The 2009 pandemic H1N1 virus induces anti-neuraminidase (NA) antibodies that cross-react with the NA of H5N1 viruses in ferrets

A miniaturized neuraminidase inhibition (NI) assay using HA-mismatched H6 reassortant viruses was performed to examine the neuraminidase (NA)-specific antibody response in ferrets immunized with live-attenuated influenza vaccine (LAIV) strains. The strains tested possessed different NAs derived from seasonal H1N1 and H3N2, 2009 pandemic H1N1, and the highly pathogenic influenza H5N1 virus. The anti-NA antibodies from the 2009 pandemic strain (A/California/7/2009) immunized ferrets cross-reacted with the NA of H5N1 but not with the NA of seasonal H1N1 viruses. The plaque size reduction assay confirmed the cross-reactivity between the NAs of A/California/7/2009 and the H5N1 virus. Sequence and structural analyses of these N1 NA proteins showed that the NA of the 2009 pandemic H1N1 strain shared at least 22 more amino acids in the head domain with the NAs of the avian H5N1 strains than with the NAs of seasonal human H1N1 viruses. Our data demonstrated LAIV-induced NA antibody responses in ferrets and cross-reactive NA antibodies induced by 2009 pandemic H1N1 and H5N1 LAIV viruses.     

Natural infection with influenza A (H3N2). The development, persistance and effect of antibodies to the surface antigens.

A technique for estimating antibodies to the neuraminidase antigens of influenza A is described. The antibody response to the haemagglutinin and neuraminidase antigens of influenza A (H3N2) was studied in a boys' public school over the four-year period 1970--4. During this time there were two outbreaks of influenza A and the effect of antibody on the result of natural challenge was investigated. No boy who had homotypic neuraminidase antibody had clinical influenza.     

Supplementation of conventional trivalent influenza vaccine with purified viral N1 and N2 neuraminidases induces a balanced immune response without antigenic competition

Influenza viruses neuraminidase (NA) were chromatographically extracted from influenza viruses A/Nanchang/933/95 H3(NC)N2(NC) [R] and A/Johannesburg/82/96 H1(JH)N1(JH) [R] and used to supplement conventional inactivated trivalent influenza vaccine. Immunization of mice with this preparation resulted in high titers of antibodies to both hemagglutinins (HA) and neuraminidases (NA); there were no significant differences in the anti-HA antibody titers between the conventional and the supplemented vaccine preparation. Likewise, there were no significant differences in anti-NA antibody titers between the supplemented vaccine and titers from mice immunized with a neuraminidase vaccine containing a mixture of N1-NA and N2-NA. There was no evidence of a diminution of the immune response to the HA components of the vaccine despite the presence of antigenically equivalent amounts of both N1-NA and N2-NAs. Homotypic and distantly related heterotypic infections for both H1, N1 and H3N2 subtypes were suppressed and greater reduction in pulmonary virus titers (PVT) were observed in the trivalent vaccine supplemented with purified neuraminidase from each subtype, N1 and N2. Effects on the influenza B viral components were not studied. Previous studies on supplementation of conventional influenza vaccine focused only on monovalent H3N2 vaccine preparations; this study demonstrates in a mouse model system that supplementation of trivalent influenza vaccine with both influenza A subtype neuraminidases, N1 and N2 is highly immunogenic for HA and NA of each subtype and efficacious in protecting against influenza from homotypic and heterotypic infectious challenges of either subtype.     

Generation of reassortant influenza vaccines by reverse genetics that allows utilization of a DIVA (Differentiating Infected from Vaccinated Animals) strategy for the control of avian influenza

Vaccination of poultry with inactivated influenza vaccine can be an effective tool in the control of avian influenza (AI). One major concern of using inactivated vaccine is vaccine-induced antibody interference with serologic surveillance and epidemiology. In the United States, low pathogenicity H5 and H7 subtype AI viruses have caused serious economic losses in the poultry industry. Most of these viruses also have the accompanying N2 subtype and no H5N1 or H7N8 subtype AI viruses have been identified in poultry in the US. In order to allow the Differentiation of Infected from Vaccinated Animals (DIVA) while maintaining maximum efficacy of the vaccine, we generated reassortant viruses by reverse genetics that contained the same H5 and H7 hemagglutinin (HA) gene as the challenge virus, but a heterologous N1 or N8 neuraminidase (NA) gene. In vaccination-challenge experiments in 2-week-old specific pathogen free chickens, reassortant influenza vaccines (rH5N1 and rH7N8) demonstrated similar antibody profiles and comparable protection rates as vaccines prepared with parent H5N2 and H7N2 viruses. Further, we were able to differentiate the sera from infected and vaccinated birds by neuraminidase inhibition test and indirect immunofluorescent antibody assay on the basis of different antibodies elicited by their NA proteins. These results demonstrate the usefulness of a reverse genetics system for the rapid generation of reassortant AI virus that allows utilization of the DIVA strategy for the control of AI infections in poultry.     

