新型甲型H1N1流感病毒HA基因DNA疫苗诱导小鼠产生中和抗体

目的 探讨新型甲型流感病毒(2009H1N1)血凝素(HA)DNA疫苗诱导小鼠产生中和抗体特性.方法 构建2009H1N1或1918甲型流感病毒(1918H1N1)HA蛋白表达质粒2009HA和1918HA,采用25μg或200μg剂量2009HA质粒免疫小鼠,以2009HA或1918HA蛋白为包被抗原,测定小鼠血清中2009HA抗体总量或交叉反应抗体含量,分别用2009H1N1和1918H1N1两种假病毒(pp)测定抗体中和活性.结果 25 μg或200μg的2009HA质粒加强免疫小鼠后,4～16周内两组小鼠血清中2009HA总抗体水平以及对2009H1N1pp的中和抗体滴度相似(P>0.05),都含有与1918HA蛋白交叉反应抗体,对1918H1N1pp的交叉中和抗体滴度相似(P>0.05).结论 小剂量2009HA质粒DNA疫苗能够诱导小鼠产生持久的高水平中和抗体,对于预防新现流感病毒具有潜在应用价值.     

Generation of human neutralizing monoclonal antibodies against the 2009 pandemic H1N1 virus from peripheral blood memory B lymphocytes

The 2009 H1N1 influenza pandemic demonstrated the significance of a global health threat to human beings. Although pandemic H1N1 vaccines have been rapidly developed, passive serotherapy may offer superior immediate protection against infections in children, the elderly and immune-compromised patients during an influenza pandemic. Here, we applied a novel strategy based on Epstein–Barr virus (EBV)-immortalized peripheral blood memory B cells to screen high viral neutralizing monoclonal antibodies (MAbs) from individuals vaccinated with the 2009 pandemic H1N1 vaccine PANFLU.1. Through a massive screen of 13 090 immortalized memory B-cell clones from three selected vaccinees, seven MAbs were identified with both high viral neutralizing capacities and hemagglutination inhibition (HAI) activities against the 2009 pandemic H1N1 viruses. These MAbs may have important clinical implications for passive serotherapy treatments of infected patients with severe respiratory syndrome, especially children, the elderly and immunodeficient individuals. Our successful strategy for generating high-affinity MAbs from EBV-immortalized peripheral blood memory B cells may also be applicable to other infectious or autoimmune diseases.     

Response of BALB/c mice to a monovalent influenza A （H1N1） 2009 split vaccine

The novel influenza A (H1N1) 2009 virus has emerged to cause the first pandemic of the twenty-first century. Disease outbreaks caused by the influenza A (H1N1) virus have prompted concerns about the potential for a pandemic and have driven the development of vaccines against this subtype of influenza A. In this study, we developed a monovalent influenza A (H1N1) split vaccine and evaluated its effects in BALB/c mice. Mice were immunized subcutaneously with 2 doses of the vaccine containing hemagglutinin (HA) alone or HA plus an aluminum hydroxide (Al(OH)₃) adjuvant. Immunization with varying doses of HA (3.75, 7.5, 15, 30, 45 or 60 µg) was performed to induce the production of neutralizing antibodies. The vaccine elicited strong hemagglutination inhibition (HI) and microneutralization, and addition of the adjuvant augmented the antibody response. A preliminary safety evaluation showed that the vaccine was not toxic at large doses (0.5 ml containing 60 µg HA+600 µg Al(OH)₃ or 60 µg HA). Moreover, the vaccine was found to be safe at a dose of 120 µg HA+1200 µg Al(OH)₃ or 120 µg HA in 1.0 ml in rats. In conclusion, the present study provides support for the clinical evaluation of influenza A (H1N1) vaccination as a public health intervention to mitigate a possible pandemic. Additionally, our findings support the further evaluation of the vaccine used in this study in primates or humans.     

