High-throughput screening of stabilizers for respiratory syncytial virus: identification of stabilizers and their effects on the conformational thermostability of viral particles.
Respiratory syncytial virus (RSV) is the leading cause of severe respiratory infection in children worldwide. Recombinant live attenuated viral preparations are one of the most promising strategies for vaccination but they typically possess poor thermostability. In this work, a library of compounds was screened and stabilizers were selected based on their ability to inhibit the aggregation of RSV perturbed at 56 degrees C. After screening and selection of excipients, the conformational stability of the RSV proteins was evaluated in the presence of potential stabilizers. The secondary and tertiary structures as well as aggregation/dissociation of RSV were monitored using circular dichroism and second derivative UV absorption spectroscopies and light scattering, respectively, as a function of temperature (10-90 degrees C). RSV membrane fluidity was also evaluated by generalized polarization of Laurdan fluorescence. Screening experiments showed that a variety of sugars, amino acids, polyols and polyanions inhibited the aggregation of viral particles. Conformational stability studies demonstrated that the addition of sugars and polyols stabilized RSV as indicated by a significant increase in the transition melting temperature (Tm) of both the secondary and tertiary structures as well as the gel to liquid crystalline membrane transition. These results should provide the basis for rational development of more physically stable formulations of live attenuated RSV vaccines.
Ausar
Salvador F
SF
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047, USA.
Espina
Marianela
M
Brock
Julie
J
Thyagarayapuran
Nagarajan
N
Repetto
Robert
R
Khandke
Lakshmi
L
Middaugh
C Russell
CR
D000596:Amino Acids / Q000737:chemistry / Q000494:pharmacology; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D002241:Carbohydrates / Q000737:chemistry / Q000494:pharmacology; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D000071228:Polyelectrolytes; D011108:Polymers / Q000737:chemistry / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D022261:Respiratory Syncytial Virus Vaccines / Q000737:chemistry / Q000494:pharmacology; D018113:Respiratory Syncytial Virus, Human / Q000737:chemistry / Q000187:drug effects*; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014764:Viral Proteins / Q000737:chemistry*
D000596:Amino Acids; D000998:Antiviral Agents; D002241:Carbohydrates; D000071228:Polyelectrolytes; D011108:Polymers; D022261:Respiratory Syncytial Virus Vaccines; D014764:Viral Proteins; C024617:polyol