Search

Background Although ferret antisera used in influenza surveillance did not detect antigenic drift of A(H1N1)pdm09 viruses during the 2015-2016 season, low vaccine effectiveness was reported in adults. We investigated the immune basis of low responses to circulating A(H1N1)pdm09 viruses after vaccination. Methods Prevaccination and postvaccination serum samples collected from >300 adults (aged 18-49 years) in 6 seasons (2010-2011 to 2015-2016) were analyzed using hemagglutination inhibition assays to evaluate the antibody responses to 13 A(H1N1) viruses circulated from 1977 to 2016. Microneutralization and serum adsorption assays were used to verify the 163K and 223R specificity of antibodies. Results Individual antibody profiles to A(H1N1) viruses revealed 3 priming patterns: USSR/77, TW/86, or NC/99 priming. More than 20% of adults had reduced titers to cell-propagated circulating 6B.1 and 6B.2 A(H1N1)pdm09 viruses compared with the A/California/07/2009 vaccine virus X-179A. Significantly reduced antibody reactivity to circulating viruses bearing K163Q was observed only in the USSR/77-primed cohort, whereas significantly lower reactivity caused by egg-adapted Q223R change was detected across all 3 cohorts. Conclusion Both 163K specificity driven by immune priming and 223R specificity from egg-adapted changes in the vaccine contributed to low responses to circulating A(H1N1)pdm09 viruses after vaccination. Our study highlights the need to incorporate human serology in influenza surveillance and vaccine strain selection.

Many broadly neutralizing antibodies (bnAbs) bind to conserved areas of the hemagglutinin (HA) stalk region and can inhibit the low pH induced HA conformational changes necessary for viral membrane fusion activity. We developed and evaluated a high-throughput virus-free and cell-free ELISA based low pH induced HA Conformational Change Inhibition Antibody Detection Assay (HCCIA) and a complementary proteinase susceptibility assay. Human serum samples (n = 150) were tested by HCCIA using H3 recombinant HA. Optical density (OD) ratios of mAb HC31 at pH 4.8 to pH 7.0 ranged from 0.87 to 0.09. Our results demonstrated that low pH induced HA conformational change inhibition antibodies (CCI) neutralized multiple H3 strains after removal of head-binding antibodies. The results suggest that HCCIA can be utilized to detect and characterize CCI in sera, that are potentially broadly neutralizing, and serves as a useful tool for evaluating universal vaccine candidates targeting the HA stalk.

The novel influenza A(H1N1)pdm09 virus caused an influenza pandemic in 2009. IgM, IgG, and IgA antibody responses to A(H1N1)pdm09 hemagglutinin (HA) following A(H1N1)pdm09 virus infection were analyzed to understand antibody isotype responses. Age-matched control sera collected from U.S. residents in 2007 and 2008 were used to establish baseline levels of cross-reactive antibodies. IgM responses often used as indicators of primary virus infection were mainly detected in young patient groups (≤5 years and 6 to 15 years old), not in older age groups, despite the genetic and antigenic differences between the HA of A(H1N1)pdm09 virus and pre-2009 seasonal H1N1 viruses. IgG and IgA responses to A(H1N1)pdm09 HA were detected in all age groups of infected persons. In persons 17 to 80 years old, paired acute- and convalescent-phase serum samples demonstrated ≥4-fold increases in the IgG and IgA responses to A(H1N1)pdm09 HA in 80% and 67% of A(H1N1)pdm09 virus-infected persons, respectively. The IgG antibody response to A(H1N1)pdm09 HA was cross-reactive with HAs from H1, H3, H5, and H13 subtypes, suggesting that infections with subtypes other than A(H1N1)pdm09 might result in false positives by enzyme-linked immunosorbent assay (ELISA). Lower sensitivity compared to hemagglutination inhibition and microneutralization assays and the detection of cross-reactive antibodies against homologous and heterologous subtype are major drawbacks for the application of ELISA in influenza serologic studies.

