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H5N1 virus infection results in ~60% mortality in patients primarily due to respiratory failure, but the underlying causes of mortality are unclear. The goal of this study is to reveal respiratory disorders occurring at the early stage of infection that may be responsible for subsequent respiratory failure and death. BALB/c mice were intranasally infected with one of two H5N1 virus strains: HK483 (lethal) or HK486 (non-lethal) virus. Pulmonary ventilation and the responses to hypoxia (HVR; 7% O2 for 3 min) and hypercapnia (HCVR; 7% CO2 for 5 min) were measured daily at 2 days prior and 1, 2, and 3 days postinfection (dpi) and compared to mortality typically by 8 dpi. At 1, 2, and 3 dpi, immunoreactivities (IR) of substance P (SP-IR) in the nodose ganglion or tyrosine hydroxylase (TH-IR) in the carotid body coupled with the nucleoprotein of influenza A (NP-IR) was examined in some mice, while arterial blood was collected in others. Our results showed that at 2 and 3 dpi: 1) both viral infections failed to alter body temperature and weight, [Formula: see text], or induce viremia while producing similarly high lung viral titers; 2) HK483, but not HK486, virus induced tachypnea and depressed HVR and HCVR without changes in arterial blood pH and gases; and 3) only HK483 virus led to NP-IR in vagal SP-IR neurons, but not in the carotid body, and increased density of vagal SP-IR neurons. In addition, all HK483, rather than HK486, mice died at 6 to 8 dpi and the earlier death was correlated with more severe depression of HVR and HCVR. Our data suggest that tachypnea and depressed HVR/HCVR occur at the early stage of lethal H5N1 viral infection associated with viral replication and increased SP-IR density in vagal neurons, which may contribute to the respiratory failure and death.

The reduced immunogenicity of the H5 hemagglutinin (HA), compared to seasonal HA serotypes, has stimulated searches for effective adjuvants to improve H5 vaccine efficacy. This study examined the immunogenicity and protective efficacy in ferrets immunized with a split-virion H5N1 vaccine combined with Advax™, a novel delta inulin-based polysaccharide adjuvant technology that has previously demonstrated ability to augment humoral and cellular immunity to co-administered antigens.Ferrets were vaccinated twice 21 days apart with 7.5 μg or 22.5 μg of a split-virion preparation of A/Vietnam/1203/2004 with or without adjuvant. An additional group received just one immunization with 22.5 μg HA plus adjuvant. Serum antibodies were measured by hemagglutination inhibition and microneutralization assays. Vaccinated animals were challenged intranasally 21 days after the last immunization with 10(6) EID(50) of the homologous strain. Morbidity was assessed by observed behavior, weight loss, temperature, cytopenias, histopathology, and viral load.No serum neutralization antibody was detected after two immunizations with unadjuvanted vaccine. Two immunizations with high or low dose adjuvanted vaccine stimulated high neutralizing antibody titers. Survival was 100% in all groups receiving adjuvanted-vaccine including the single dose group, compared to 67% survival with unadjuvanted vaccine, and 0% survival in saline or adjuvant-alone controls. Minimal morbidity was seen in all animals receiving adjuvanted vaccine, and was limited to rhinorrhea and mild thrombocytopenia, without fever, weight loss, or reduced activity. H5N1 virus was cleared from the nasal wash by day 4 post-challenge only in animals receiving adjuvanted vaccine which also prevented viral invasion of the brain in most animals.In this initial study, Advax™ adjuvant formulations improved the protective efficacy of a split-virion H5N1 vaccine as measured by significantly enhanced immunogenicity, survival, and reduced morbidity.

Highly pathogenic avian influenza (HPAI) has an approximate 60% fatality rate in humans. In the United States, a vaccine for HPAI has been manufactured and stockpiled using FDA approved methods for seasonal vaccines. Compared to seasonal vaccines, the HPAI vaccine is less immunogenic in humans despite similar production methods. We developed a mouse model of influenza vaccination to examine differences in the host immune response to these vaccines. Mice that received the seasonal vaccine produced a robust neutralizing antibody response whereas no neutralizing antibodies were detected following HPAI vaccination. Dendritic cells (DCs) cultured with HPAI vaccine had decreased expression of activation markers CD40 and CD86, and produced significantly lower levels of cytokines IL-6, IL-12, and TNF-α compared to those treated with seasonal vaccine. C-type lectin receptors have been implicated as a class of pattern recognition receptors in innate immunity and immunization. Pretreatment with mannan diminished cytokine induction by DCs in a dose dependent manner following seasonal but not HPAI vaccine treatment, suggesting a role for C-type lectin receptors in DC activation by influenza vaccines. These findings implicate a potential mechanism for attenuated DC function following HPAI vaccination and may explain the lack of immunogenicity of the currently approved HPAI vaccine.

Highly pathogenic avian influenza (HPAI) is an emerging infectious virus with an approximate 60% fatality rate in humans. In the United States, a vaccine for H5N1 has been developed and stockpiled in case of a pandemic. This vaccine demonstrated low immunogenicity in human clinical trials with only 22% of recipients developing protective antibody titers. Previous studies using a lethal challenge ferret model have shown that supplementing the H5N1 vaccine with an alum adjuvant can improve immunogenicity and survival. We developed a lethal challenge mouse model of H5N1 vaccination to examine differences in innate immune responses to the vaccine alone or in the presence of an alum adjuvant. Mice treated with the adjuvanted vaccine showed significantly lower weight loss and higher survival than mice treated with the nonadjuvanted vaccine. Further, mice treated with adjuvanted vaccine had higher neutralizing antibody titers when compared to mice that received the nonadjuvanted vaccine. In vitro, dendritic cells (DCs) cultured with the adjuvanted vaccine had increased expression of the activation markers CD40 and CD86 compared to DCs treated with the nonadjuvanted vaccine. Interestingly, neither treatment induced activation of Toll-like receptors or the production of cytokines IL-6, IL-12, or TNF-α. Taken together, our results suggest that the alum adjuvant rescues immunogenicity and improves survival in the host.