Pharmacokinetics of the Long-Acting Basal Insulin LY2605541 in Subjects With Varying Degrees of Renal Function.

The pharmacokinetics of LY2605541 (basal insulin peglispro), a novel long-acting basal insulin analogue, was evaluated in 5 groups of subjects with varying degrees of renal function based on creatinine clearance: normal renal function (>80 mL/min), mild renal impairment (51-80 mL/min), moderate renal impairment (30-50 mL/min), severe renal impairment (<30 mL/min), or end-stage renal disease (ESRD) requiring hemodialysis. Serial blood samples for pharmacokinetic analyses were collected up to 12 days following a single 0.33 U/kg subcutaneous dose of LY2605541. The apparent clearance (CL/F) and half-life across groups were not affected by renal function. Cmax values were lower in subjects with increasing severity of renal impairment; however, the small decrease in Cmax did not affect the overall exposure. Regression analysis showed that LY2605541 clearance is independent of renal function (slope = 0.000863; P = .885). The mean fraction of LY2605541 eliminated by a single hemodialysis session was 13% in subjects with ESRD. LY2605541 was generally well tolerated in healthy subjects and those with renal impairment following a single 0.33 U/kg subcutaneous dose. Given these data, no dose adjustment of LY2605541 based on pharmacokinetics is recommended in renal impairment or in patients undergoing hemodialysis.

Manipulation of the infectious bronchitis coronavirus genome for vaccine development and analysis of the accessory proteins.

Infectious bronchitis coronavirus (IBV) is the cause of the single most economically costly infectious disease of domestic fowl in the UK--and probably so in many countries that have a developed poultry industry. A major reason for its continued dominance is its existence as many serotypes, determined by the surface spike protein (S), cross-protection being poor. Although controlled to some degree by live and inactivated vaccines, a new generation of IB vaccines is called for. Reverse genetic or 'infectious clone' systems, which allow the manipulation of the IBV genome, are key to this development. New vaccines would ideally be: genetically stable (i.e. maintain a stable attenuated phenotype); administered in ovo; and be flexible with respect to the source of the spike protein gene. Rational attenuation of IBV requires the identification of genes that are simultaneously not essential for replication and whose absence would reduce pathogenicity. Being able to modify a 'core' vaccine strain to make it applicable to a prevailing serotype requires a procedure for doing so, and the demonstration that 'spike-swapping' is sufficient to induce good immunity. We have demonstrated that four small IBV proteins, encoded by genes 3 and 5, are not essential for replication; failure to produce these proteins had little detrimental affect on the titre of virus produced. Our current molecularly cloned IBV, strain Beaudette, is non-pathogenic, so we do not know what effect the absence of these proteins would have on pathogenicity. That said, plaque size and composition of various gene 3/5 recombinant IBVs in cell culture, and reduced output and ciliostasis in tracheal organ cultures, shows that they are less aggressive than the wild-type Beaudette. Consequently these genes remain targets for rational attenuation. We have recently obtained evidence that one or more of the 15 proteins encoded by gene 1 are also determinants of pathogenicity. Hence gene 1 is also a target for rational attenuation. Replacing the S protein gene of Beaudette with that from the pathogenic M41 strain resulted in a recombinant virus that was still non-pathogenic but which did induce protection against challenge with M41. We have since made other 'spike-swapped' recombinants, including ones with chimaera S genes. Uniquely, our molecular clone of Beaudette is benign when administered to 18-day-old embryos, even at high doses, and induces immunity after this route of vaccination. Taken together, our results point to the creation of a new generation of IB vaccines, based on rational modification of the genome, as being a realisable objective.

Neither the RNA nor the proteins of open reading frames 3a and 3b of the coronavirus infectious bronchitis virus are essential for replication.

Gene 3 of infectious bronchitis virus is tricistronic; open reading frames (ORFs) 3a and 3b encode two small nonstructural (ns) proteins, 3a and 3b, of unknown function, and a third, structural protein E, is encoded by ORF 3c. To determine if either the 3a or the 3b protein is required for replication, we first modified their translation initiation codons to prevent translation of the 3a and 3b proteins from recombinant infectious bronchitis viruses (rIBVs). Replication in primary chick kidney (CK) cells and in chicken embryos was not affected. In chicken tracheal organ cultures (TOCs), the recombinant rIBVs reached titers similar to those of the wild-type virus, but in the case of viruses lacking the 3a protein, the titer declined reproducibly earlier. Translation of the IBV E protein is believed to be initiated by internal entry of ribosomes at a structure formed by the sequences corresponding to ORFs 3a and 3b. To assess the necessity of this mechanism, we deleted most of the sequence representing 3a and 3b to produce a gene in which ORF 3c (E) was adjacent to the gene 3 transcription-associated sequence. Western blot analysis revealed that the recombinant IBV produced fivefold less E protein. Nevertheless, titers produced in CK cells, embryos, and TOCs were similar to those of the wild-type virus, although they declined earlier in TOCs, probably due to the absence of the 3a protein. Thus, neither the tricistronic arrangement of gene 3, the internal initiation of translation of E protein, nor the 3a and 3b proteins are essential for replication per se, suggesting that these proteins are accessory proteins that may have roles in vivo.

Recombinant infectious bronchitis coronavirus Beaudette with the spike protein gene of the pathogenic M41 strain remains attenuated but induces protective immunity.

We have replaced the ectodomain of the spike (S) protein of the Beaudette strain (Beau-R; apathogenic for Gallus domesticus chickens) of avian infectious bronchitis coronavirus (IBV) with that from the pathogenic M41 strain to produce recombinant IBV BeauR-M41(S). We have previously shown that this changed the tropism of the virus in vitro (R. Casais, B. Dove, D. Cavanagh, and P. Britton, J. Virol. 77:9084-9089, 2003). Herein we have assessed the pathogenicity and immunogenicity of BeauR-M41(S). There were no consistent differences in pathogenicity between the recombinant BeauR-M41(S) and its apathogenic parent Beau-R (based on snicking, nasal discharge, wheezing, watery eyes, rales, and ciliostasis in trachea), and both replicated poorly in trachea and nose compared to M41; the S protein from the pathogenic M41 had not altered the apathogenic nature of Beau-R. Both Beau-R and BeauR-M41(S) induced protection against challenge with M41 as assessed by absence of recovery of challenge virus and nasal exudate. With regard to snicking and ciliostasis, BeauR-M41(S) induced greater protection (seven out of nine chicks [77%]; assessed by ciliostasis) than Beau-R (one out of nine; 11%) but less than M41 (100%). The greater protection induced by BeauR-M41(S) against M41 may be related to the ectodomain of the spike protein of Beau-R differing from that of M41 by 4.1%; a small number of epitopes on the S protein may play a disproportionate role in the induction of immunity. The results are promising for the prospects of S-gene exchange for IBV vaccine development.