Please cite this paper as: Fry et al. (2012) The first cases of 2009 pandemic influenza A (H1N1) virus infection in the United States: a serologic investigation demonstrating early transmission. Influenza and Other Respiratory Viruses 6(3), e48–e53. Background The first two laboratory-confirmed cases of 2009 pandemic influenza A (H1N1) virus (H1N1pdm09) infection were detected in San Diego (SD) and Imperial County (IC) in southern California, April 2009. Objectives To describe H1N1pdm09 infections and transmission early in the 2009 H1N1 pandemic. Patients/Methods We identified index case-patients from SD and IC with polymerase chain reaction (PCR)-confirmed H1N1pdm09 infections and investigated close contacts for a subset of case-patients from April 17–May 6, 2009. Acute and convalescent serum was collected. Serologic evidence for H1N1pdm09 infection was determined by microneutralization and hemagglutination inhibition assays. Results Among 75 close contacts of seven index case-patients, three reported illness onset prior to patient A or B, including two patient B contacts and a third with no links to patient A or B. Among the 69 close contacts with serum collected >14 days after the onset of index case symptoms, 23 (33%) were seropositive for H1N1pdm09, and 8 (35%) had no fever, cough, or sore throat. Among 15 household contacts, 8 (53%) were seropositive for H1N1pdm09. The proportion of contacts seropositive for H1N1pdm09 was highest in persons aged 5–24 years (50%) and lowest in persons aged ≥50 years (13%) (P = 0·07). Conclusions By the end of April 2009, before H1N1pdm09 was circulating widely in the community, a third of persons with close contact to confirmed H1N1pdm09 cases had H1N1pdm09 infection in SD and IC. Three unrelated clusters during March 21–30 suggest that transmission of H1N1pdm09 had begun earlier in southern California.

Background Understanding transmissibility of influenza viruses within households is critical for guiding public health response to pandemics. We studied serologically confirmed infection and disease among household contacts of index case patients with 2009 pandemic influenza A (H1N1) virus (pH1N1) infection in a setting of minimal community pH1N1 transmission. Methods We defined index case patients as students and staff of a New York City high school with laboratory-confirmed pH1N1 infection during the earliest phase of the pH1N1 outbreak in April 2009. We visited households of index case patients twice, once in early May and again in June/July 2009. At each visit, household members (both index case patents and household contacts) provided serum samples and completed questionnaires about illness and possible risk factors. Serologic testing was performed using microneutralization and hemagglutination-inhibition assays. Results Of 79 eligible household contacts in 28 households, 19% had serologically confirmed pH1N1 infection, and 28% of those infected were asymptomatic. Serologically confirmed infection varied by age among household contacts: 36% of contacts younger than 10 years were infected, compared with 46% of contacts age 10-18 years, 8% of contacts aged 19-54 years, and 22% of contacts aged 55 years and older. Conclusions Infection rates were high for household contacts of persons with confirmed pH1N1, particularly for contacts aged 10-18 years, and asymptomatic infection was common. Efforts to reduce household transmission during influenza pandemics are important adjuncts to strategies to reduce community illness.

Matrix protein 2 (M2) of influenza A viruses is an attractive target for the development of broadly cross-protective influenza vaccines and therapeutic antibodies. The available evidence suggests that antibodies reactive to the natural tetrameric form of M2 proteins, rather than those to synthetic peptides of M2 ectodomain (M2e), best correlate with M2-mediated immune protection. However, the current ability to quantify strain-specific and/or subtype-cross-reactive M2 antibodies against the natural form of M2 antigens from influenza A viruses of different host origin is limited. In the present study, we generated a panel of 293FT transfected cell lines stably expressing full-length tetrameric forms of M2 molecules from human, avian and the swine-origin 2009 pandemic H1N1 influenza A virus, respectively, and developed an M2-293FT cell line-based flow cytometric assay (M2-FCA). Side-by-side comparison of M2-FCA with a synthetic M2e peptide-based indirect ELISA (M2e-ELISA) reveals that M2-FCA is highly efficient in quantifying both M2e sequence-specific and cross-reactive antibodies to the native form of M2 antigens. In contrast, promiscuity was evident when specificity and cross-reactivity of anti-M2 antibodies were assessed by M2e-ELISA. These results demonstrate that M2-FCA represents a rapid, simple and sensitive method to quantitatively assess specificity and cross-reactivity of anti-M2 antibodies after infection or vaccination.

Increasing resistance to currently available influenza antivirals highlights the need to develop alternate approaches for the prevention and/or treatment of influenza. DAS181 (Fludase), a novel sialidase fusion protein that enzymatically removes sialic acids on respiratory epithelium, exhibits potent antiviral activity against influenza A and B viruses. Here, we use a mouse model to evaluate the efficacy of DAS181 treatment against a highly pathogenic avian influenza H5N1 virus. When used to treat mice daily beginning 1 day before infection with A/Vietnam/1203/2004(H5N1) virus, DAS181 treatment at 1 mg/kg/day protected 100% of mice from fatal disease, prevented viral dissemination to the brain, and effectively blocked infection in 70% of mice. DAS181 at 1 mg/kg/day was also effective therapeutically, conferring enhanced survival of H5N1 virus-challenged mice when treatment was begun 72 h after infection. This notable antiviral activity underscores the potential utility of DAS181 as a new class of drug that is effective against influenza viruses with pandemic potential.

