Minor changes in the hemagglutinin of influenza A(H1N1)2009 virus alter its antigenic properties

Background

The influenza A(H1N1)2009 virus has been the dominant type of influenza A virus in Finland during the 2009–2010 and 2010–2011 epidemic seasons. We analyzed the antigenic characteristics of several influenza A(H1N1)2009 viruses isolated during the two influenza seasons by analyzing the amino acid sequences of the hemagglutinin (HA), modeling the amino acid changes in the HA structure and measuring antibody responses induced by natural infection or influenza vaccination.

Methods/Results

Based on the HA sequences of influenza A(H1N1)2009 viruses we selected 13 different strains for antigenic characterization. The analysis included the vaccine virus, A/California/07/2009 and multiple California-like isolates from 2009–2010 and 2010–2011 epidemic seasons. These viruses had two to five amino acid changes in their HA1 molecule. The mutation(s) were located in antigenic sites Sa, Ca1, Ca2 and Cb region. Analysis of the antibody levels by hemagglutination inhibition test (HI) indicated that vaccinated individuals and people who had experienced a natural influenza A(H1N1)2009 virus infection showed good immune responses against the vaccine virus and most of the wild-type viruses. However, one to two amino acid changes in the antigenic site Sa dramatically affected the ability of antibodies to recognize these viruses. In contrast, the tested viruses were indistinguishable in regard to antibody recognition by the sera from elderly individuals who had been exposed to the Spanish influenza or its descendant viruses during the early 20^th century.

Conclusions

According to our results, one to two amino acid changes (N125D and/or N156K) in the major antigenic sites of the hemagglutinin of influenza A(H1N1)2009 virus may lead to significant reduction in the ability of patient and vaccine sera to recognize A(H1N1)2009 viruses.     

Prevalence of Antibodies against Avian Influenza A (H5N1) Virus among Cullers and Poultry Workers in Ho Chi Minh City, 2005

Background

Between 2003 and 2005, highly pathogenic avian influenza A (H5N1) viruses caused large scale outbreaks in poultry in the Ho Chi Minh City area in Vietnam. We studied the prevalence of antibodies against H5N1 in poultry workers and cullers who were active in the program in Ho Chi Minh City in 2004 and 2005.

Methodology/Principal Findings

Single sera from 500 poultry workers and poultry cullers exposed to infected birds were tested for antibodies to avian influenza H5N1, using microneutralization assays and hemagglutination inhibition assay with horse blood. All sera tested negative using microneutralization tests. Three samples showed a 1∶80 titer in the hemagglutination inhibition assay.

Conclusions/Significance

This study provides additional support for the low transmissibility of clade 1 H5N1 to humans, but limited transmission to highly exposed persons cannot be excluded given the presence of low antibody titers in some individuals.     

Viral infection of cynomolgus macaque (Macaca irus)

The antiviral antibody against 14 viruses was studied with sera from 178 cynomolgus macaques. The viruses were employed, taking into consideration the probability of natural infection with the viruses in the habitats of the macaques and by contact with humans.Results on influenza and group B arboviruses were significant. 1) No hemagglutination inhibition (HI) antibody against influenza A2 (Adachi) was found but antibody against influenza B (Setagaya) was found in about 50% of the sera tested every year. 2) The results of the HI test with group B arboviruses indicated that macaques were infected with dengue type 2 and Japanese encephalitis viruses.Macaques also showed a high proportion of sero-positive cases and high antibody titers against herpes simplex, measles, and SV5 viruses.Antibody against at least one of the viruses tested was present in 170 of the 178 sera tested.     

Reliability of Pseudotyped Influenza Viral Particles in Neutralizing Antibody Detection

Background

Current influenza control strategies require an active surveillance system. Pseudotyped viral particles (pp) together with the evaluation of pre-existing immunity in a population might satisfy this requirement. However, the reliability of using pp in neutralizing antibody (nAb) detection are undefined.

