A promoter mutation in the haemagglutinin segment of influenza A virus generates an effective candidate live attenuated vaccine

Abstract

Background: Annual seasonal and pandemic influenza vaccines need to be produced in a very tight time frame. Haemagglutinin (HA) is the major immunogenic component of influenza vaccines, and there is a lot of interest in improving candidate vaccine viruses.

Objectives: It has been shown elsewhere that mutations introduced in the non-coding region of influenza genome segments can upregulate protein expression. Our objective was to assess a virus based on the laboratory strain A/PR/8/34 (PR8) containing a modified 3' non-coding region of RNA segment 4 (haemagglutinin).

Methods: NIBRG-93 was generated using reverse genetics. HA protein expression and growth properties were assessed. The virus phenotype suggested that it could be a candidate for use as a live attenuated vaccine, so in vivo studies were performed to assess its suitability.

Results: NIBRG-93 virus has enhanced haemagglutinin production and is significantly attenuated. Electron microscopy (EM) shows that the modified virus produces a large proportion of 'virus-like particles' that consist of budded cell membrane covered in HA but lacking M1 protein. The virus was shown to be attenuated in mice and offered complete protection against lethal challenge.

Conclusions: We demonstrate that NIBRG-93 is an effective live attenuated vaccine virus protecting mice against lethal challenge and reducing virus shedding.

Keywords: Candidate vaccine virus; haemagglutinin; influenza; live attenuated; promoter mutation; vaccine.

Figure 1

Yield analysis of NIBRG-93. (A) Quantitation of haemagglutinin (HA) content. Quantitation was carried out using image quant software. For each sample, the HA content was calculated as described in Materials and methods. (B) Total protein yield (Dark grey bars). The total protein yield for each virus was measured using a BCA assay as described in Materials and methods. HA protein yield (light grey bars). The yield of HA (mg HA/100 eggs) was calculated based on total protein and the %HA (as shown in panel A). For all data in A and B, each virus was tested on at least 3 occasions and error bars show standard deviations. (C) SDS-PAGE analysis of virus concentrates. Samples were reduced and deglycosylated; major virus proteins are identified: 1, NP; 2, HA1; 3, M1; and 4, HA2. (D) Scan of gel lanes using image quant software. Viral proteins are indicated by numbers as above. (E) Ratio of M1 to NP. Quantitation was carried out using image quant software.

Figure 2

Growth characteristics of PR8 and NIBRG-93. (A) Titre of viruses in eggs at 35°C. Pale grey bars show pfu/ml and dark grey bars show haemagglutinin titre. (B) Titre of PR8 (dark grey) and NIBRG-93 (light grey) in eggs at various temperatures. Each data point is the average of at least three repeats and error bars show standard deviation.

Figure 3

Morphology of virus particles. Virus particles were visualised using cryo-electron microscopy. Representative electron micrographs of wild-type PR8 (A) and NIBRG-93 (B) are shown. Scale bar (in white) denotes 100 nm.

Figure 4

Attenuation of NIBRG-93 in vivo. (A) Weight loss and (B) survival following infection with PR8 or NIBRG-93. 15 mice were inoculated IN with 10^5·5 TCID₅₀ wild-type A/PR/8/34, three groups of 35 mice were inoculated IN with 10^4·5, 10^5·5 or 10^6·5 TCID₅₀ NIBRG-93. Two groups of 15 mice were used as control groups and were inoculated IM with 15ugHA/mouse inactivated PR8 or PBS only. After inoculation, and throughout the entire course of the study, mice were weighed and observed daily for clinical signs of disease. Error bars show standard deviations.

Figure 5

Titration of virus recovered from tissues and nasal washes. Virus recovery was assessed using TCID₅₀ titration of samples. (A) Virus recovery from lungs. (B) Virus recovery from nasal turbinates. (C) Virus recovery from lungs and nasal turbinates day 30 post-inoculation (2 days post-challenge). (D) Percentage of animals with virus recovered from nasal washes. (E) TCID₅₀ of virus recovered from positive nasal washes.

Figure 6

Protection from viral challenge. (A) Weight loss and (B) survival following challenge with wt PR8. At day 28 post-inoculation, mice were challenged with a lethal dose of 10^5·5 TCID₅₀ wt PR8 virus. Mice were weighed and observed for clinical signs of disease. Error bars show standard deviations.

Figure 7

Haemagglutination inhibition antibody titres from blood samples pre-immunisation, post-immunisation and post-challenge. Data shown are geometric mean titres of antibody responses from all animals within a group.