Profiling of humoral immune responses to influenza viruses by using protein microarray

The emergence of pandemic A(H1N1) 2009 influenza showed the importance of rapid assessment of the degree of immunity in the population, the rate of asymptomatic infection, the spread of infection in households, effects of control measures, and ability of candidate vaccines to produce a response in different age groups. A limitation lies in the available assay repertoire: reference standard methods for measuring antibodies to influenza virus are haemagglutination inhibition (HI) assays and virus neutralization tests. Both assays are difficult to standardize and may be too specific to assess possible partial humoral immunity from previous exposures. Here, we describe the use of antigen-microarrays to measure antibodies to HA1 antigens from seven recent and historical seasonal H1, H2 and H3 influenza viruses, the A(H1N1) 2009 pandemic influenza virus, and three avian influenza viruses. We assessed antibody profiles in 18 adult patients infected with A(H1N1) 2009 influenza virus during the recent pandemic, and 21 children sampled before and after the pandemic, against background reactivity observed in 122 persons sampled in 2008, a season dominated by seasonal A(H1N1) influenza virus. We show that subtype-specific and variant-specific antibody responses can be measured, confirming serological responses measured by HI. Comparison of profiles from persons with similar HI response showed that the magnitude and broadness of response to individual influenza subtype antigens differs greatly between individuals. Clinical and vaccination studies, but also exposure studies, should take these findings into consideration, as they may indicate some level of humoral immunity not measured by HI assays.     

A novel monoclonal antibody effective against lethal challenge with swine-lineage and 2009 pandemic H1N1 influenza viruses in mice

The HA protein of the 2009 pandemic H1N1 viruses (H1N1pdm) is antigenically closely related to the HA of classical North American swine H1N1 influenza viruses (cH1N1). Since 1998, through mutation and reassortment of HA genes from human H3N2 and H1N1 influenza viruses, swine influenza strains are undergoing substantial antigenic drift and shift. In this report we describe the development of a novel monoclonal antibody (S-OIV-3B2) that shows high hemagglutination inhibition (HI) and neutralization titers not only against H1N1pdm, but also against representatives of the α, β, and γ clusters of swine-lineage H1 influenza viruses. Mice that received a single intranasal dose of S-OIV-3B2 were protected against lethal challenge with either H1N1pdm or cH1N1 virus. These studies highlight the potential use of S-OIV-3B2 as effective intranasal prophylactic or therapeutic antiviral treatment for swine-lineage H1 influenza virus infections.     

Protective immunity against H5N1 influenza virus by a single dose vaccination with virus-like particles

We generated influenza virus-like particles (VLPs) containing the wild type (WT) H5 hemagglutinin (HA) from A/Viet Nam/1203/04 virus or a mutant H5 HA with a deletion of the multibasic cleavage motif. VLPs containing mutant H5 HA were found to be as immunogenic as VLPs containing WT HA. A single intramuscular vaccination with either type of H5 VLPs provided complete protection against lethal challenge. In contrast, the recombinant H5 HA vaccine was less immunogenic and vaccination even with a 5 fold higher dose did not induce protective immunity. VLP vaccines were superior to the recombinant HA in inducing T helper type 1 immune responses, hemagglutination inhibition titers, and antibody secreting cells, which significantly contribute to inducing protective immunity after a single dose vaccination. This study provides insights into the potential mechanisms of improved immunogenicity by H5 VLP vaccines as an approach to improve the protective efficacy against potential pandemic viruses.     

Seasonal H1N1 2007 influenza virus infection is associated with elevated pre‐exposure antibody titers to the 2009 pandemic influenza A (H1N1) virus

The new influenza strain detected in humans in April 2009 has caused the first influenza pandemic of the 21st century. A cross‐reactive antibody response, in which antibodies against seasonal H1N1 viruses neutralized the 2009 pandemic influenza A (H1N1) virus (2009 pH1N1), was detected among individuals aged >60 years. However, factors other than age associated with such a cross‐reactive antibody response are poorly documented. Our objective was to examine factors potentially associated with elevated pre‐exposure viro‐neutralization and hemagglutination‐inhibition antibody titers against the 2009 pH1N1. We also studied factors associated with antibody titers against the 2007 seasonal H1N1 virus. One hundred subjects participating in an influenza cohort were selected. Sera collected in 2008 were analysed using hemagglutination inhibition and viro‐neutralization assays for the 2009 pH1N1 virus and the 2007 seasonal H1N1 virus. Viro‐neutralization results were explored using a linear mixed‐effect model and hemagglutination‐inhibition results using linear‐regression models for interval‐censored data. Elevated antibody titers against 2009 pH1N1 were associated with seasonal 2007 H1N1 infection (viro‐neutralization, p 0.006; hemagglutination‐inhibition, p 0.018). Elevated antibody titers were also associated with age in the viro‐neutralization assay (p <0.0001). Seasonal 2007 H1N1 infection is an independent predictor of elevated pre‐exposure antibody titers against 2009 pH1N1 and may have contributed to lowering the burden of the 2009 pH1N1 pandemic.     