Avian H5N1 influenza viruses currently circulating in southeast Asia could potentially cause the next pandemic. However, currently licensed human vaccines are subtype-specific and do not protect against these H5N1 viruses. We aimed to develop an influenza vaccine and assessed its immunogenicity and efficacy to confer protection in BALB/c mice.We developed an egg-independent strategy to combat the avian influenza virus, because the virus is highly lethal to chickens and the maintenance of a constant supply of embryonated eggs would be difficult in a pandemic. We used a replication-incompetent, human adenoviral-vector-based, haemagglutinin subtype 5 influenza vaccine (HAd-H5HA), which induces both humoral and cell-mediated immune responses against avian H5N1 influenza viruses isolated from people.Immunisation of mice with HAd-H5HA provided effective protection from H5N1 disease, death, and primary viral replication (p0.0001) against antigenically distinct strains of H5N1 influenza viruses. Unlike the recombinant H5HA vaccine, which is based on a traditional subunit vaccine approach, HAd-H5HA vaccine induced a three-fold to eight-fold increase in HA-518-epitope-specific interferon-gamma-secreting CD8 T cells (p=0.01).Our findings highlight the potential of an Ad-vector-based delivery system, which is both egg-independent and adjuvant-independent and offers stockpiling options for the development of a pandemic influenza vaccine.

The outbreak of avian influenza H5N1 in Hong Kong in 1997 raised concerns about the potential for the H5 subtype to cause a human pandemic. In 2001 a new H5N1 virus, A/Duck Meat/Anyang/AVL-1/2001 (A/Dkmt), was isolated from imported duck meat in Korea. The pathogenesis of this virus was investigated in mice. A/Dkmt virus had low infectivity but was lethal for mice at high doses, and at lethal doses, the virus replicated in the brains of infected mice. A/Dkmt virus cross-reacted poorly with ferret antisera raised against human H5N1 viruses, but prior infection with A/Dkmt virus protected mice from death after secondary infection with human H5N1 virus.

The microneutralization assay is commonly used to detect antibodies to influenza virus, and multiple protocols are used worldwide. These protocols differ in the incubation time of the assay as well as in the order of specific steps, and even within protocols there are often further adjustments in individual laboratories. The impact these protocol variations have on influenza serology data is unclear. Thus, a laboratory comparison of the 2-day enzyme-linked immunosorbent assay (ELISA) and 3-day hemagglutination (HA) microneutralization (MN) protocols, using A(H1N1)pdm09, A(H3N2), and A(H5N1) viruses, was performed by the CONSISE Laboratory Working Group. Individual laboratories performed both assay protocols, on multiple occasions, using different serum panels. Thirteen laboratories from around the world participated. Within each laboratory, serum sample titers for the different assay protocols were compared between assays to determine the sensitivity of each assay and were compared between replicates to assess the reproducibility of each protocol for each laboratory. There was good correlation of the results obtained using the two assay protocols in most laboratories, indicating that these assays may be interchangeable for detecting antibodies to the influenza A viruses included in this study. Importantly, participating laboratories have aligned their methodologies to the CONSISE consensus 2-day ELISA and 3-day HA MN assay protocols to enable better correlation of these assays in the future.

Avian influenza A H9N2 viruses are widespread among domestic poultry and were recently isolated from humans with respiratory illness in China. Two antigenically and genetically distinct groups of H9N2 viruses (G1 and G9) are prevalent in China. To evaluate a strategy for vaccination, we compared G1 and G9 viruses for their relative immunogenicity and cross-protective efficacy. Infection of BALB/c mice with representative viruses of either group protected against subsequent challenge with the homologous or heterologous H9N2 virus in the absence of detectable cross-reactive serum hemagglutination inhibition antibody. Mice injected intramuscularly with inactivated G1 whole virus vaccine were completely protected from challenge with either H9N2 virus. In contrast, mice administered inactivated G9 vaccine were only partially protected against heterologous challenge with the G1 virus. These results have implications for the development of human vaccines against H9N2 viruses, a priority for pandemic preparedness.

The first outbreak of avian influenza A (H5N1) occurred among humans in Hong Kong in 1997. To estimate the risk of person-to-person transmission, a retrospective cohort study was conducted to compare the prevalence of H5N1 antibody among health care workers (HCWs) exposed to H5N1 case-patients with the prevalence among nonexposed HCWs. Information on H5N1 case-patient and poultry exposures and blood samples for H5N1-specific antibody testing were collected. Eight (3.7%) of 217 exposed and 2 (0.7%) of 309 nonexposed HCWs were H5N1 seropositive (P=.01). The difference remained significant after controlling for poultry exposure (P=.01). This study presents the first epidemiologic evidence that H5N1 viruses were transmitted from patients to HCWs. Human-to-human transmission of avian influenza may increase the chances for the emergence of a novel influenza virus with pandemic potential.

