Prevention of influenza virus shedding and protection from lethal H1N1 challenge using a consensus 2009 H1N1 HA and NA adenovirus vector vaccine

Abstract

Vaccines against emerging pathogens such as the 2009 H1N1 pandemic virus can benefit from current technologies such as rapid genomic sequencing to construct the most biologically relevant vaccine. A novel platform (Ad5 [E1-, E2b-]) has been utilized to induce immune responses to various antigenic targets. We employed this vector platform to express hemagglutinin (HA) and neuraminidase (NA) genes from 2009 H1N1 pandemic viruses. Inserts were consensuses sequences designed from viral isolate sequences and the vaccine was rapidly constructed and produced. Vaccination induced H1N1 immune responses in mice, which afforded protection from lethal virus challenge. In ferrets, vaccination protected from disease development and significantly reduced viral titers in nasal washes. H1N1 cell mediated immunity as well as antibody induction correlated with the prevention of disease symptoms and reduction of virus replication. The Ad5 [E1-, E2b-] should be evaluated for the rapid development of effective vaccines against infectious diseases.

Fig. 1

CMI dose response titration of Ad5 [E1-, E2b-]-HA and Ad5 [E1-, E2b-]-NA. Ad5 naïve BALB/c mice (n = 5/group) were immunized three times every two weeks using a dose of 10⁸, 10⁹ or 10¹⁰ VP Ad5 [E1-, E2b-]-HA (a), Ad5 [E1-, E2b-]-NA (b) or Ad5 [E1-, E2b-]-HA + NA (c and d) or buffer alone (controls). Fourteen days after the final immunization, splenocytes from the mice were assessed by ELISpot analysis for IFN-γ (black bars) or IL-2 (striped bars) secretion following antigen peptide stimulation. The greatest induction of CMI was achieved using 10¹⁰ VP of the vector. For positive controls, splenocytes were pulsed with concanavalin A in all ELISpot assays (data not shown). The error bars depict the standard error of the mean (SEM).

Fig. 2

Immune protection from lethal H1N1 challenge in mice. Mice (n = 15) were immunized with Ad5 [E1-, E2b-]-HA + NA (2 × 10¹⁰ VP total) once (●), twice (■) or three times (▲) at a two week interval. A fourth group of mice were immunized three times at a two week interval with 10¹⁰ VP of Ad5 [E1-, E2b-]-HA alone (▼). Mice immunized with Ad5-null, an Ad5 vector with no transgene insert, were used as a vector control group (○) and non-immunized mice were used as experimental controls (◆). Forty-six days after the last vaccination, mice were inoculated intranasally with 10⁵ PFU/animal of Influenza H1N1 A/CA/07/2009 and weight loss (a) and survival (b) were recorded and evaluated. p < 0.01; log rank test between vaccinated and vector control mice survival and p < 0.01; log rank test, between vaccinated and experimental control mice survival by Mantel–Cox analysis.

Fig. 3

Photomicrographs of the lung sections of H1N1 challenged mice. Lung sections from mice at 6 days post challenge were hematoxylin and eosin stained. Lungs from mice immunized one (a), two (b) or three times (c) with Ad5 [E1-, E2b-]-HA + NA (2 × 10¹⁰ VP total), three times with Ad5 [E1-, E2b-]-HA alone (d) exhibited no signs of H1N1 infection and the airway epithelium remained intact (original magnification 40×). Lung samples from vector control mice (e) and non-immunized mice (f) had dense granulocytic and lymphocytic cell infiltrates.