Elicitation of broadly neutralizing influenza antibodies in animals with previous influenza exposure

The immune system responds to influenza infection by producing neutralizing antibodies to the viral surface protein, hemagglutinin (HA), which regularly changes its antigenic structure. Antibodies that target the highly conserved stem region of HA neutralize diverse influenza viruses and can be elicited through vaccination in animals and humans. Efforts to develop universal influenza vaccines have focused on strategies to elicit such antibodies; however, the concern has been raised that previous influenza immunity may abrogate the induction of such broadly protective antibodies. We show here that prime-boost immunization can induce broadly neutralizing antibody responses in influenza-immune mice and ferrets that were previously infected or vaccinated. HA stem-directed antibodies were elicited in mice primed with a DNA vaccine and boosted with inactivated vaccine from H1N1 A/New Caledonia/20/1999 (1999 NC) HA regardless of preexposure. Similarly, gene-based vaccination with replication-defective adenovirus 28 (rAd28) and 5 (rAd5) vectors encoding 1999 NC HA elicited stem-directed neutralizing antibodies and conferred protection against unmatched 1934 and 2007 H1N1 virus challenge in influenza-immune ferrets. Indeed, previous exposure to certain strains could enhance immunogenicity: The strongest HA stem-directed immune response was observed in ferrets previously infected with a divergent 1934 H1N1 virus. These findings suggest that broadly neutralizing antibodies against the conserved stem region of HA can be elicited through vaccination despite previous influenza exposure, which supports the feasibility of developing stem-directed universal influenza vaccines for humans.     

B Cell Response and Hemagglutinin Stalk-Reactive Antibody Production in Different Age Cohorts following 2009 H1N1 Influenza Virus Vaccination

The 2009 pandemic H1N1 (pH1N1) influenza virus carried a swine-origin hemagglutinin (HA) that was closely related to the HAs of pre-1947 H1N1 viruses but highly divergent from the HAs of recently circulating H1N1 strains. Consequently, prior exposure to pH1N1-like viruses was mostly limited to individuals over the age of about 60 years. We related age and associated differences in immune history to the B cell response to an inactivated monovalent pH1N1 vaccine given intramuscularly to subjects in three age cohorts: 18 to 32 years, 60 to 69 years, and ≥70 years. The day 0 pH1N1-specific hemagglutination inhibition (HAI) and microneutralization (MN) titers were generally higher in the older cohorts, consistent with greater prevaccination exposure to pH1N1-like viruses. Most subjects in each cohort responded well to vaccination, with early formation of circulating virus-specific antibody (Ab)-secreting cells and ≥4-fold increases in HAI and MN titers. However, the response was strongest in the 18- to 32-year cohort. Circulating levels of HA stalk-reactive Abs were increased after vaccination, especially in the 18- to 32-year cohort, raising the possibility of elevated levels of cross-reactive neutralizing Abs. In the young cohort, an increase in MN activity against the seasonal influenza virus A/Brisbane/59/07 after vaccination was generally associated with an increase in the anti-Brisbane/59/07 HAI titer, suggesting an effect mediated primarily by HA head-reactive rather than stalk-reactive Abs. Our findings support recent proposals that immunization with a relatively novel HA favors the induction of Abs against conserved epitopes. They also emphasize the need to clarify how the level of circulating stalk-reactive Abs relates to resistance to influenza.     

