Live, attenuated influenza A H5N1 candidate vaccines provide broad cross-protection in mice and ferrets - PubMed (original) (raw)

Comparative Study

Live, attenuated influenza A H5N1 candidate vaccines provide broad cross-protection in mice and ferrets

Amorsolo L Suguitan Jr et al. PLoS Med. 2006 Sep.

Abstract

Background: Recent outbreaks of highly pathogenic influenza A H5N1 viruses in humans and avian species that began in Asia and have spread to other continents underscore an urgent need to develop vaccines that would protect the human population in the event of a pandemic.

Methods and findings: Live, attenuated candidate vaccines possessing genes encoding a modified H5 hemagglutinin (HA) and a wild-type (wt) N1 neuraminidase from influenza A H5N1 viruses isolated in Hong Kong and Vietnam in 1997, 2003, and 2004, and remaining gene segments derived from the cold-adapted (ca) influenza A vaccine donor strain, influenza A/Ann Arbor/6/60 ca (H2N2), were generated by reverse genetics. The H5N1 ca vaccine viruses required trypsin for efficient growth in vitro, as predicted by the modification engineered in the gene encoding the HA, and possessed the temperature-sensitive and attenuation phenotypes specified by the internal protein genes of the ca vaccine donor strain. More importantly, the candidate vaccines were immunogenic in mice. Four weeks after receiving a single dose of 10(6) 50% tissue culture infectious doses of intranasally administered vaccines, mice were fully protected from lethality following challenge with homologous and antigenically distinct heterologous wt H5N1 viruses from different genetic sublineages (clades 1, 2, and 3) that were isolated in Asia between 1997 and 2005. Four weeks after receiving two doses of the vaccines, mice and ferrets were fully protected against pulmonary replication of homologous and heterologous wt H5N1 viruses.

Conclusions: The promising findings in these preclinical studies of safety, immunogenicity, and efficacy of the H5N1 ca vaccines against antigenically diverse H5N1 vaccines provide support for their careful evaluation in Phase 1 clinical trials in humans.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Replication of H5N1 2004 ca and 2004 wt Viruses in the Brains of Mice

The H5N1 2004 ca virus, unlike the H5N1 2004 wt parent virus, is undetectable in the brain. Influenza A/Beijing/95 ca, an H1N1 ca reassortant virus, was also undetectable in the brains of mice. The lower limit of detection is indicated by the dashed horizontal line. Four mice per group were euthanized at each time point (days 2, 4, 6, 8, 10, and 12). Data from days 6 through 12 are combined in the figure because virus was not detected in any of the brain tissues harvested on these days.

Figure 2

Figure 2. Survival of Mice Immunized with a Single Dose of H5N1 ca Vaccine Candidates Following Lethal Challenge with H5N1 1997 wt or H5N1 2004 wt Viruses

Groups of eight mice that received a single dose of 106 TCID50 of the H5N1 1997 ca (▪), H5N1 2003 ca (▵), H5N1 2004 ca (^), or AA ca (○) viruses, or were mock-immunized (L-15 medium; ♦) were challenged with the H5N1 1997 wt (A–C) or H5N1 2004 wt virus (D–F) at 50 (A and D), 500 (B and E), or 5,000 (C and F) LD50. The mice were monitored daily for 21 d p.i. Immunization with the H5N1 1997 ca, H5N1 2003 ca, and H5N1 2004 ca viruses resulted in 100% survival of the mice following challenge, so the symbols for these groups are superimposed.

Figure 3

Figure 3. Replication of H5N1 wt Challenge Viruses in Mice Immunized with One or Two Doses of H5N1 ca Viruses

(A and B) Mice that received a single dose of 106 TCID50 of the immunizing virus were challenged with 105 TCID50 of H5N1 1997 wt, H5N1 2003 wt, or H5N1 2004 wt viruses on day 28 p.i. Replication of challenge virus is shown in the lungs (A) and nasal turbinates (B). (C and D) Mice that received two doses of the immunizing virus 4 wk apart were challenged with the H5N1 wt viruses on day 56 p.i. Replication of challenge virus is shown in the lungs (C) and nasal turbinates (D). Lungs were harvested 2 d following challenge, homogenized, and titrated on MDCK cells. Groups of mock-immunized (L-15) mice and mice immunized with the nonlethal 2003 wt virus (single dose) or with AA ca were included as control groups. Four mice were evaluated in each group. The lower limit of detection is indicated by the dashed horizontal line. Log-transformed viral titers were compared using the Mann-Whitney U test. * p ≤ 0.05 compared to mock-immunized mice.

Figure 4

Figure 4. Immunization of Mice with Two Doses of H5N1 ca Viruses Provides Protection against Replication of Homologous and Heterologous H5N1 wt Viruses in the Lungs

Mice that received two doses of 106 TCID50 of H5N1 1997 ca, H5N1 2003 ca, or H5N1 2004 ca viruses 4 wk apart were challenged on day 56 p.i. with 105 TCID50 of homologous and heterologous H5N1 wt viruses, including A/Indonesia/05/2005, a clade 2 H5N1 wt virus. The lungs were harvested from these mice 4 d later and were homogenized and titered on MDCK cells. Four mice were evaluated in each group. The lower limit of detection is indicated by the dashed horizontal line. * p < 0.02 compared to mock-immunized mice.

Figure 5

Figure 5. Efficacy in Ferrets of Two Doses of H5N1 ca Viruses against Challenge with Homologous and Heterologous H5N1 wt Viruses

Ferrets were immunized with two doses of 107 TCID50 of H5N1 1997 ca or H5N1 2004 ca viruses 1 mo apart and challenged 1 mo later with 107 TCID50 of either H5N1 1997 wt, H5N1 2004 wt, or A/Indonesia/05/2005 (H5N1) wt. In order to determine the contribution of the internal protein genes of the AA ca virus to protection, an additional group of ferrets was immunized with H1N1 A/New Caledonia/99 ca and challenged with H5N1 1997 wt. These ferrets were not challenged with H5N1 2004 wt or A/Indonesia/05/2005 (H5N1) wt. Lungs, nasal turbinates, and brains were harvested 3 d after challenge and were homogenized and titered on MDCK cells. Three ferrets per group were evaluated. The lower limit of detection is indicated by the dashed line. *p < 0.05 compared to mock-immunized ferrets.

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