New pre-pandemic influenza vaccines: an egg- and adjuvant-independent human adenoviral vector strategy induces long-lasting protective immune responses in mice

Abstract

Highly pathogenic avian H5N1 influenza viruses that are currently circulating in southeast Asia may acquire the potential to cause the next influenza pandemic. A number of alternate approaches are being pursued to generate cross-protective, dose-sparing, safe, and effective vaccines, as traditional vaccine approaches, i.e., embryonated egg-grown, are not immunogenic. We developed a replication-incompetent adenoviral vector-based, adjuvant- and egg-independent pandemic influenza vaccine strategy as a potential alternative to conventional egg-derived vaccines. In this paper, we address suboptimal dose and longevity of vaccine-induced protective immunity and demonstrate that a vaccine dose as little as 1 x 10(6) plaque-forming unit (PFU) is sufficient to induce protective immune responses against a highly pathogenic H5N1 virus. Furthermore, the vaccine-induced humoral and cellular immune responses and protective immunity persisted at least for a year.

Figure 1. Preventive vaccination strategies for pandemic preparedness

In preparation for a pandemic, a number of vaccine approaches are currently being investigated in preclinical and clinical trials, where each approach is depicted in one of the six circles surrounding the core, described here as pandemic influenza vaccines. Other factors affecting the outcome of a successful vaccine are range of dose, including dose-limiting strategies and immunization schedule, and formulation, with adjuvants or other immunostimulatory compounds, in efforts to meet the preferred profile for an effective vaccine. RG, reverse genetics. VSV, vesicular stomatitis virus.

Figure 2. HAd-H5HA vaccine induces neutralizing antibody and HI titers in a dose-dependent manner

BALB/c mice were immunized i.m. with varying doses of HAd-H5HA vaccine starting from 10⁹ PFU twice 4 weeks apart as indicated on the X axis. An empty vector group (HAd-ΔE1E3) was included as a negative control. Four weeks post-boost, sera were obtained for detection of neutralizing (solid bars) and hemagglutinating (empty bars) antibodies by virus microneutralization assay and HI assay using horse red blood cells, respectively.

Figure 3. HAd-H5HA vaccine induces protection against lethal challenge in a dose-dependent manner

BALB/c mice were immunized i.m. with five consecutive log dilutions (▲10⁹ ; ● 10⁸ ; △10⁷ ; X 10⁶ ; and □10⁵ PFU) of HAd-H5HA vaccine twice 4 weeks apart. An empty vector group (○) was included as a negative control. Four weeks post-boost, the mice were challenged with a 10 LD₅₀ of A/Hong Kong/483/97 and monitored for clinical signs and weight loss for 14 days. The figure shows (a) percent survival after lethal challenge and (b) morbidity as determined by weight loss.

Figure 4. HAd-H5HA vaccine induces durable serological responses

(a) BALB/c mice were immunized i.m. (solid bars) or i.n. (empty bars) with 10⁸ PFU of HAd-H5HA or i.m. with empty vector (hatched bars) twice 4 weeks apart. Sera were obtained 4, 28, and 48 weeks post-boost for detection of antibodies by HI assay using horse red blood cells. (b) After 48 weeks, three additional groups of naive mice were immunized on the same schedule as those immunized before. Sera were obtained 4 weeks post-boost from all six groups of mice (4 weeks and 56 weeks) to determine neutralizing antibody titers by virus microneutralization assay.

Figure 5. HAd-H5HA vaccine induces long-term persistence of epitope-specific CD8+ T-cell responses

BALB/c mice were immunized i.m. or i.n. with 10⁸ PFU of HAd-H5HA or i.m. with empty vector twice 4 weeks apart and aged for 55 weeks. Three additional groups of mice were immunized on the same schedule as those immunized 48 weeks before. Spleens were harvested from four mice in each group at 4 and 55 weeks post-boost and passed through screens to obtain a single-cell suspension for each mouse. Splenocytes (1 × 10⁶ ) were resuspended in phosphate-buffered saline with 2% bovine serum albumin and 0.05% sodium azide (staining buffer) and blocked for 5 min on ice with Fc block, followed by PE (phycoerythrin)-labeled HA518 or NP147 epitope-specific pentamer and anti-CD8 antibody conjugated with APC (allophycocyanin) and analyzed by flow cytometry, with a tight gate around the CD8 + T-cell population. Data are expressed as box and whisker plots (interquartile ranges). *P<0.001.

Figure 6. HAd-H5HA vaccine confers long-term protective immunity

(a) BALB/c mice were immunized i.m. (■) or i.n. (●) with 10⁸ PFU of HAd-H5HA or i.m. with empty vector (▲) twice 4 weeks apart and aged for an additional 56 weeks. (b) Forty-eight weeks after the first three groups were immunized, three additional groups of mice were immunized on the same schedule as those immunized before. All groups of mice were challenged with 50 LD₅₀ of A/Hong Kong/483/97 and monitored for clinical signs and weight loss for 14 days.