珠海市斗门区人群甲型H1N1流感病毒抗体影响因素分析

目的了解珠海市斗门区人群甲型H1N1流感病毒感染状况及抗体水平,探讨影响普通人群血清甲型H1N1流感病毒抗体阳性的因素。方法采用多阶段随机抽样方法,从斗门区2个镇选取480人进行问卷调查,并采集血液标本进行甲型H1N1流感病毒血凝抑制（HI）检测。结果甲型H1N1流感抗体阳性人数为110人,阳性率为22.92%（110/480）。单因素和多因素二项Logistic分析均显示职业、接种甲型H1N1流感疫苗、调查前曾患过感冒与血清甲型H1N1流感病毒抗体阳性有关。结论珠海市斗门区人群甲型H1N1流感病毒抗体水平不高。接种甲型H1N1流感疫苗是预防甲型H1N1流感的重要手段。     

国产流感裂解疫苗接种人体后安全性和免疫原性评价

目的:评价变更后流感毒株生产的国产流感裂解疫苗安全性和免疫原性。方法:对宁波市宁海县559例分组接种流感裂解疫苗者进行临床指标(局部反应、全身反应)的监测,并抽取其中213例,用流感病毒HI抗体测定法对接种者免疫前后的抗体滴度进行测定,比较免疫前后抗体阳转率及几何平均滴度(GMT)。结果:观察对象接种疫苗后发热反应发生率为1.22%,全身其他反应发生率为1.22%,局部反应发生率为1.07%,且以轻度反应为主。H1N1型、H3N2型、B型免后抗体阳转率分别为84.04%、85.45%、83.57%;不同年龄组免后抗体滴度的增长倍数,H1N1型在5.50～16.00倍之间,H3N2型在13.16～39.33倍之间,B型在7.28～24.85倍之间,各年龄组各抗体型别免疫前后GMT差别均有统计学意义。结论:国产流感裂解疫苗具有良好的安全性和免疫原性。     

Randomized comparative study of the serum antihemagglutinin and antineuraminidase antibody responses to six licensed trivalent influenza vaccines

Background

Serum antibody to the hemagglutinin (HA) surface protein of influenza virus induced by influenza vaccination is a correlate of protection against influenza. The neuraminidase (NA) protein is also on the surface of the virus; antibody to it has been shown to impair virus release from infected cells and to reduce the intensity of influenza infections in animal models and in humans challenged with infectious virus. Recently we have shown that NA inhibiting antibody can independently contribute to immunity to naturally-occurring influenza immunity in the presence of antibody to the HA.

Purpose

The present study was conducted to evaluate induction of antibody to the NA and the HA by commercially available influenza vaccines.

Methods

Healthy young adults were vaccinated with one of five commercially available trivalent inactivated vaccines or live influenza vaccine. Frequencies of serum antibody and fold geometric mean titer (GMT) increases four weeks later were measured to each of the three vaccine viruses (A/H1N1, A/H3N2, B) in hemagglutination-inhibition (HAI) and neutralization (neut) assays. Frequency and fold GMT increase in neuraminidase-inhibition (NI) antibody titers were measured to the influenza A viruses (A/H1N1, A/H3N2).

Results

No significant reactogenicity occurred among the vaccinated subjects. The Fluvirin inactivated vaccine induced more anti-HA antibody responses and a higher fold GMT increase than the other inactivated vaccines but there were no major differences in response frequencies or fold GMT increase among the inactivated vaccines. Both the frequency of antibody increase and fold GMT increase were significantly lower for live vaccine than for any inactivated vaccine in HAI and neut assays for all three vaccine viruses. Afluria inactivated vaccine induced more N1 antibody and Fluarix induced more N2 antibody than the other vaccines but all inactivated vaccines induced serum NI antibody. The live vaccine failed to elicit any NI responses for the N2 NA of A/H3N2 virus and frequencies were low for the N1 of A/H1N1 virus.