Methodology/Principal Findings

Pseudotyped particles of A(H1N1)pmd09 (A/California/7/2009) and HPAI H5N1 (A/Anhui/1/2005), as well as their reassortants, were generated. The reliability of using these pp in nAb detection were compared concurrently with the corresponding viruses by a hemagglutination inhibition test, as well as ELISA-, cytopathic effect-, and fluorescence-based microneutralization assays. In the qualitative detection on nAbs, the pp and their corresponding viruses were in complete agreement, with an R² value equal to or near 1 in two different populations. In the quantitative detection on nAbs, although the geometric mean titers (95% confidence interval) differed between the pp and viruses, no significant difference was observed. Furthermore, humoral immunity against the reassortants was evaluated; our results indicated strong consistency between the nAbs against reassortant pp and those against naïve pp harboring the same hemagglutinin.

Conclusion/Significance

The pp displayed high reliability in influenza virus nAb detection. The use of reassortant pp is a safe and convenient strategy for characterizing emerging influenza viruses and surveying the disease burden.     

Immunosuppression induced by respiratory viruses (influenza virus, adenovirus) in mice

The influence of respiratory viruses (adenovirus, influenza virus) on humoral immune response to heterologous T-dependent and T-independent antigens was studied. It was shown that inoculation of mice by the influenza virus (A/PR8/34-A/PR/8) 3 days before sheep red blood cells administration led to the inhibition of antibody forming cell (AFC) and immunoglobulin, forming cell (IFC) increase on 69% and 59% respectively. Adenovirus type 6 induced the similar suppression of AFC and IFC formation. Thus, viruses induced immuno-suppression, which was polyclonal. It was also shown that virus of one strain (type) could inhibit immune response to another strain (type) of virus. The immune response to T-independent antigen was not suppressed. The virus-induced immunosuppression was dependent on: 1) the infectivity of respiratory viruses, 2) the route of virus and heterologous antigen injection, and 3) the interval between the viruses and antigen inoculation.     

A pan-H1 anti-hemagglutinin monoclonal antibody with potent broad-spectrum efficacy in vivo

Seasonal epidemics caused by antigenic variations in influenza A virus remain a public health concern and an economic burden. The isolation and characterization of broadly neutralizing anti-hemagglutinin monoclonal antibodies (MAb) have highlighted the presence of highly conserved epitopes in divergent influenza A viruses. Here, we describe the generation and characterization of a mouse monoclonal antibody designed to target the conserved regions of the hemagglutinin of influenza A H1 viruses, a subtype that has caused pandemics in the human population in both the 20th and 21st centuries. By sequentially immunizing mice with plasmid DNA encoding the hemagglutinin of antigenically different H1 influenza A viruses (A/South Carolina/1/1918, A/USSR/92/1977, and A/California/4/2009), we isolated and identified MAb 6F12. Similar to other broadly neutralizing MAb previously described, MAb 6F12 has no hemagglutination inhibition activity against influenza A viruses and targets the stalk region of hemagglutinins. As designed, it has neutralizing activity against a divergent panel of H1 viruses in vitro, representing 79 years of antigenic drift. Most notably, MAb 6F12 prevented gross weight loss against divergent H1 viruses in passive transfer experiments in mice, both in pre- and postexposure prophylaxis regimens. The broad but specific activity of MAb 6F12 highlights the potent efficacy of monoclonal antibodies directed against a single subtype of influenza A virus.     

Evolutionary pattern of the hemagglutinin gene of influenza B viruses isolated in Japan: cocirculating lineages in the same epidemic season.

The unexpectedly low efficacy of influenza vaccine during school outbreaks of influenza B virus in the spring of 1987 in Japan was probably attributable to a poor antibody response of vaccinees to the epidemic viruses. An antigenic analysis of the causative B viruses isolated in 1987 and 1988 showed much variation in hemagglutination inhibition patterns. The nucleotide sequences that code for the HA1 domain of B/Fukuoka/c-27/81, B/Ibaraki/2/85, B/Nagasaki/1/87, and B/Yamagata/16/88 viruses were determined and compared with those of the previously reported hemagglutinin genes. The nucleotide sequences of the hemagglutinin gene of a new variant, B/Yamagata/16/88, had only 93.4% homology with those of two other viruses from the same epidemic. An analysis of nucleotide and amino acid substitutions of the hemagglutinin genes of influenza B viruses revealed that new and some old variants could cocirculate in the same epidemic. A phylogenetic tree constructed by the neighbor-joining method allowed estimation of an evolutionary rate of 2.3 x 10(-3) synonymous (silent) substitutions per nucleotide site per year in the hemagglutinin gene.     