A pandemic H1N1 influenza virus-like particle vaccine induces cross-protection in mice

Influenza virus-like particles (VLPs) represent promising alternative vaccines. However, it is necessary to demonstrate that influenza VLPs confer cross-protection against antigenically distinct viruses. In this study, a VLP vaccine comprising hemagglutinin (HA) and M1 from the A/California/04/2009 (H1N1) were used and its ability to induce cross-protective efficacy against heterologous viruses A/PR/8/34 (H1N1) and A/New Caledonia/20/99 (H1N1) in mice was assessed. Vaccination with 2009 H1 VLPs induced significantly higher levels of IgG cross-reactive with these heterologous viruses after the second boost compared to after the prime or first boost. Lung virus titers also decreased significantly and the lung cross-reactive IgG response after lethal virus challenge was significantly greater in immunized mice compared to naïve mice. Vaccinated mice showed 100% protection against A/PR/8/34 and A/Caledonia/20/99 viruses with only moderate body weight loss and induction of cross-reactive recall, IgG antibody-secreting cell responses. The variations in HA amino acid sequences and antigenic sites were determined and correlated with induction of cross-protective immunity. These results indicate that VLPs can be used as an effective vaccine that confers cross-protection against antigenically distinct viruses.     

Comparative analysis of antibody induction and protection against influenza virus infection by DNA immunization with HA,HAe, and HA1 in mice

Plasmid DNA vaccines are considered alternatives to inactivated influenza virus vaccines to control influenza. Vaccination with a hemagglutinin (HA)-, HA ectodomain (HAe)-, or HA subunit 1 (HA1)-based vaccine can stimulate protective immunity in animals. The aim of this study was to compare their capacity to induce an antibody response and protection against influenza virus infection in mice after DNA vaccination. We constructed three expression vectors encoding full-length HA, HAe, or HA1 of the A/California/07/2009 influenza A virus and designed three animal experiments: (i) BALB/c mice were immunized twice with 30 μg of the HA, HAe, or HA1 DNA vaccine with high-voltage electroporation (100 V), and 3 weeks after boosting, they were challenged with a lethal dose of virus. (ii) Immunization and challenge were as in experiment i, but with low-voltage electroporation (10 V). (iii) Mice were immunized once with 50 μg of DNA and challenged 1 week later. The immunogenic effects of the three DNA vaccines were evaluated in terms of antibody titer, survival rate, bodyweight change, and lung viral titer. In all three experiments, both HA and HAe induced higher antibody and neutralization titers than HA1. Following challenge with a lethal mouse-adapted homologous virus, both HA and HAe reduced the viral titers in lung washes or offered better protection from weight loss than HA1 in experiments ii and iii. Thus, HA1 induces a lower immune response than HA or HAe when used as a DNA vaccination. Our data should be valuable in choosing the optimal candidate vaccine when faced with the threat of pandemic influenza.     

无锡市流感病毒抗体水平调查

目的调查无锡市人群中甲型、乙型流感病毒抗体水平和新甲型H1N1流感病毒传人前后人群中抗体水平,并对新甲型H1N1流感病毒传人1年后自然人群中成人抗体水平与接种新甲型H1N1流感疫苗后1年的成人抗体水平进行比较。方法收集2008年9月至2009年5月、2010年9月至2011年1月无锡市不同年龄段人群血清和接种新甲型HIN1流感疫苗1年的成人血清,用血凝抑制（HI）试验测定抗体,并比较不同时间段各人群中的流感抗体阳性率、保护率和几何平均滴度（GMT）。结果新甲型H1N1流感病毒传入前,无锡市自然人群的HI抗体阳性率为2．86％（4／140）,保护率为0．71％（1／140）,GMT为5．23。新甲型H1N1流感病毒传入1年后,自然人群的HI抗体阳性率为66．33％,保护率为37．76％、GMT为19．17;其中成人HI抗体阳性率、保护率和GMT分别为50．00％、19．44％和13．09。接种新甲型H1N1流感疫苗的成人1年后HI抗体阳性率、保护率和GMT分别为61．36％、22．73％和14．14,与自然人群中成人在流感病毒传入1年后的抗体水平差异无统计学意义（P均〉0．05）。无锡市人群中甲型与乙型流感病毒HI抗体水平分别为：H1N1病毒抗体阳性率为55．00％,保护率为35．00％,GMT16．90;H3N2抗体阳性率为86．40％,保护率为84．30％,GMT为58．56。结论新甲型H1N1流感病毒传入无锡市1年后,自然人群中新甲型H1N1流感病毒抗体阳性率、保护率和GMT均已达到季节性流感抗体水平。同时人群中已有一定水平的甲型、乙型流感病毒抗体,近期不会发生较大的季节性流感疫情。     