Conclusions

Trivalent inactivated influenza vaccines with similar HA dosage induce similar serum anti-HA antibody responses in healthy adults. Current inactivated vaccines all induce serum anti-NA antibody to the N1 and N2 NA proteins but some are better than others for N1 or N2. The live vaccine, Flumist, was a poor inducer of either anti-HA or anti-NA serum antibody compared to inactivated vaccine in the healthy adults. In view of the capacity for contributing to immunity to influenza in humans, developing guidelines for NA content and induction of NA antibody is desirable.     

A high dosage influenza vaccine induced significantly more neuraminidase antibody than standard vaccine among elderly subjects

Antibody to the neuraminidase (NA) antigen of influenza viruses has been shown to correlate with immunity to influenza in humans and animal models. In a previous report, we showed that an inactivated influenza vaccine containing 60 μg of the hemagglutinin (HA) of each strain induced significantly more serum anti-HA antibody among elderly persons than did the standard vaccine containing 15 μg of the HA of each component. We developed a lectin-based assay for anti-NA antibody and used it to measure anti-NA antibody responses among subjects who had participated in that study. The high dosage vaccine contained eight times as much NA activity as the standard vaccine and induced a significantly higher frequency of antibody responses and higher mean postvaccination anti-NA titers to the N1 and N2 of the A/H1N1 and A/H3N2 viruses in the vaccines than did the standard vaccine. Ensuring an increased antibody response to the NA antigen in inactivated influenza virus vaccines should increase the protection against influenza. An increased quantity of the NA antigen in the vaccine will ensure an increased response.     

Antibodies cross-reactive to influenza A (H3N2) variant virus and impact of 2010-11 seasonal influenza vaccine on cross-reactive antibodies - United States

Since August 2011, a total of 12 human infections with influenza A (H3N2) variant viruses with genes from avian, swine, and human viruses (i.e., A [H3N2]v) that had acquired the M gene from influenza A (H1N1)pdm09 virus have been reported to CDC. Eleven of the cases occurred in children aged <10 years. In six cases, no history of recent exposure to swine was noted, suggesting that human-to-human transmission had occurred. This new gene constellation for A (H3N2)v viruses and its temporal association with an increase in human cases of A (H3N2)v highlight the need to better understand the risk for human infection with these viruses and the extent to which current seasonal vaccines might elicit cross-reactive antibodies to them. CDC conducted a preliminary analysis to evaluate the age-specific presence of serum cross-reactive antibody in U.S. populations vaccinated or not vaccinated with the 2010-11 seasonal trivalent influenza vaccine (TIV). The results indicated that 1) little or no cross-reactive antibody to A (H3N2)v exists among children aged <10 years, 2) immunization with the 2010-11 TIV had no impact on cross-reactive antibody levels in those aged <3 years, 3) cross-reactive antibody was detected in 20%-30% of those aged ≥10 years, and 4) among adults, vaccination with TIV provided a modest boost to the level of cross-reactive A (H3N2)v antibodies. Receipt of seasonal influenza vaccine continues to be recommended to protect against circulating human influenza viruses for all age groups and might provide limited protection against A (H3N2)v infection in the adult population. A vaccine virus specific for A (H3N2)v has been developed and could be used to produce an H3N2v vaccine, if needed.     

Antibody response to influenza immunization in two consecutive epidemic seasons in patients with renal diseases

The aim of this study was to assess antihemagglutinin and antineuraminidase antibody kinetics in 26 patients with renal diseases vaccinated against influenza in two consecutive epidemic seasons. Antibody responses were measured before immunization and 1, 3 and 6 months after immunization. Antihemagglutinin (HI) antibodies were determined by the hemagglutinin inhibition test and antineuraminidase (NI) antibody levels by the neuraminidase inhibition test. After vaccination HI and NI antibody titers significantly increased when compared with the pre-vaccination levels. Three months after vaccination the protection rates ranged from 50 to 61.5% in the 1995/96 season and 100% for all antigens in the 1996/97 season. Response rates ranged from 50 to 57.7% and 93.8 to 100% respectively. Significantly higher humoral response was recorded in the 1996/97 season than in the 1995/96 season. No serious adverse reactions were observed in the vaccinated patients and no symptoms of influenza or influenza-like infection were noted. In spite of some doubts about the safety and efficacy of influenza vaccination in patients from high-risk groups, the results of this study showed that many of them are able to produce HI antibodies in titers which are sufficient to protect against the influenza infection.     