Antigenic drift in H5N1 avian influenza virus in poultry is driven by mutations in major antigenic sites of the hemagglutinin molecule analogous to those for human influenza virus

H5N1 highly pathogenic avian influenza virus has been endemic in poultry in Egypt since 2008, notwithstanding the implementation of mass vaccination and culling of infected birds. Extensive circulation of the virus has resulted in a progressive genetic evolution and an antigenic drift. In poultry, the occurrence of antigenic drift in avian influenza viruses is less well documented and the mechanisms remain to be clarified. To test the hypothesis that H5N1 antigenic drift is driven by mechanisms similar to type A influenza viruses in humans, we generated reassortant viruses, by reverse genetics, that harbored molecular changes identified in genetically divergent viruses circulating in the vaccinated population. Parental and reassortant phenotype viruses were antigenically analyzed by hemagglutination inhibition (HI) test and microneutralization (MN) assay. The results of the study indicate that the antigenic drift of H5N1 in poultry is driven by multiple mutations primarily occurring in major antigenic sites at the receptor binding subdomain, similarly to what has been described for human influenza H1 and H3 subtype viruses.     

Serological Evidence and Risk Factors for Swine Influenza Infections among Chinese Swine Workers in Guangdong Province

During July to September 2014, we performed a controlled, cross-sectional, seroepidemiologic study among 203 swine workers and 115 control subjects in Guangdong Province. Sera were tested using a hemagglutination inhibition assay against locally-isolated swine H3N2 and H1N1 viruses and commercially-obtained human influenza viral antigens. We found swine workers had a greater prevalence and odds of seropositivity against the swine H3N2 virus (17.3% vs. 7.0%; adjusted OR, 3.4; 95% CI, 1.1 -10.7). Younger age, self-report of a respiratory illness during the last 12 months, and seropositivity against seasonal H3N2 virus were identified as significant risk factors for seropositivity against swine H3N2 virus. As swine workers in China may be exposed to novel influenza viruses, it seems prudent for China to conduct special surveillance for such viruses among them. It also seems wise to offer such workers seasonal influenza vaccines with a goal to reduce cross-species influenza virus transmission.     

Filament-Producing Mutants of Influenza A/Puerto Rico/8/1934 (H1N1) Virus Have Higher Neuraminidase Activities than the Spherical Wild-Type

Influenza virus exhibits two morphologies – spherical and filamentous. Strains that have been grown extensively in laboratory substrates are comprised predominantly of spherical virions while clinical or low passage isolates produce a mixture of spheres and filamentous virions of varying lengths. The filamentous morphology can be lost upon continued passage in embryonated chicken eggs, a common laboratory substrate for influenza viruses. The fact that the filamentous morphology is maintained in nature but lost in favor of a spherical morphology in ovo suggests that filaments confer a selective advantage within the infected host that is not necessary for growth in laboratory substrates. Indeed, we have recently shown that filament-producing variant viruses are selected upon passage of the spherical laboratory strain A/Puerto Rico/8/1934 (H1N1) [PR8] in guinea pigs. Toward determining the nature of the selective advantage conferred by filaments, we sought to identify functional differences between spherical and filamentous particles. We compared the wild-type PR8 virus to two previously characterized recombinant PR8 viruses in which single point mutations within M1 confer a filamentous morphology. Our results indicate that these filamentous PR8 mutants have higher neuraminidase activities than the spherical PR8 virus. Conversely, no differences were observed in HAU:PFU or HAU:RNA ratios, binding avidity, sensitivity to immune serum in hemagglutination inhibition assays, or virion stability at elevated temperatures. Based on these results, we propose that the pleomorphic nature of influenza virus particles is important for the optimization of neuraminidase functions in vivo.     

