Enhanced Immunogenicity, Mortality Protection, and Reduced Viral Brain Invasion by Alum Adjuvant with an H5N1 Split-Virion Vaccine in the Ferret

Pre-pandemic development of an inactivated, split-virion avian influenza vaccine is challenged by the lack of pre-existing immunity and the reduced immunogenicity of some H5 hemagglutinins compared to that of seasonal influenza vaccines. Identification of an acceptable effective adjuvant is needed to improve immunogenicity of a split-virion avian influenza vaccine.No serum antibodies were detected after vaccination with unadjuvanted vaccine, whereas alum-adjuvanted vaccination induced a robust antibody response. Survival after unadjuvanted dose regimens of 30 µg, 7.5 µg and 1.9 µg (21-day intervals) was 64%, 43%, and 43%, respectively, yet survivors experienced weight loss, fever and thrombocytopenia. Survival after unadjuvanted dose regimen of 22.5 µg (28-day intervals) was 0%, suggesting important differences in intervals in this model. In contrast to unadjuvanted survivors, either dose of alum-adjuvanted vaccine resulted in 93% survival with minimal morbidity and without fever or weight loss. The rarity of brain inflammation in alum-adjuvanted survivors, compared to high levels in unadjuvanted vaccine survivors, suggested that improved protection associated with the alum adjuvant was due to markedly reduced early viral invasion of the ferret brain.Alum adjuvant significantly improves efficacy of an H5N1 split-virion vaccine in the ferret model as measured by immunogenicity, mortality, morbidity, and brain invasion

Severe acute respiratory syndrome-coronavirus infection in aged nonhuman primates is associated with modulated pulmonary and systemic immune responses

BACKGROUND: Many respiratory viruses disproportionately impact the elderly. Likewise, advanced age correlated with more adverse disease outcomes following severe acute respiratory syndrome coronavirus (SARS-CoV) infection in humans. We used an aged African green monkey SARS-CoV infection model to better understand age-related mechanisms of increased susceptibility to viral respiratory infections. Nonhuman primates are critical translational models for such research given their similarities to humans in immune-ageing as well as lung structure. RESULTS: Significant age- and infection-dependent differences were observed in both systemic and mucosal immune compartments. Peripheral lymphocytes, specifically CD8 T and B cells were significantly lower in aged monkeys pre- and post- SARS-CoV infection, while neutrophil and monocyte numbers were not impacted by age or infection status. Serum proinflammatory cytokines were similar in both age groups, whereas significantly lower levels of IL-1beta, IL-18, IL-6, IL-12 and IL-15 were detected in the lungs of SARS-CoV-infected aged monkeys at either 5 or 10 days post infection. Total lung leukocyte numbers and relative frequency of CD8 T cells, B cells, macrophages and dendritic cells were greatly reduced in the aged host during SARS-CoV infection, despite high levels of chemoattractants for many of these cells in the aged lung. Dendritic cells and monocytes/macrophages showed age-dependent differences in activation and chemokine receptor profiles, while the CD8 T cell and B cell responses were significantly reduced in the aged host. In examination of viral titers, significantly higher levels of SARS-CoV were detected in the nasal swabs early, at day 1 post infection, in aged as compared to juvenile monkeys, but virus levels were only slightly higher in aged animals by day 3. Although there was a trend of higher titers in respiratory tissues at day 5 post infection, this did not reach statistical significance and virus was cleared from all animals by day 10, regardless of age. CONCLUSIONS: This study provides unique insight into how several parameters of the systemic and mucosal immune response to SARS-CoV infection are significantly modulated by age. These immune differences may contribute to deficient immune function and the observed trend of higher SARS-CoV replication in aged nonhuman primates

Lack of Innate Interferon Responses during SARS Coronavirus Infection in a Vaccination and Reinfection Ferret Model