Age distribution of patients with medically-attended illnesses caused by sequential variants of influenza A/H1N1: comparison to age-specific infection rates, 1978-1989

Since influenza A/H1N1 viruses reappeared during the 1977-1978 season, this subtype has contributed 27% of 6,609 documented influenza infections of persons with acute respiratory disease presenting to clinics serving as surveillance sites of the Influenza Research Center in Houston for the 12-year period ending June 1989. Wide differences in the distribution of H1N1 viruses occurred by age group: more than 50% of H1N1 infections were detected among persons aged 10-34 years, compared with 28% for influenza A/H3N2 and 35% for influenza B. Over age 35 years, the contribution of H1N1 viruses dropped to only 4%, compared with 20% and 16% for influenza A/H3N2 and influenza B, respectively. When birth dates of persons with positive cultures were examined, it was found that most of the H1N1-positive persons were born after 1950. Concurrently, longitudinal studies of families and other adults under intensive surveillance for infection, including cultures of all respiratory illnesses and tests for serum antibody rise over the respiratory disease season, revealed appreciable infection rates for adults born before 1950. Furthermore, the annual peak of hospitalization of older persons with pneumonia and other acute respiratory illnesses was significantly correlated with the peak of H1N1 virus activity in 1978-1979, a year when H1N1 viruses were the only influenza viruses prevalent. These observations indicate that many persons infected with influenza A/H1N1 viruses that circulated from 1946 through 1953 have immunity which has persisted for more than 25 years but this immunity is not complete. Reinfection that may result in serious illness in older vulnerable adults does occur but with lower frequency than with influenza A/H3N2 infection. Currently prevalent H1N1 variants are antigenically different from those that circulated in the 1950s; however, older adults readily acquire immunity to these new variants--perhaps as a result of immunologic priming that occurred in childhood.     

上海市人群甲型流感病毒H1、H3、H5、H9抗体血清学监测

[目的 ]　了解上海市人群对甲 3型、甲 1型流感病毒和禽流感病毒H9、H5的抗体 ,探讨甲 3型、甲 1型流感流行的趋势和禽流感病毒H9、H5抗体存在情况。　 [方法 ]　应用常规血凝抑制试验微量半加敏法对上海市不同年龄组人群进行甲型流感抗体血清学监测。　 [结果 ]　对A/SH/1/98(H3N2 )毒株的抗体阳性率为 96.2 0 % ;对A/SH /7/99(H1N1)毒株的抗体阳性率为 72 .60 % ;对H9毒株的抗体阳性率为 7.5 6% ;对H5毒株的抗体阳性率仅 0 .75 %。　 [结论 ]　A/SH/1/98(H3N2 )类毒株已不可能在上海再度引起流行 ;A/SH/7/99(H1N1)类毒株今后在人群中 ,特别在 0～ 4岁年龄组中可能存在散在性发生或局限性小爆发 ;本市人群曾受到禽流感H 9病毒的感染 ,不能排除由本市禽类中的H9病毒感染及人的可能 ;受禽流感H5毒株感染只是个别现象。禽流感病毒在本市禽类中的感染状况及对人的影响需进一步研究     

婴幼儿流行性感冒病毒抗体的检出率与新近感染分析

目的了解流行性感冒（流感）病毒流行后婴幼儿获得新近感染的情况。方法选择出生后3～4月龄、10～11月龄时两次暴露于流感病毒甲1（H1N1）亚型流行高峰的13月龄幼儿,和出生后5～6月龄时暴露于H1N1亚型流行高峰的8月龄婴儿的血清,测定流感病毒抗体。结果两次暴露于流感病毒H1N1亚型流行高峰的13月龄组中,H1N1亚型流感病毒的抗体阳性率为31.47%;暴露1次H1N1亚型流行高峰的8月龄组,抗体阳性率为26.42%;差异无统计学意义（χ2=1.207,P〉0.05）。结论13月龄组的幼儿可能在≤4月龄的那次H1N1亚型的暴露中,由于胎传抗体的保护而很少被感染,而8月龄组的婴儿是≥5月龄时才暴露,比13月龄组≤4月龄时的那次暴露更易被流感病毒感染。     

Serological studies of influenza viruses in pigs in Great Britain 1991-2.