Synthetic sialylphosphatidylethanolamine derivatives bind to human influenza A viruses and inhibit viral infection

We synthesized the sialylphosphatidylethanolamine (sialyl PE) derivatives Neu5Ac-PE, (Neu5Ac)2-PE, Neu5Ac-PE (amide) and Neu5Ac-PE (methyl). We examined the anti-viral effects of the derivatives on human influenza A virus infection by ELISA/virus-binding, hemagglutination inhibition, hemolysis inhibition and neutralization assays. The sialyl PE derivatives that we examined bound to A/Aichi/2/68, A/Singapore/1/57 and A/Memphis/1/71 strains of H3N2 subtype, but not to A/PR/8/34 strain of H1N1 subtype. The derivatives inhibited viral hemagglutination and hemolysis of human erythrocytes with A/Aichi/2/68 and A/Singapore/1/57 (H3N2), but not with A/PR/8/34 (H1N1). The inhibitory activity of the (Neu5Ac)2-PE derivative was the strongest of all sialyl PE derivatives (IC50, 35 M to 40 M). Sialyl PE derivatives also inhibited the infection of A/Aichi/2/68 in MDCK cells. Complete inhibition was observed at a concentration between 0.3 to 1.3 mM. IC50 of (Neu5Ac)2-PE was 15 M in A/Aichi/2/68 strain. Taken together, the synthetic sialyl PE derivatives may be effective reagents against infection of some types of influenza A viruses.     

Pre-Existing Immunity with High Neutralizing Activity to 2009 Pandemic H1N1 Influenza Virus in Shanghai Population

Pre-existing immunity is an important factor countering the pandemic potential of an emerging influenza virus strain. Thus, studying of pre-existing immunity to the 2009 pandemic H1N1 virus (2009 H1N1) will advance our understanding of the pathogenesis and epidemiology of this emerging pathogen. In the present study, sera were collected from 486 individuals in a hospital in Shanghai, China, before the 2009 H1N1 influenza pandemic. The serum anti-hemagglutinins (HA) antibody, hemagglutination inhibition (HI) antibody and neutralizing antibody against the 2009 H1N1 were assayed. Among this population, 84.2%, 14.61% and 26.5% subjects possessed anti-HA antibody, HI antibody and neutralizing antibody, respectively. Although neutralizing antibody only existed in those sera with detectable anti-HA antibody, there was no obvious correlation between the titers of anti-HA and neutralizing antibody. However, the titers of anti-HA and neutralizing antibody against seasonal H1N1 virus were highly correlated. In the same population, there was no correlation between titers of neutralizing antibody against 2009 H1N1 and seasonal H1N1. DNA immunization performed on mice demonstrated that antibodies to the HA of 2009 pandemic and seasonal H1N1 influenza viruses were strain-specific and had no cross-neutralizing activity. In addition, the predicted conserved epitope in the HA of 2009 H1N1 and recently circulating seasonal H1N1 virus, GLFGAIAGFIE, was not an immunologically valid B-cell epitope. The data in this report are valuable for advancing our understanding of 2009 H1N1 influenza virus infection.     

Microneutralization Assay Titres Correlate with Protection against Seasonal Influenza H1N1 and H3N2 in Children

Although the microneutralization (MN) assay has been shown to be more sensitive than the hemagglutination inhibition (HAI) assay for the measurement of humoral immunity against influenza viruses, further evidence relating MN titres to protective efficacy against infection is needed. Serum antibodies against seasonal H1N1 and H3N2 influenza were measured in children and adolescents (n = 656) by MN and hemagglutination inhibition (HAI) assays. Compared to HAI, the MN assay is more sensitive in detecting serum antibodies and estimates of protective effectiveness against PCR-confirmed infection were higher for both subtypes. Given our findings, the MN assay warrants further consideration as a formal tool for the routine evaluation of vaccine-induced antibody responses.     

