The mechanisms responsible for the virulence of the highly pathogenic avian influenza (HPAI) and of the 1918 pandemic influenza virus in humans remain poorly understood. To identify crucial components of the early host response during these infections by using both conventional and functional genomics tools, we studied 34 cynomolgus macaques (Macaca fascicularis) to compare a 2004 human H5N1 Vietnam isolate with 2 reassortant viruses possessing the 1918 hemagglutinin (HA) and neuraminidase (NA) surface proteins, known conveyors of virulence. One of the reassortants also contained the 1918 nonstructural (NS1) protein, an inhibitor of the host interferon response. Among these viruses, HPAI H5N1 was the most virulent. Within 24 h, the H5N1 virus produced severe bronchiolar and alveolar lesions. Notably, the H5N1 virus targeted type II pneumocytes throughout the 7-day infection, and induced the most dramatic and sustained expression of type I interferons and inflammatory and innate immune genes, as measured by genomic and protein assays. The H5N1 infection also resulted in prolonged margination of circulating T lymphocytes and notable apoptosis of activated dendritic cells in the lungs and draining lymph nodes early during infection. While both 1918 reassortant viruses also were highly pathogenic, the H5N1 virus was exceptional for the extent of tissue damage, cytokinemia, and interference with immune regulatory mechanisms, which may help explain the extreme virulence of HPAI viruses in humans.1918 pandemic ͉ functional genomics ͉ H5N1

The pathogenicity and transmission of influenza A viruses are likely determined in part by replication efficiency in human cells, which is the net effect of complex virus-host interactions. H5N1 avian, H1N1 seasonal, and H1N1 2009 pandemic influenza virus strains were compared by infecting human differentiated bronchial epithelial cells in air-liquid interface cultures at relatively low virus particle/cell ratios. Differential equation and computational models were used to characterize the in vitro kinetic behaviors of the three strains. The models were calibrated by fitting experimental data in order to estimate difficult-to-measure parameters. Both models found marked differences in the relative values of p, the virion production rate per cell, and R 0 , an index of the spread of infection through the monolayer, with the values for the strains in the following rank order (from greatest to least): pandemic strain, followed by seasonal strain, followed by avian strain, as expected. In the differential equation model, which treats virus and cell populations as well mixed, R 0 and p varied proportionately for all 3 strains, consistent with a primary role for productivity. In the spatially explicit computational model, R 0 and p also varied proportionately except that R 0 derived for the pandemic strain was reduced, consistent with constrained viral spread imposed by multiple host defenses, including mucus and paracrine antiviral effects. This synergistic experimental-computational strategy provides relevant parameters for identifying and phenotyping potential pandemic strains.Influenza viruses cause annual epidemics and occasional pandemics, and the recent pandemic due to the swine-origin 2009 (H1N1) virus provides a fresh opportunity to explore in vitro and in vivo measures of pathogenicity and transmission among different strains (12,23,31). Comparative analysis of virulence and transmission in animal models has been informative (21,24,30), although the sensitivity of this approach in identifying a pandemic strain is not yet clear. The ultimate goal of linking detailed phenotype with detailed genotype is impeded by a limited repertoire of tools to characterize the strain phenotype.Pathogenicity and transmission are in part functions of virus replication, but the complex kinetics of replication are not necessarily reflected in simple counts of virus particles harvested from infected mucosa in vivo or from cell monolayers in vitro. Strains differ dramatically in their entry and replication efficiencies within specific cell types, and they display various potencies in suppressing cell defenses (43). Strains also differ in their efficiencies of person-to-person transmission as measured in closed populations and expressed as the basic reproductive number R 0 (12). The kinetics of viral replication in respiratory mucosal epithelial cells may reflect critical determinants in person-to-person transmission.Using a seasonal strain as a reference point, we compared its replication kinetics to that of an avian strain (as a ...

Immunotherapeutic approaches to cancer should focus on novel undertakings that modulate immune responses by synergistic enhancement of antitumor immunological parameters. Cancer vaccines should preferably be composed of multiple defined tumor Ag-specific B and T cell epitopes. To develop a multiepitope vaccine, 12 high ranking B cell epitopes were identified from the extracellular domain of the human epidermal growth factor receptor-2 (HER-2) oncoprotein by computer-aided analysis. Four novel HER-2 B cell epitopes were synthesized as chimeras with a promiscuous T cell epitope (aa 288–302) from the measles virus fusion protein (MVF). Two chimeric peptide vaccines, MVF HER-2316–339 and MVF HER-2485–503 induced high levels of Abs in outbred rabbits, which inhibited tumor cell growth. In addition, Abs induced by a combination of two vaccines, MVF HER-2316–339 and MVF HER-2628–647 down-modulated receptor expression and activated IFN-γ release better than the individual vaccines. Furthermore, this multiepitope vaccine in combination with IL-12 caused a significant reduction (p = 0.004) in the number of pulmonary metastases induced by challenge with syngeneic tumor cells overexpressing HER-2. Peptide Abs targeting specific sites in the extracellular domain may be used for exploring the oncoprotein’s functions. The multiepitope vaccine may have potential application in the treatment of HER-2-associated cancers.

Highly pathogenic avian influenza A (HPAI), subtype H5N1, remains an emergent threat to the human population. While respiratory disease is a hallmark of influenza infection, H5N1 has a high incidence of neurological sequelae in many animal species and sporadically in humans. We elucidate the temporal/spatial infection of H5N1 in the brain of ferrets following a low dose, intranasal infection of two HPAI strains of varying neurovirulence and lethality. A/Vietnam/1203/2004 (VN1203) induced mortality in 100% of infected ferrets while A/Hong Kong/483/1997 (HK483) induced lethality in only 20% of ferrets, with death occurring significantly later following infection. Neurological signs were prominent in VN1203 infection, but not HK483, with seizures observed three days post challenge and torticollis or paresis at later time points. VN1203 and HK483 replication kinetics were similar in primary differentiated ferret nasal turbinate cells, and similar viral titers were measured in the nasal turbinates of infected ferrets. Pulmonary viral titers were not different between strains and pathological findings in the lungs were similar in severity. VN1203 replicated to high titers in the olfactory bulb, cerebral cortex, and brain stem; whereas HK483 was not recovered in these tissues. VN1203 was identified adjacent to and within the olfactory nerve tract, and multifocal infection was observed throughout the frontal cortex and cerebrum. VN1203 was also detected throughout the cerebellum, specifically in Purkinje cells and regions that coordinate voluntary movements. These findings suggest the increased lethality of VN1203 in ferrets is due to increased replication in brain regions important in higher order function and explains the neurological signs observed during H5N1 neurovirulence.

Background 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. Methods 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 106 EID50 of the homologous strain. Morbidity was assessed by observed behavior, weight loss, temperature, cytopenias, histopathology, and viral load. Results 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. Conclusions In this initial study, Advax™ adjuvant formulations improved the protective efficacy of a split-virion H5N1vaccine as measured by significantly enhanced immunogenicity, survival, and reduced morbidity.