The first documented outbreak of human respiratory disease caused by avian influenza A (H5N1) viruses occurred in Hong Kong in 1997. The kinetics of the antibody response to the avian virus in H5N1-infected persons was similar to that of a primary response to human influenza A viruses; serum neutralizing antibody was detected, in general, >/=14 days after symptom onset. Cohort studies were conducted to assess the risk of human-to-human transmission of the virus. By use of a combination of serologic assays, 6 of 51 household contacts, 1 of 26 tour group members, and none of 47 coworkers exposed to H5N1-infected persons were positive for H5 antibody. One H5 antibody-positive household contact, with no history of poultry exposure, provided evidence that human-to-human transmission of the avian virus may have occurred through close physical contact with H5N1-infected patients. In contrast, social exposure to case patients was not associated with H5N1 infection.

During 1997 in Hong Kong, 18 human cases of respiratory illness, including 6 fatalities, were caused by highly pathogenic avian influenza A (H5N1) viruses. Since H5 viruses had previously been isolated only from avian species, the outbreak raised questions about the ability of these viruses to cause severe disease and death in humans. To better understand the pathogenesis and immunity to these viruses, we have used the BALB/c mouse model. Four H5N1 viruses replicated equally well in the lungs of mice without prior adaptation but differed in lethality for mice. H5N1 viruses that were highly lethal for mice were detected in multiple organs, including the brain. This is the first demonstration of an influenza A virus that replicates systemically in a mammalian species and is neurotropic without prior adaptation. The mouse model was also used to evaluate a strategy of vaccination against the highly pathogenic avian H5N1 viruses, using an inactivated vaccine prepared from nonpathogenic A/Duck/Singapore-Q/F119-3/97 (H5N3) virus that was antigenically related to the human H5N1 viruses. Mice administered vaccine intramuscularly, with or without alum, were completely protected from lethal challenge with H5N1 virus. Protection from infection was also observed in 70% of animals administered vaccine alone and 100% of mice administered vaccine with alum. The protective effect of vaccination correlated with the level of virus-specific serum antibody. These results suggests a strategy of vaccine preparedness for rapid intervention in future influenza pandemics that uses antigenically related nonpathogenic viruses as vaccine candidates.

Swine origin 2009 H1N1 influenza virus has spread globally to cause the first influenza pandemic of the 21st century. Serological studies can improve our understanding of the extent of human infection and risk factors associated with the transmission of this pandemic virus. The “gold standard” for serodiagnosis of human influenza virus infection is the detection of seroconversion between acute- and convalescent-stage samples. However, the timing of seroepidemiological investigations often precludes the collection of truly acute-phase sera, requiring development of serological criteria for evaluating convalescent-phase sera that optimize detection of true positives and true negatives. To guide seroepidemiological investigations into the spread of the novel 2009 pandemic H1N1 virus, we characterized serum antibody responses to 2009 H1N1 virus in 87 individuals with confirmed viral infection and 227 nonexposed U.S. individuals using microneutralization (MN) and hemagglutination inhibition (HI) assays. Sensitivity and specificity were determined for each assay alone and in combination for detection of 2009 H1N1 virus-specific antibodies in convalescent-phase sera. Although the HI assay was more specific for detecting antibody to 2009 H1N1, the MN assay was more sensitive, particularly for detecting low-titer seroconversions. A combination of titers (MN ≥ 40 and HI ≥ 20) provided the highest sensitivity (90%) and specificity (96%) for individuals aged

Background: Memory CD8 T cells to influenza A viruses are widely detectable in healthy human subjects and broadly crossreactive for serologically distinct influenza A virus subtypes. However, it is not clear to what extent such pre-existing cellular immunity can provide cross-subtype protection against novel emerging influenza A viruses. Methodology/Principal Findings: We show in the mouse model that naturally occurring sequence variations of the conserved nucleoprotein of the virus significantly impact cross-protection against lethal disease in vivo. When priming and challenge viruses shared identical sequences of the immunodominant, protective NP366/D b epitope, strong cross-subtype protection was observed. However, when they did not share complete sequence identity in this epitope, cross-protection was considerably reduced. Contributions of virus-specific antibodies appeared to be minimal under these circumstances. Detailed analysis revealed that the magnitude of the memory CD8 T cell response triggered by the NP366/D b variants was significantly lower than those triggered by the homologous NP366/D b ligand. It appears that strict specificity of a dominant public TCR to the original NP366/D b ligand may limit the expansion of cross-reactive memory CD8 T cells to the NP366/D b variants. Conclusions/Significance: Pre-existing CD8 T cell immunity may provide substantial cross-protection against heterosubtypic influenza A viruses, provided that the priming and the subsequent challenge viruses share the identical sequences of the immunodominant, protective CTL epitopes.