Model-based inference of neutralizing antibody avidities against influenza virus

To assess the response to vaccination, quantity (concentration) and quality (avidity) of neutralizing antibodies are the most important parameters. Specifically, an increase in avidity indicates germinal center formation, which is required for establishing long-term protection. For influenza, the classical hemagglutination inhibition (HI) assay, however, quantifies a combination of both, and to separately determine avidity requires high experimental effort. We developed from first principles a biophysical model of hemagglutination inhibition to infer IgG antibody avidities from measured HI titers and IgG concentrations. The model accurately describes the relationship between neutralizing antibody concentration/avidity and HI titer, and explains quantitative aspects of the HI assay, such as robustness to pipetting errors and detection limit. We applied our model to infer avidities against the pandemic 2009 H1N1 influenza virus in vaccinated patients (n = 45) after hematopoietic stem cell transplantation (HSCT) and validated our results with independent avidity measurements using an enzyme-linked immunosorbent assay with urea elution. Avidities inferred by the model correlated with experimentally determined avidities (ρ = 0.54, 95% CI = [0.31, 0.70], P < 10⁻⁴). The model predicted that increases in IgG concentration mainly contribute to the observed HI titer increases in HSCT patients and that immunosuppressive treatment is associated with lower baseline avidities. Since our approach requires only easy-to-establish measurements as input, we anticipate that it will help to disentangle causes for poor vaccination outcomes also in larger patient populations. This study demonstrates that biophysical modelling can provide quantitative insights into agglutination assays and complement experimental measurements to refine antibody response analyses.     

A monoclonal antibody targeting a highly conserved epitope in influenza B neuraminidase provides protection against drug resistant strains

All influenza viral neuraminidases (NA) of both type A and B viruses have only one universally conserved sequence located between amino acids 222–230. A monoclonal antibody against this region has been previously reported to provide broad inhibition against all nine subtypes of influenza A NA; yet its inhibitory effect against influenza B viral NA remained unknown. Here, we report that the monoclonal antibody provides a broad inhibition against various strains of influenza B viruses of both Victoria and Yamagata genetic lineage. Moreover, the growth and NA enzymatic activity of two drug resistant influenza B strains (E117D and D197E) are also inhibited by the antibody even though these two mutations are conformationally proximal to the universal epitope. Collectively, these data suggest that this unique, highly-conserved linear sequence in viral NA is exposed sufficiently to allow access by inhibitory antibody during the course of infection; it could represent a potential target for antiviral agents and vaccine-induced immune responses against diverse strains of type B influenza virus.     

A Prospective Study of Romanian Agriculture Workers for Zoonotic Influenza Infections

Background

In this prospective study we sought to examine seroepidemiological evidence for acute zoonotic influenza virus infection among Romanian agricultural workers.

Methods

Sera were drawn upon enrollment (2009) and again at 12 and 24 months from 312 adult agriculture workers and 51 age-group matched controls. Participants were contacted monthly for 24 months and queried regarding episodes of acute influenza-like illnesses (ILI). Cohort members meeting ILI criteria permitted respiratory swab collections as well as acute and convalescent serum collection. Serologic assays were performed against 9 avian, 3 swine, and 3 human influenza viruses.

Results

During the two-year follow-up, a total of 23 ILI events were reported. Two subjects'' specimens were identified as influenza A by rRT-PCR. During the follow-up period, three individuals experienced elevated microneutralization antibody titers ≥1∶80 against three (one each) avian influenza viruses: A/Teal/Hong Kong/w312/97(H6N1), A/Hong Kong/1073/1999(H9N2), or A/Duck/Alberta/60/1976(H12N5). However, none of these participants met the criteria for poultry exposure. A number of subjects demonstrated four-fold increases over time in hemagglutination inhibition (HI) assay titers for at least one of the three swine influenza viruses (SIVs); however, it seems likely that two of these three responses were due to cross-reacting antibody against human influenza. Only elevated antibody titers against A/Swine/Flanders/1/1998(H3N2) lacked evidence for such confounding. In examining risk factors for elevated antibody against this SIV with multiple logistic regression, swine exposure (adjusted OR = 1.8, 95% CI 1.1–2.8) and tobacco use (adjusted OR = 1.8; 95% CI 1.1–2.9) were important predictors.

Conclusions

While Romania has recently experienced multiple incursions of highly pathogenic avian influenza among domestic poultry, this cohort of Romanian agriculture workers had sparse evidence of avian influenza virus infections. In contrast, there was evidence, especially among the swine exposed participants, of infections with human and one swine H3N2 influenza virus.     