Evaluation of neutralizing efficacy of monoclonal antibodies specific for 2009 pandemic H1N1 influenza A virus in vitro and in vivo

Pandemic influenza A virus (H1N1) 2009 poses a serious public-health challenge worldwide. To characterize the neutralizing epitopes of this virus, we generated a panel of eight monoclonal antibodies (mAbs) against the HA of the A/California/07/2009 virus. The antibodies were specific for the 2009 pdm H1N1 HA, as the antibodies displayed HA-specific ELISA, hemagglutination inhibition (HAI) and neutralization activity. One mAb (mAb12) showed significantly higher HAI and neutralizing titers than the other mAbs. We mapped the antigenic epitopes of the HA by characterizing escape mutants of a 2009 H1N1 vaccine strain (NYMC X-179A). The amino acid changes suggested that these eight mAbs recognized HA antigenic epitopes located in the Sa, Sb, Ca1 and Ca2 sites. Passive immunization with mAbs showed that mAb12 displayed more efficient neutralizing activity in vivo than the other mAbs. mAb12 was also found to be protective, both prophylactically and therapeutically, against a lethal viral challenge in mice. In addition, a single injection of 10 mg/kg mAb12 outperformed a 5-day course of treatment with oseltamivir (10 mg/kg/day by gavage) with respect to both prophylaxis and treatment of lethal viral infection. Taken together, our results showed that mouse-origin mAbs displayed neutralizing effectiveness in vitro and in vivo. One mAb in particular (mAb12) recognized an epitope within the Sb site and demonstrated outstanding neutralizing effectiveness.     

Preexisting Antibody Response against 2009 Pandemic Influenza H1N1 Viruses in the Taiwanese Population

A novel pandemic influenza H1N1 (pH1N1) virus spread rapidly across the world in 2009. Due to the important role of antibody-mediated immunity in protection against influenza infection, we used an enzyme-linked immunosorbent assay-based microneutralization test to investigate cross-reactive neutralizing antibodies against the 2009 pH1N1 virus in 229 stored sera from donors born between 1917 and 2008 in Taiwan. The peak of cumulative geometric mean titers occurred in donors more than 90 years old and declined sharply with decreasing age. Sixteen of 27 subjects (59%) more than 80 years old had cross-reactive antibody titers of 160 or more against the 2009 pH1N1 virus, whereas none of the donors from age 9 to 49 had an antibody titer of 160 or more. Interestingly, 2 of 51 children (4%) from 6 months to 9 years old had an antibody titer of 40. We further tested the antibody responses in 9 of the 51 pediatric sera to three endemic seasonal influenza viruses isolated in 2006 and 2008 in Taiwan, and the results showed that only the 2 sera from children with antibody responses to the 2009 pH1N1 virus had high titers of neutralizing antibody against recent seasonal influenza virus strains. Our study shows the presence of some level of cross-reactive antibody in Taiwanese persons 50 years old or older, and the elderly subjects who may already have been exposed to the 1918 virus had high titers of neutralizing antibody to the 2009 pH1N1 virus. Our data also indicate that natural infection with the Taiwan 2006 and 2008 seasonal H1N1 viruses may induce a cross-reactive antibody response to the 2009 pH1N1 virus.Influenza A viruses have caused several pandemics during the past century and continue to cause epidemics around the world yearly. Pandemics are typically caused by the introduction of a virus with a hemagglutinin (HA) subtype that is new to human populations (14). In 2009, a novel pandemic influenza H1N1 (pH1N1) virus of swine origin spread rapidly and has caused variable disease globally via interhuman transmission (2, 3).The 2009 pH1N1 virus contains a unique combination of gene segments from both the North American and Eurasian swine lineages and is antigenically distinct from any known seasonal human influenza virus (14). Since H1N1 influenza A viruses have been circulating in human populations for decades, much of the world has encountered these viruses repeatedly, either through infection or through vaccination. Under the threat of a pandemic outbreak, however, a major concern is whether preexisting immunity can provide some protection from the novel 2009 pH1N1 virus.Recent reports from the United States suggested that 33% of individuals over the age of 60 years had neutralization antibodies to the novel 2009 pH1N1 virus, probably due to previous exposure to antigenically similar H1N1 viruses (1, 7). In Japan, however, appreciable neutralization antibodies against the 2009 pH1N1 virus were found only in individuals more than 90 years old (9). The differences in geographical location and vaccination programs against influenza in 1976 may account for the different age distributions of neutralization antibodies in the two countries. In the early 1900s, Taiwan had had a close relationship with Japan historically and geographically. The prevalence of influenza in Taiwan may be quite similar to that in Japan. In recent years, however, sequence analysis of epidemic influenza virus strains revealed that the Taiwanese strains usually circulate in Taiwan prior to their circulation in many other countries, including Japan. (16). The differences between the studies from United States and Japan, and the unique epidemic situation in Taiwan, highlight the need for us to assess the level of preexisting immunity in the Taiwanese population.In this study, we measured the titers of neutralizing antibodies against the 2009 pH1N1 virus in sera obtained from previous influenza infection or vaccination of different age groups. In addition, we also assessed the antibodies against the local seasonal H1N1 strains isolated in Taiwan in 2006 and 2008 (A/Taiwan/N86/06, A/Taiwan/N94/08, and A/Taiwan/N510/08) to evaluate whether there is a cross-reactive antibody response between recent local strains and the 2009 pH1N1 virus.     