Influenza vaccines capable of inducing cross-reactive or heterotypic immunity could be an important first line of prevention against a novel subtype virus. Influenza virus-like particles (VLPs) displaying functional viral proteins are effective vaccines against replication-competent homologous virus, but their ability to induce heterotypic immunity has not been adequately tested. To measure VLP vaccine efficacy against a known influenza pandemic virus, recombinant VLPs were generated from structural proteins of the 1918 H1N1 virus. Mucosal and traditional parenteral administrations of H1N1 VLPs were compared for the ability to protect against the reconstructed 1918 virus and a highly pathogenic avian H5N1 virus isolated from a fatal human case. Mice that received two intranasal immunizations of H1N1 VLPs were largely protected against a lethal challenge with both the 1918 virus and the H5N1 virus. In contrast, mice that received two intramuscular immunizations of 1918 VLPs were only protected against a homologous virus challenge. Mucosal vaccination of mice with 1918 VLPs induced higher levels of cross-reactive immunoglobulin G (IgG) and IgA antibodies than did parenteral vaccination. Similarly, ferrets mucosally vaccinated with 1918 VLPs completely survived a lethal challenge with the H5N1 virus, while only a 50% survival rate was observed in parenterally vaccinated animals. These results suggest a strategy of VLP vaccination against a pandemic virus and one that stimulates heterotypic immunity against an influenza virus strain with threatening pandemic potential.

BACKGROUND: The development of new therapeutic targets and strategies to control highly pathogenic avian influenza (HPAI) H5N1 virus infection in humans is urgently needed. Broadly cross-neutralizing recombinant human antibodies obtained from the survivors of H5N1 avian influenza provide an important role in immunotherapy for human H5N1 virus infection and definition of the critical epitopes for vaccine development. METHODOLOGY/PRINCIPAL FINDINGS: We have characterized two recombinant baculovirus-expressed human antibodies (rhAbs), AVFluIgG01 and AVFluIgG03, generated by screening a Fab antibody phage library derived from a patient recovered from infection with a highly pathogenic avian influenza A H5N1 clade 2.3 virus. AVFluIgG01 cross-neutralized the most of clade 0, clade 1, and clade 2 viruses tested, in contrast, AVFluIgG03 only neutralized clade 2 viruses. Passive immunization of mice with either AVFluIgG01 or AVFluIgG03 antibody resulted in protection from a lethal H5N1 clade 2.3 virus infection. Furthermore, through epitope mapping, we identify two distinct epitopes on H5 HA molecule recognized by these rhAbs and demonstrate their potential to protect against a lethal H5N1 virus infection in a mouse model. CONCLUSIONS/SIGNIFICANCE: Importantly, localization of the epitopes recognized by these two neutralizing and protective antibodies has provided, for the first time, insight into the human antibody responses to H5N1 viruses which contribute to the H5 immunity in the recovered patient. These results highlight the potential of a rhAbs treatment strategy for human H5N1 virus infection and provide new insight for the development of effective H5N1 pandemic vaccines.

We generated a high-growth 7:1 reassortant (Len17/H5) that contained the hemagglutinin (HA) gene from non-pathogenic A/Duck/Potsdam/1402-6/86 (H5N2) virus and other genes from the cold-adapted (ca) attenuated A/Leningrad/134/17/57 (H2H2) strain. Len17/H5 demonstrated an attenuated phenotype in mice and did not infect chickens. Mice administered Len17/H5 either as a live-attenuated intranasal vaccine or as an inactivated intramuscular vaccine were substantially protected from lethal challenge with highly pathogenic A/Hong Kong/483/97 (H5N1) virus and were protected from pulmonary infection with antigenically distinct A/Hong Kong/213/2003 (H5N1) virus. The cross-protective effect correlated with the levels of virus-specific mucosal IgA and/or serum IgG antibodies. Our results suggest a new strategy of using classical genetic reassortment between a high-growth ca H2N2 strain and antigenically related non-pathogenic avian viruses to prepare live-attenuated and inactivated vaccines for influenza pandemic.

The recent emergence of highly pathogenic avian influenza virus (HPAI) strains in poultry and their subsequent transmission to humans in Southeast Asia have raised concerns about the potential pandemic spread of lethal disease. In this paper we describe the development and testing of an adenovirus-based influenza A virus vaccine directed against the hemagglutinin (HA) protein of the A/Vietnam/1203/2004 (H5N1) (VN/1203/04) strain isolated during the lethal human outbreak in Vietnam from 2003 to 2005. We expressed different portions of HA from a recombinant replication-incompetent adenoviral vector, achieving vaccine production within 36 days of acquiring the virus sequence. BALB/c mice were immunized with a prime-boost vaccine and exposed to a lethal intranasal dose of VN/1203/04 H5N1 virus 70 days later. Vaccination induced both HA-specific antibodies and cellular immunity likely to provide heterotypic immunity. Mice vaccinated with full-length HA were fully protected from challenge with VN/1203/04. We next evaluated the efficacy of adenovirus-based vaccination in domestic chickens, given the critical role of fowl species in the spread of HPAI worldwide. A single subcutaneous immunization completely protected chickens from an intranasal challenge 21 days later with VN/1203/04, which proved lethal to all control-vaccinated chickens within 2 days. These data indicate that the rapid production and subsequent administration of recombinant adenovirus-based vaccines to both birds and high-risk individuals in the face of an outbreak may serve to control the pandemic spread of lethal avian influenza.