Samples from a sow serum bank representative of the pig population of Great Britain collected during 1991-2, were examined for antibodies to influenza A, B and C viruses, using viruses which had been isolated from a variety of hosts. For influenza A viruses there was evidence of the continued circulation of classical swine H1N1 virus (26%) seroprevalence), and human H3N2 viruses (39%) which are antigenically most closely-related to A/Port Chalmers/1/73 virus. In addition antibodies were detected to A/swine/England/201635/92 (8%), a strain of H3N2 virus which appears to have arisen by antigenic drift from conventional H3N2 swine strains. Specific antibodies (2%) were detected to an H1N1 virus (A/swine/England/195852/92) related most closely to avian H1N1 strains. In tests with human H1N1 and H3N2 viruses, excluding isolates from pigs, the highest seroprevalence was detected to the prevailing strains from the human population. Serological tests with avian H4 and H10, human H2, equine 1 and 2 influenza A viruses were all negative. Seven pigs seropositive by haemagglutination-inhibition, virus neutralization and immunoblotting assays for antibody to influenza B virus, were randomly distributed geographically suggesting that influenza B viruses may be transmitted to pigs but fail to spread. The seroprevalence to influenza C viruses was 9.9% indicating that these viruses are widespread in pigs. These results provide further evidence that the pig can be infected by a number of influenza viruses, some of which may have significance in the epidemiology of human influenza.     

Antibody status to influenza A/Singapore/1/57(H2N2) in Finland during a period of outbreaks caused by H3N2 and H1N1 subtype viruses

The incidence of haemagglutination inhibition (HI) antibody (titre greater than or equal to 12) to influenza A/Singapore/1/57(H2N2) in sera collected from a Finnish population in the summer of 1981 was 58%. Subjects born after 1968 were essentially seronegative, and a comparable low HI antibody status was also recorded among the elderly, the lowest being in people born during the period 1901-10. A small increase in antibody titre to the H2N2 virus was observed in the different age groups after infections with the H3N2, but not the H1N1, subtype influenza A viruses. The heterotypic response, which could be due to HI or NA antibodies, was restricted almost exclusively to subjects already exhibiting this antibody in acute phase sera. Moreover, the anamnestic boosting was not as strong as that described in earlier studies from samples collected at the beginning of the present era of H3N2 viruses. At population level, the HI antibody status to H2N2 was about the same at the beginning and end of the follow-up period which covered eight epidemic seasons. The results are discussed with respect to the doctrine of 'original antigenic sin' and to the threat of re-emergence of the H2N2 viruses.     

Oseltamivir-Resistant Influenza A(H1N1)pdm09 Viruses,United States, 2013–14

We report characteristics of oseltamivir-resistant influenza A(H1N1)pdm09 viruses and patients infected with these viruses in the United States. During 2013–14, fifty-nine (1.2%) of 4,968 analyzed US influenza A(H1N1)pdm09 viruses had the H275Y oseltamivir resistance–conferring neuraminidase substitution. Our results emphasize the need for local surveillance for neuraminidase inhibitor susceptibility among circulating influenza viruses.     

Antibody status to influenza A/Singapore/1/57(H2N2) in Finland during a period of outbreaks caused by H3N2 and H1N1 subtype viruses.

The incidence of haemagglutination inhibition (HI) antibody (titre greater than or equal to 12) to influenza A/Singapore/1/57(H2N2) in sera collected from a Finnish population in the summer of 1981 was 58%. Subjects born after 1968 were essentially seronegative, and a comparable low HI antibody status was also recorded among the elderly, the lowest being in people born during the period 1901-10. A small increase in antibody titre to the H2N2 virus was observed in the different age groups after infections with the H3N2, but not the H1N1, subtype influenza A viruses. The heterotypic response, which could be due to HI or NA antibodies, was restricted almost exclusively to subjects already exhibiting this antibody in acute phase sera. Moreover, the anamnestic boosting was not as strong as that described in earlier studies from samples collected at the beginning of the present era of H3N2 viruses. At population level, the HI antibody status to H2N2 was about the same at the beginning and end of the follow-up period which covered eight epidemic seasons. The results are discussed with respect to the doctrine of ''original antigenic sin'' and to the threat of re-emergence of the H2N2 viruses.     