In terms of its highly pathogenic nature, there remains a significant need to further define the immune pathology of SARS-coronavirus (SARS-CoV) infection, as well as identify correlates of immunity to help develop vaccines for severe coronaviral infections. Here we use a SARS-CoV infection-reinfection ferret model and a functional genomics approach to gain insight into SARS immunopathogenesis and to identify correlates of immune protection during SARS-CoV-challenge in ferrets previously infected with SARS-CoV or immunized with a SARS virus vaccine. We identified gene expression signatures in the lungs of ferrets associated with primary immune responses to SARS-CoV infection and in ferrets that received an identical second inoculum. Acute SARS-CoV infection prompted coordinated innate immune responses that were dominated by antiviral IFN response gene (IRG) expression. Reinfected ferrets, however, lacked the integrated expression of IRGs that was prevalent during acute infection. The expression of specific IRGs was also absent upon challenge in ferrets immunized with an inactivated, Al(OH)3-adjuvanted whole virus SARS vaccine candidate that protected them against SARS-CoV infection in the lungs. Lack of IFN-mediated immune enhancement in infected ferrets that were previously inoculated with, or vaccinated against, SARS-CoV revealed 9 IRG correlates of protective immunity. This data provides insight into the molecular pathogenesis of SARS-CoV and SARS-like-CoV infections and is an important resource for the development of CoV antiviral therapeutics and vaccines

Lack of Innate Interferon Responses during SARS Coronavirus Infection in a Vaccination and Reinfection Ferret Model

In terms of its highly pathogenic nature, there remains a significant need to further define the immune pathology of SARS-coronavirus (SARS-CoV) infection, as well as identify correlates of immunity to help develop vaccines for severe coronaviral infections. Here we use a SARS-CoV infection-reinfection ferret model and a functional genomics approach to gain insight into SARS immunopathogenesis and to identify correlates of immune protection during SARS-CoV-challenge in ferrets previously infected with SARS-CoV or immunized with a SARS virus vaccine. We identified gene expression signatures in the lungs of ferrets associated with primary immune responses to SARS-CoV infection and in ferrets that received an identical second inoculum. Acute SARS-CoV infection prompted coordinated innate immune responses that were dominated by antiviral IFN response gene (IRG) expression. Reinfected ferrets, however, lacked the integrated expression of IRGs that was prevalent during acute infection. The expression of specific IRGs was also absent upon challenge in ferrets immunized with an inactivated, Al(OH)(3)-adjuvanted whole virus SARS vaccine candidate that protected them against SARS-CoV infection in the lungs. Lack of IFN-mediated immune enhancement in infected ferrets that were previously inoculated with, or vaccinated against, SARS-CoV revealed 9 IRG correlates of protective immunity. This data provides insight into the molecular pathogenesis of SARS-CoV and SARS-like-CoV infections and is an important resource for the development of CoV antiviral therapeutics and vaccines

Number of regulated genes in different functional categories following primary infection and reinfection.

<p>Number of regulated genes in different functional categories with at least 1.5-fold change and a significant t-test of p<0.05 (↑ upregulated, ↓ downregulated).</p

Expression of selected significantly changed genes over time in SARS-CoV infected–reinfected ferret lungs.

a<p>Mean gene expression ratios (log2) for selected genes is relative to the mock-infected ferret gene expression at 2 DPI.</p>b<p>Statistical significance of gene expression differences over time is determined by EDGE analysis as described in the Methods.</p>c<p><i>Canis familiaris</i> UniGene Build #11 (April, 2005) identifiers as per the Affymetrix GeneChip Canine Genome 2.0 Array probe library.</p

IFN responses in SARS-CoV infected-reinfected ferret lungs.

<p>(A) Fifty IRGs selected by pathway analysis are shown in a one-way hierarchical cluster. (B) IPA canonical IFN-signaling pathway analysis at 3 DPI and 3 DPR. All genes are significantly differently expressed (EDGE analysis: ≥2-fold change in at least one time point, p≤0.05, and q≤0.1).</p

Lung histopathology to SARS-CoV challenge following reinfection.

<p>Histological lung sections (5 µm) were obtained from multiple lung lobes at 7 d postchallenge and stained by hematoxylin and eosin. Representative micrographs from uninfected (A), SARS infected alone (B), or SARS infected and re-infected (C and D) are shown. Primary SARS-CoV infection produced inflammation and the appearance of lung immune cells primarily surrounding small-to-medium bronchial airways at 7 days following challenge (B). Ferrets that received infection-reinfection were largely protected from lung histopathology (C).</p

Viral burden in lung tissue.

<p>Viral burdens in sections of lung were determined by the TCID₅₀ method, as outlined in the materials and methods section. Log10 virus titres are shown on the vertical axis vs. study day on the horizontal axis. Note that ferrets were infected on study day 1, and reinfected ferrets were also inoculated on study day 30. Mock infected animals received an intranasal instillation of serum-free media on study day 1 and had undetectable virus. Values shown represent group means of 3–4 ferrets per group, and error bars show standard deviation.</p