Since 1997, outbreaks of highly pathogenic (HP) H5N1 and circulation of H9N2 viruses among domestic poultry in Asia have posed a threat to public health. To better understand the extent of transmission of avian influenza viruses (AIV) to humans in Asia, we conducted a cross-sectional virologic study in live bird markets (LBM) in Hanoi, Vietnam, in October 2001. Specimens from 189 birds and 18 environmental samples were collected at 10 LBM. Four influenza A viruses of the H4N6 ( n = 1), H5N2 ( n = 1), and H9N3 ( n = 2) subtypes were isolated from healthy ducks for an isolation frequency of over 30% from this species. Two H5N1 viruses were isolated from healthy geese. The hemagglutinin (HA) genes of these H5N1 viruses possessed multiple basic amino acid motifs at the cleavage site, were HP for experimentally infected chickens, and were thus characterized as HP AIV. These HA genes shared high amino acid identities with genes of other H5N1 viruses isolated in Asia during this period, but they were genetically distinct from those of H5N1 viruses isolated from poultry and humans in Vietnam during the early 2004 outbreaks. These viruses were not highly virulent for experimentally infected ducks, mice, or ferrets. These results establish that HP H5N1 viruses with properties similar to viruses isolated in Hong Kong and mainland China circulated in Vietnam as early as 2001, suggest a common source for H5N1 viruses circulating in these Asian countries, and provide a framework to better understand the recent widespread emergence of HP H5N1 viruses in Asia.

An unprecedented outbreak of H5N1 highly pathogenic avian influenza (HPAI) has been reported for poultry in eight different Asian countries, including South Korea, since December 2003. A phylogenetic analysis of the eight viral genes showed that the H5N1 poultry isolates from South Korea were of avian origin and contained the hemagglutinin and neuraminidase genes of the A/goose/Guangdong/1/96 (Gs/Gd) lineage. The current H5N1 strains in Asia, including the Korean isolates, share a gene constellation similar to that of the Penfold Park, Hong Kong, isolates from late 2002 and contain some molecular markers that seem to have been fixed in the Gs/Gd lineage virus since 2001. However, despite genetic similarities among recent H5N1 isolates, the topology of the phylogenetic tree clearly differentiates the Korean isolates from the Vietnamese and Thai isolates which have been reported to infect humans. A representative Korean isolate was inoculated into mice, with no mortality and no virus being isolated from the brain, although high titers of virus were observed in the lungs. The same isolate, however, caused systemic infections in chickens and quail and killed all of the birds within 2 and 4 days of intranasal inoculation, respectively. This isolate also replicated in multiple organs and tissues of ducks and caused some mortality. However, lower virus titers were observed in all corresponding tissues of ducks than in chicken and quail tissues, and the histological lesions were restricted to the respiratory tract. This study characterizes the molecular and biological properties of the H5N1 HPAI viruses from South Korea and emphasizes the need for comparative analyses of the H5N1 isolates from different countries to help elucidate the risk of a human pandemic from the strains of H5N1 HPAI currently circulating in Asia.

Influenz a viruses cause annual epidemics and occasional pandemics of acute respiratory disease. Improved vaccines that can overcome the decline in immune function with aging and/or can induce broader immunity to novel pandemic strains are a high priority. To design improved vaccines for the elderly, we need to better understand the effects of age on both innate and adaptive immunity. In a murine model, we have determined that defects in antigen-presenting cell (APC) expression of pattern-recognition molecules, costimulatory molecules, and cytokine production may play an important role in the reduced clonal expansion of T cells in aging. The use of immunomodulators such as adjuvants may overcome some of the defects of aging immunity and may also be, useful in the development of improved vaccines for avian influenza A subtypes that pose a pandemic threat. Several novel strategies including the use of ISCOM-formulated vaccines, mucosal delivery, or DNA vaccination provided cross-subtype protection that could provide an important component of immunity in the event of a pandemic.