Comparative efficacy of North American and antigenically matched reverse genetics derived H5N9 DIVA marker vaccines against highly pathogenic Asian H5N1 avian influenza viruses in chickens

Highly pathogenic (HP) H5N1 avian influenza has become endemic in several countries in Asia and Africa, and vaccination is being widely used as a control tool. However, there is a need for efficacious vaccines preferably utilizing a DIVA (differentiate infected from vaccinated animals) marker strategy to allow for improved surveillance of influenza in vaccinated poultry. Using a reverse genetics approach, we generated Asian rgH5N9 vaccine strain deriving the hemagglutinin gene from A/chicken/Indonesia/7/2003 (H5N1) with modification of the cleavage site to be low pathogenic (LP) and N9 neuraminidase gene from the North American LP A/turkey/Wisconsin/1968 (H5N9) virus. The recombinant rgH5N9, A/turkey/Wisconsin/1968 (H5N9) A/chicken/Hidalgo/232/1994 (H5N2), and wild type HP A/chicken/Indonesia/7/2003 (H5N1) viruses were used to prepare inactivated oil-emulsified whole virus vaccines. Two weeks after vaccination, chickens were challenged with either Asian HP H5N1 viruses, A/chicken/Indonesia/7/2003 (W.H.O. clade 2.1) or A/chicken/Supranburi Thailand/2/2004 (W.H.O. clade 1.0). The H5 HA1 of the North American vaccine strains exhibited 12% amino acid differences including amino acid changes in the major antigenic sites as compared to the Asian HP H5N1 challenge viruses, serologically exhibited substantial antigenic difference, but still provided 100% protection from mortality. However, challenge virus shedding was significantly higher in chickens immunized with antigenically distinct American lineage vaccines as compared to the antigenically matched Asian rgH5N9 and the wild type Asian H5N1 vaccine. The antibody response to the heterologous subtype neuraminidase proteins were discriminated in vaccinated and infected chickens using a rapid fluorescent 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid sodium salt as substrate for neuraminidase inhibition assay. This study demonstrates the value of using a vaccine containing antigenically matched H5 hemagglutinin for control of HP H5N1 avian influenza in poultry and the potential utility of a heterologous neuraminidase as a DIVA marker.     

Influenza virus-like particles comprised of the HA, NA, and M1 proteins of H9N2 influenza virus induce protective immune responses in BALB/c mice

Avian influenza viruses represent a growing threat for an influenza pandemic. To develop recombinant vaccine for avian influenza of the H9N2 subtype, we expressed in insect cells virus-like particles (VLPs) consisting of three structural proteins of influenza A/Hong Kong/1073/99 (H9N2) virus. Upon infection of Sf9 cells with recombinant baculoviruses, the hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins were co-expressed in the infected cells, self-assembled, and released into the culture medium as VLPs of 80–120 nm in diameter. VLPs exhibited functional characteristics of influenza virus including hemagglutination and neuraminidase activities. In BALB/c mice, VLPs elicited serum antibodies specific for influenza A/Hong Kong/1073/99 (H9N2) virus and inhibited replication of the influenza virus after challenge. Thus, VLPs represent a potential strategy for the development of human vaccines against avian influenza H9N2 viruses.     

Neutralizing antibody but not hemagglutination antibody provides accurate evaluation for protective immune response to H5N1 avian influenza virus in vaccinated rabbits

In order to develop an animal model and an assay method to evaluate protective immune response to H5N1 avian influenza vaccination, H5N1 avian influenza vaccine was prepared. New Zealand rabbits were assigned to receive two doses of vaccine with different hemagglutinin (HA) dosage. The sera from vaccinated rabbits was evaluated to determine antibody titer and specificity using different tested methods including hemagglutination inhibition assay (HI), neutralizing assay (NT), cross-HI assay, cross-single immunodiffusion assay and cross-neutralization assay. The titer of HI antibody from rabbits immunized with different doses of HA were no less than 1:40 among groups 14 days after the first immunization. Whereas the NT antibody titer was less than 1:10 among groups 14 days after the first immunization. NT antibodies can be detected 14 days after the second immunization in rabbits immunized at HA doses higher than 6 μg, and the NT antibody titers were equal to or higher than 1:40. A good concentration-dependent NT antibody response can be detected in the vaccinated rabbits 14 days after the second immunization, and in contrast, no concentration-dependent relationship can be seen for HA antibody. The cross-HI test showed sera from vaccinated rabbits could cross react with influenza A H5N1 virus with the titers higher than 1:40. No cross reaction among different types (influenza A/H1N1 virus, influenza A/H3N2 virus, influenza B virus and influenza A/H5N1 virus) can be detected in the sera using the single immunodiffusion assay and using NT antibody test. This showed NT antibody test was demonstrated as a more accurate assay method for evaluating vaccination and quality of the vaccine than HI antibody test.