Genetic and Antigenic Typing of Seasonal Influenza Virus Breakthrough Cases from a 2008-2009 Vaccine Efficacy Trial

Estimations of the effectiveness of vaccines against seasonal influenza virus are guided by comparisons of the antigenicities between influenza virus isolates from clinical breakthrough cases with strains included in a vaccine. This study examined whether the prediction of antigenicity using a sequence analysis of the hemagglutinin (HA) gene-encoded HA1 domain is a simpler alternative to using the conventional hemagglutination inhibition (HI) assay, which requires influenza virus culturing. Specimens were taken from breakthrough cases that occurred in a trivalent influenza virus vaccine efficacy trial involving >43,000 participants during the 2008-2009 season. A total of 498 influenza viruses were successfully subtyped as A(H3N2) (380 viruses), A(H1N1) (29 viruses), B(Yamagata) (23 viruses), and B(Victoria) (66 viruses) from 603 PCR- or culture-confirmed specimens. Unlike the B strains, most A(H3N2) (377 viruses) and all A(H1N1) viruses were classified as homologous to the respective vaccine strains based on their HA1 domain nucleic acid sequence. HI titers relative to the respective vaccine strains and PCR subtyping were determined for 48% (182/380) of A(H3N2) and 86% (25/29) of A(H1N1) viruses. Eighty-four percent of the A(H3N2) and A(H1N1) viruses classified as homologous by sequence were matched to the respective vaccine strains by HI testing. However, these homologous A(H3N2) and A(H1N1) viruses displayed a wide range of relative HI titers. Therefore, although PCR is a sensitive diagnostic method for confirming influenza virus cases, HA1 sequence analysis appeared to be of limited value in accurately predicting antigenicity; hence, it may be inappropriate to classify clinical specimens as homologous or heterologous to the vaccine strain for estimating vaccine efficacy in a prospective clinical trial.     

Comparison of protection against H5N1 influenza virus in mouse offspring provided by maternal vaccination with HA DNA and inactivated vaccine

H5N1 influenza virus is one of the viruses that can potentially cause an influenza pandemic. Protection of newborns against influenza virus infection could be effectively provided by maternal immunization. In this study, female mice were immunized with H5N1 HA DNA vaccine or inactivated whole-virion vaccine, and the protection provided by maternal antibodies in their offspring against a lethal homologous influenza virus challenge was compared. The results showed that maternal antibodies, whether induced by a DNA vaccine or an inactivated vaccine, could completely protect offspring aged 1-4 weeks from a lethal influenza virus challenge. Breast-feeding was the major route of transfer for maternal antibodies. Milk-derived antibodies were able to effectively protect the offspring aged 1-4 weeks from lethal influenza virus infection, whereas maternal antibodies transferred through the placenta only partially protected the offspring 1-2 weeks of age. The milk- and placenta-transferred IgG2a antibody levels in offspring from their mothers, whether vaccinated with DNA vaccine or inactivated vaccine, were higher than the IgG1 levels. Our results indicated that maternal vaccination with HA DNA, as well as with whole-virion inactivated vaccine, could offer effective protection to offspring against H5N1 influenza virus infection.     