Avian influenza A viruses are the ancestral origin of all human influenza viruses. The outbreak of highly pathogenic (HP) avian H5N1 in Hong Kong in 1997 highlighted the potential of these viruses to infect and cause severe disease in humans. Since 1999, HP H5N1 viruses were isolated several times from domestic poultry in Asia. In 2001, a HP H5N1 virus, A/Duck/Anyang/AVL-1/2001 (Dk/Anyang), was isolated from imported frozen duck meat in Korea. Because of this novel source of HP H5N1 virus isolation, concerns were raised about the potential for human exposure and infection; we therefore compared the Dk/Anyang virus with HP H5N1 viruses isolated from humans in 1997 in terms of antigenicity and pathogenicity for mammals. At high doses, Dk/Anyang virus caused up to 50% mortality in BALB/c mice, was isolated from the brains and lymphoid organs of mice, and caused lymphopenia. Overall Dk/Anyang virus was substantially less pathogenic for mice than the H5N1 virus isolated from a fatal human case in 1997. Likewise, Dk/Anyang virus was apathogenic for ferrets. Dk/Anyang virus was antigenically distinguishable by hemagglutination-inhibition (HI) assay from human H5N1 viruses isolated in 1997 and avian H5N1 viruses isolated in 2001 in Hong Kong. Nevertheless, prior infection with Dk/Anyang virus protected mice from death after secondary infection with HP human H5N1 viruses. These results indicate that compared with HP human H5N1 viruses, Dk/Anyang virus is substantially less pathogenic for mammalian species. Nevertheless, the novel source of isolation of this avian H5N1 virus must be considered when evaluating the potential risk to public health.

Avian influenza A H5N1 viruses similar to those that infected humans in Hong Kong in 1997 continue to circulate in waterfowl and have reemerged in poultry in the region, raising concerns that these viruses could reappear in humans. The currently licensed trivalent inactivated influenza vaccines contain hemagglutinin (HA) and neuraminidase genes from epidemic strains in a background of internal genes derived from the vaccine donor strain, A/Puerto Rico/8/34 (PR8). Such reassortant candidate vaccine viruses are currently not licensed for the prevention of human infections by H5N1 influenza viruses. A transfectant H5N1/PR8 virus was generated by plasmid-based reverse genetics. The removal of the multibasic amino acid motif in the HA gene associated with high pathogenicity in chickens, and the new genotype of the H5N1/PR8 transfectant virus, attenuated the virus for chickens and mice without altering the antigenicity of the HA. A Formalin-inactivated vaccine prepared from this virus was immunogenic and protected mice from subsequent wild-type H5N1 virus challenge. This is the first successful attempt to develop an H5N1 vaccine seed virus resembling those used in currently licensed influenza A vaccines with properties that make it a promising candidate for further evaluation in humans.

In 1997, outbreaks of highly pathogenic influenza A (H5N1) among poultry coincided with 18 documented human cases of H5N1 illness. Although exposure to live poultry was associated with human illness, no cases were documented among poultry workers (PWs). To evaluate the potential for avian-to-human transmission of H5N1, a cohort study was conducted among 293 Hong Kong government workers (GWs) who participated in a poultry culling operation and among 1525 PWs. Paired serum samples collected from GWs and single serum samples collected from PWs were considered to be anti-H5 antibody positive if they were positive by both microneutralization and Western blot testing. Among GWs, 3% were seropositive, and 1 seroconversion was documented. Among PWs, approximately 10% had anti-H5 antibody. More-intensive poultry exposure, such as butchering and exposure to ill poultry, was associated with having anti-H5 antibody. These findings suggest an increased risk for avian influenza infection from occupational exposure.

Background: In 1997, highly pathogenic avian influenza A H5N1 viruses caused 18 human cases of respiratory illness and six deaths in Hong Kong. The genes of the H5N1 viruses isolated from humans were derived from avian influenza viruses, with no evidence of reassortment with human influenza viruses. The molecular determinants of human pathogenesis were not evident. Methods: The BALB/c mouse was used as a mammalian model to evaluate the biological and molecular basis of human H5N1 virus pathogenesis. Molecular correlates of H5N1 virus pathogenicity for mice were sought by analyzing the nucleotide sequence of human H5N1 viruses. Results: Based on high and low lethality for mice, the pathogenicity phenotype of 15 human H5N1 viruses was characterized; nine viruses displayed a high, five a low, and one an intermediate pathogenicity phenotype. H5N1 viruses with a high pathogenicity phenotype replicated in multiple solid organs and caused depletion of peripheral blood leukocytes. Sequence analysis determined that five specific amino acids in four proteins correlated with pathogenicity in mice. Conclusions: Alone or in combination, these specific residues are the likely determinants of virulence of human H5N1 influenza viruses in this mammalian model.