Comparison of the replication of influenza A viruses in Chinese ring-necked pheasants and chukar partridges

We investigated the replication and transmission of avian influenza A viruses in two species thought to be intermediate hosts in the spread of influenza A viruses in live poultry markets: Chinese ring-necked pheasants and chukar partridges. All 15 hemagglutinin subtypes replicated in pheasants, and most subtypes transmitted to na?ve contact pheasants, primarily via the fecal-oral route. Many viruses were shed from the gastrointestinal tract of experimentally inoculated pheasants for 14 days or longer. Virus was isolated from the cloacal swabs of one contact pheasant for an unprecedented 45 days. Chukar partridges were less susceptible to infection with avian influenza viruses. The viruses that replicated in chukar partridges were isolated for 7 days after experimental inoculation, predominantly from the respiratory tract. We detected high neutralizing antibody titers with correspondingly low levels of serum hemagglutination inhibition antibody titers in pheasants and chukar partridges when chicken red blood cells were used in serological analyses. When horse erythrocytes were used, antibody titers were comparable to those obtained by using the neutralization assay. More importantly, the results suggested that pheasants can serve as a reservoir of influenza virus. Because of their continuous asymptomatic infection and longer stay in the markets, pheasants are ideal "carriers" of influenza A viruses. Their continued presence in live markets contributes to the perpetuation and genetic interaction of influenza viruses there. On the basis of our findings, it does not make good sense to ban quail but not pheasants from the live markets.     

Profiling of Humoral Response to Influenza A(H1N1)pdm09 Infection and Vaccination Measured by a Protein Microarray in Persons with and without History of Seasonal Vaccination

Background

The influence of prior seasonal influenza vaccination on the antibody response produced by natural infection or vaccination is not well understood.

Methods

We compared the profiles of antibody responses of 32 naturally infected subjects and 98 subjects vaccinated with a 2009 influenza A(H1N1) monovalent MF59-adjuvanted vaccine (Focetria®, Novartis), with and without a history of seasonal influenza vaccination. Antibodies were measured by hemagglutination inhibition (HI) assay for influenza A(H1N1)pdm09 and by protein microarray (PA) using the HA1 subunit for seven recent and historic H1, H2 and H3 influenza viruses, and three avian influenza viruses. Serum samples for the infection group were taken at the moment of collection of the diagnostic sample, 10 days and 30 days after onset of influenza symptoms. For the vaccination group, samples were drawn at baseline, 3 weeks after the first vaccination and 5 weeks after the second vaccination.

Results

We showed that subjects with a history of seasonal vaccination generally exhibited higher baseline titers for the various HA1 antigens than subjects without a seasonal vaccination history. Infection and pandemic influenza vaccination responses in persons with a history of seasonal vaccination were skewed towards historic antigens.

Conclusions

Seasonal vaccination is of significant influence on the antibody response to subsequent infection and vaccination, and further research is needed to understand the effect of annual vaccination on protective immunity.     

An Anti-Influenza Virus Antibody Inhibits Viral Infection by Reducing Nucleus Entry of Influenza Nucleoprotein

To date, four main mechanisms mediating inhibition of influenza infection by anti-hemagglutinin antibodies have been reported. Anti-globular-head-domain antibodies block either influenza virus receptor binding to the host cell or progeny virion release from the host cell. Anti-stem region antibodies hinder the membrane fusion process or induce antibody-dependent cytotoxicity to infected cells. In this study we identified a human monoclonal IgG₁ antibody (CT302), which does not inhibit both the receptor binding and the membrane fusion process but efficiently reduced the nucleus entry of viral nucleoprotein suggesting a novel inhibition mechanism of viral infection by antibody. This antibody binds to the subtype-H3 hemagglutinin globular head domain of group-2 influenza viruses circulating throughout the population between 1997 and 2007.     

细胞表达流感病毒抗体CR6261 VH与scFv对流感病毒感染的影响

由于新发现的流感病毒抗体CR6261可以中和多种亚型的流感病毒,而且是其VH区结合于流感病毒蛋白HA的保守区,因此该抗体有望成为一种广谱的流感治疗性抗体而备受关注。通过构建真核表达载体pCR6261VH、pCR6261VH-GFP、pCR6261scFv,成功地筛选到在细胞膜上稳定表达CR6261VH、CR6261VH-GFP与CR6261scFv的单克隆细胞系。用流感病毒去感染稳定细胞系,间隔12 h取上清检测病毒血凝效价。结果表明：稳定表达CR6261scFv和CR6261VH-GFP细胞系能够降低病