Live attenuated influenza virus vaccines by computer-aided rational design - PubMed (original) (raw)

Live attenuated influenza virus vaccines by computer-aided rational design

Steffen Mueller et al. Nat Biotechnol. 2010 Jul.

Abstract

Despite existing vaccines and enormous efforts in biomedical research, influenza annually claims 250,000-500,000 lives worldwide, motivating the search for new, more effective vaccines that can be rapidly designed and easily produced. We applied the previously described synthetic attenuated virus engineering (SAVE) approach to influenza virus strain A/PR/8/34 to rationally design live attenuated influenza virus vaccine candidates through genome-scale changes in codon-pair bias. As attenuation is based on many hundreds of nucleotide changes across the viral genome, reversion of the attenuated variant to a virulent form is unlikely. Immunization of mice by a single intranasal exposure to codon pair-deoptimized virus conferred protection against subsequent challenge with wild-type (WT) influenza virus. The method can be applied rapidly to any emerging influenza virus in its entirety, an advantage that is especially relevant when dealing with seasonal epidemics and pandemic threats, such as H5N1- or 2009-H1N1 influenza.

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Figures

Fig. 1. Characterization in tissue culture of synthetic codon pair-deoptimized Influenza viruses

(A) Plaque phenotypes on MDCK cells of PR8 wild type virus and synthetic PR8 derivatives, carrying one (NPMin, HAMin, PB1Min), two (NP/HAMin; HA/PB1Min) or three (PR83F) deoptimized gene segments. (B) Growth kinetics of PR8 wild type virus and three synthetic PR8 derivatives in MDCK cells after infection with 0.001 MOI of the indicated viruses. All combinations grew within log 8-9 although only PR8-HAMin, PR8-NP/HAMin and PR83F are shown as compared to wild type. (C) Analysis of Influenza virus protein expression in infected cells. MDCK cells were infected with PR8 wild type virus or synthetic PR8 derivatives, carrying one deoptimized gene segment each (NPMin, HAMin, PB1Min), as indicated. Western blot (WB) analysis of proteins extracted from whole cell lysates was carried out with PB1, NP, HA, or actin antibodies. Actin was used to indicate equal protein loading.

Fig. 2. Attenuation of codon pair-deoptimized Influenza virus PR83F in BALB/c mice

(A) Body weight curve following intranasal infection with 104 PFU of PR8 wild type (triangles), 104 PFU of deoptimized PR83F (diamonds), or mock-infected (saline; squares). The average of 5 mice per time point and standard deviations are indicated. Wild type-infected mice did not survive beyond day 5 (indicated by a cross). (B) Virus titer in whole lung homogenate after infection with either 103 PFU PR8 wild type (squares), or deoptimized PR83F (circles). Average of three mice per time point. * On day 9 post infection, PR83F was no longer detectable (below 40 PFU/lung).

Fig. 3. Immune responses and protection

(A, B) Vaccine Margin of Safety for PR8 wild type and deoptimized PR83F viruses. The left ordinate indicates the percentage of animals surviving the primary inoculation (black squares) with (A) PR83F or (B) wt PR8, at doses ranging between 100 to 106 PFU. After 28 days, the surviving, vaccinated animals were challenged with a single 1000 × lethal dose 50 of PR8 wild type virus. Disease and survival were monitored (right ordinate; open circles) for (A) PR83F- and (B) PR8-vaccinated mice. (C) Virus load in mouse lungs following wild type challenge of PR83F-vaccinated animals. 28 days following a single intranasal vaccination with 104 PFU PR83F mice were challenged with 1000 × LD50 of PR8 wt virus. Three days thereafter the level of challenge virus in lung homogenates was determined. (D) ELSIA determination of influenza-specific serum antibodies. 28 days after a primary infection, serum was collected, and anti-influenza IgG serum antibody titers were determined from animals that had received a primary inoculation of 0.01 × LD50 (black diamonds) or 0.001 × LD50 of PR83F (black circles), 0.01 × LD50 of PR8 (white squares), or saline (black triangles). ELISA antibody titer against PR8 virus antigen is expressed as the lowest reciprocal serum dilution that resulted in a positive ELISA signal (5 standard deviation above background).

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Live attenuated influenza virus vaccines by computer-aided rational design - PubMed (original) (raw)