Previously, we observed that several virulent influenza A (H5N1) viruses which caused severe or fatal disease in humans were also lethal in BALB/c mice following dissemination of the virus to solid organs, including the brain. In contrast, one particular human H5N1 virus was nonlethal in mice and showed no evidence of systemic spread. To compare H5N1 viruses of varying pathogenicity for their ability to alter the mammalian immune system, mice were infected with either influenza A/Hong Kong/483/97 (HK/483) (lethal) or A/Hong Kong/486/97 (HK/486) (nonlethal) virus and monitored for lymphocyte depletion in the blood, lungs, and lymphoid tissue. Intranasal infection with HK/483 resulted in a significant decrease in the total number of circulating leukocytes evident as early as day 2 postinfection. Differential blood counts demonstrated up to an 80% drop in lymphocytes by day 4 postinfection. In contrast, nonlethal HK/486-infected mice displayed only a transient drop of lymphocytes during the infectious period. Analysis of lung and lymphoid tissue from HK/483-infected mice demonstrated a reduction in the number of CD4+and CD8+T cells and reduced synthesis of the cytokines interleukin-1β and gamma interferon and the chemokine macrophage inflammatory protein compared with HK/486-infected mice. In contrast, the cytokine and chemokine levels were increased in the brains of mice infected with HK/483 but not HK/486. Evidence of apoptosis in the spleen and lung of HK/483-infected mice was detected in situ, suggesting a mechanism for lymphocyte destruction. These results suggest that destructive effects on the immune system may be one factor that contributes to the pathogenesis of H5N1 viruses in mammalian hosts.

The use of adjuvants is one approach to improve influenza vaccine immunogenicity and efficacy, particularly in aged populations. The response of BALB/c mice to subcutaneously administered formalin-inactivated whole influenza virus vaccine in the presence or absence of a nonionic block copolymer adjuvant CRL1005 was evaluated. In young adult naive mice, the copolymer adjuvant significantly enhanced virus-specific IgG and hemagglutination-inhibition (HI) antibody responses and augmented the production of IL-2 following vaccination. Influenza vaccine formulated with 2.5 mg CRL1005 significantly enhanced the protective efficacy of the inactivated vaccine in the upper and lower respiratory tract. In mice previously infected with influenza virus or naive aged mice, inactivated vaccine administered with the copolymer adjuvant substantially enhanced the serum HI antibody response to inactivated influenza vaccine and significantly reduced lung virus titers following subsequent challenge with live virus compared with mice administered vaccine alone. These results suggest that the copolymer adjuvant warrants further investigation as a potential adjuvant for use in human vaccination against influenza.

Alternative strategies for vaccination against influenza that elicit both systemic antibody and mucosal IgA responses are needed to improve the efficacy in protection against infection. This study demonstrated that oral delivery of inactivated influenza vaccine with the heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli elicited the spectrum of humoral and cell-mediated responses in BALB/c mice critical for the protection and recovery from influenza virus infection. Coadministration of LT with oral influenza vaccine increased antiviral serum IgG and mucosal IgA responses compared with administration of oral influenza vaccine alone. Serum hemagglutination-inhibition and neutralizing antibodies were also augmented by LT. The adjuvant potentiated protection from infection with influenza A H3N2 viruses in mouse lower and upper respiratory tracts, enabling the use of lower doses of oral vaccine. Coadministration of LT with oral inactivated influenza vaccine induced influenza virus-specific proliferative T cells, interleukin-2 production, and major histocompatibility complex class I-restricted cytotoxic T cells.

BACKGROUND: The frequency of avian influenza A virus infections among poultry workers is not well understood. METHODS: A seroprevalence study of market poultry workers and persons without occupational poultry exposure was conducted during 2001 in Hanoi, Vietnam. Sera were tested for avian influenza H5 and H9 antibodies by microneutralization and Western blot assays. RESULTS: Seroprevalence of H5 and H9 antibodies was 4% and 3% in poultry workers and 1% and 3.5% in non-poultry workers, respectively. CONCLUSIONS: Seroprevalence of H5 and H9 antibodies was low among Hanoi market poultry workers in 2001, but can serve as a baseline for additional studies.

Avian H7 is one of several influenza A virus subtypes that have the potential to cause pandemics. Herein we describe preclinical results following administration of an investigational H7N1 inactivated detergent-split virion vaccine adjuvanted with the AS03 Adjuvant System. The adjuvanted H7N1 vaccine was highly immunogenic compared to the non-adjuvanted H7N1 vaccine in unprimed mice with less than 100 ng of hemagglutinin antigen per dose. In addition, compared to the non-adjuvanted vaccine, the AS03-adjuvanted H7N1 vaccine also induced robust HI and VN antibody responses that cross-reacted with other H7 subtypes, including recently emerged H7N9 virus. These H7 data from the preclinical mouse model add to the existing H5 data to suggest that AS03 adjuvant technology may be generally effective for formulating antigen-sparing detergent-split virion vaccines against intrinsically sub-immunogenic avian influenza A virus subtypes.