     

“Armed” oncolytic herpes simplex viruses for brain tumor therapy

Genetically engineered, conditionally replicating herpes simplex viruses type 1 (HSV-1) are promising therapeutic agents for brain tumors and other solid cancers. They can replicate in situ, spread and exhibit oncolytic activity via a direct cytocidal effect. One of the advantages of HSV-1 is the capacity to incorporate large and/or multiple transgenes within the viral genome. Oncolytic HSV-1 can therefore be “armed” to add certain functions. Recently, the field of armed oncolytic HSV-1 has drastically advanced, due to development of recombinant HSV-1 generation systems that utilize bacterial artificial chromosome and multiple DNA recombinases. Because antitumor immunity is induced in the course of oncolytic activities of HSV-1, transgenes encoding immunomodulatory molecules have been most frequently used for arming. Other armed oncolytic HSV-1 include those that express antiangiogenic factors, fusogenic membrane glycoproteins, suicide gene products, and proapoptotic proteins. Provided that the transgene product does not interfere with viral replication, such arming of oncolytic HSV-1 results in augmentation of antitumor efficacy. Immediate-early viral promoters are often used to control the arming transgenes, but strict-late viral promoters have been shown useful to restrict the expression in the late stage of viral replication when desirable. Some armed oncolytic HSV-1 have been created for the purpose of noninvasive in vivo imaging of viral infection and replication. Development of a wide variety of armed oncolytic HSV-1 will lead to an establishment of a new genre of therapy for brain tumors as well as other cancers.Key words: oncolytic virus therapy, gene therapy, herpes simplex virus, viral vectors, G47Δ, G207, antitumor immunity     

Cloning, sequencing, and expression of the meso-diaminopimelate dehydrogenase gene from Bacillus sphaericus

The gene encoding the meso-diaminopimelate dehydrogenase of Bacillus sphaericus was cloned into E. coli cells and its complete DNA sequence was determined. The meso-diaminopimelate dehydrogenase gene consisted of 978 nucleotides and encoded 326 amino acid residues corresponding to the subunit of the dimeric enzyme. The amino acid sequence deduced from the nucleotide sequence of the enzyme gene of B. sphaericus showed 50% identity with those of the enzymes from Corynebacterium glutamicum and Brevibacterium flavum. The enzyme gene from B. sphaericus was highly expressed in E. coli cells. We purified the enzyme to homogeneity from a transformant with 76% recovery. The N-terminal amino acid of both the enzyme from B. sphaericus and the transformant were serine, indicating that the N-terminal methionine is removed by post-translational modification in B. sphaericus and E. coli cells.