Impact of glycosylation on the immunogenicity of a DNA-based influenza H5 HA vaccine

Avian H5N1 influenza viruses isolated from humans in Hong Kong in 1997 were divided into two antigenic groups based on the presence or absence of a potential glycosylation site at amino acid residues 154-156 in the HA1 region of the viral hemagglutinin (HA) surface glycoprotein. To assess the impact of glycosylation on the immunogenicity of an HA-expressing DNA vaccine, a series of plasmid vaccine constructs that differed in the presence of potential glycosylation sites at amino acid residues 154-156, 165-167, and 286-288 were used to immunize BALB/c mice. Postvaccination serum IgG, hemagglutination inhibition, and neutralizing antibody titers as well as the morbidity and mortality following a lethal H5N1 viral challenge did not vary significantly among any of the experimental groups. We conclude that the glycosylation pattern of the influenza virus HA1 domain has little impact on the murine antibody response raised to a DNA vaccine encoding the H5 HA, thereby minimizing the concern that the pattern of glycosylation sites encoded by the vaccine match those of closely related H5 viruses.     

Classical swine H1N1 influenza viruses confer cross protection from swine-origin 2009 pandemic H1N1 influenza virus infection in mice and ferrets

The hemagglutinin of the 2009 pandemic H1N1 influenza virus is a derivative of and is antigenically related to classical swine but not to seasonal human H1N1 viruses. We compared the A/California/7/2009 (CA/7/09) virus recommended by the WHO as the reference virus for vaccine development, with two classical swine influenza viruses A/swine/Iowa/31 (sw/IA/31) and A/New Jersey/8/1976 (NJ/76) to establish the extent of immunologic cross-reactivity and cross-protection in animal models. Primary infection with 2009 pandemic or NJ/76 viruses elicited antibodies against the CA/7/09 virus and provided complete protection from challenge with this virus in ferrets; the response in mice was variable and conferred partial protection. Although ferrets infected with sw/IA/31 virus developed low titers of cross-neutralizing antibody, they were protected from pulmonary replication of the CA/7/09 virus. The data suggest that prior exposure to antigenically related H1N1 viruses of swine-origin provide some protective immunity against the 2009 pandemic H1N1 virus.     

Identification of a linear epitope on the haemagglutinin protein of pandemic A/H1N1 2009 influenza virus using monoclonal antibodies

A novel influenza A/H1N1 virus, emerging from Mexico and the United States in the spring of 2009, caused the pandemic human infection of 2009-2010. The haemagglutinin (HA) glycoprotein is the major surface antigen of influenza A virus and plays an important role in viral infection. In this study, three hybridoma cell lines secreting specific monoclonal antibodies (Mabs) against the HA protein of pandemic influenza A/H1N1 2009 virus were generated with the recombinant plasmid pCAGGS-HA as an immunogen. Using Pepscan analysis, the binding sites of these Mabs were identified in a linear region of the HA protein. Further, refined mapping was conducted using truncated peptides expressed as GST-fusion proteins in E. coli. We found that the ²⁵⁰VPRYA²⁵⁴ motif was the minimal determinant of the linear epitope that could be recognized by the Mabs. Alignment with sequences from the databases showed that the amino acid residues of this epitope were highly conserved among all pandemic A/H1N1 2009 viruses as well as the classical swine H1N1 viruses isolated to date. These results provide additional insights into the antigenic structure of the HA protein and virus-antibody interactions at the amino acid level, which may assist in the development of specific diagnostic methods for influenza viruses.     

IgM,IgG, and IgA Antibody Responses to Influenza A(H1N1)pdm09 Hemagglutinin in Infected Persons during the First Wave of the 2009 Pandemic in the United States

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.