Evaluation of influenza virus-like particles and Novasome adjuvant as candidate vaccine for avian influenza

The development of safe and effective vaccines for avian influenza viruses is a priority for pandemic preparedness. Adjuvants improve the efficacy of vaccines and may allow antigen sparing during a pandemic. We have previously shown that influenza virus-like particles (VLPs) comprised of HA, NA, and M1 proteins represent a candidate vaccine for avian influenza H9N2 virus [Pushko P, Tumpey TM, Fang Bu, Knell J, Robinson R, Smith G. Influenza virus-like particles comprised of the HA, NA, and M1 proteins of H9N2 influenza virus induce protective immune responses in BALB/c mice. Vaccine 2005;23(50):5751-9]. In this study, an H9N2 VLP vaccine and recombinant HA (rH9) vaccine were evaluated in three animal models. The H9N2 VLP vaccine protected mice and ferrets from challenge with A/Hong Kong/1073/99 (H9N2) virus. Novasome adjuvant improved immunogenicity and protection. Positive effect of the adjuvant was also detected using the rH9 vaccine. The results have implications for the development of safe and effective vaccines for avian influenza viruses with pandemic potential.     

Generation and evaluation of a high-growth reassortant H9N2 influenza A virus as a pandemic vaccine candidate

H9N2 subtype avian influenza viruses (AIVs) are widely distributed in avian species and were isolated from humans in Hong Kong and Guangdong province, China in 1999 raising concern of their potential for pandemic spread. We generated a high-growth reassortant virus (G9/PR8) that contains the hemagglutinin (HA) and neuraminidase (NA) genes from the H9N2 avian influenza virus A/chicken/Hong Kong/G9/97 (G9) and six internal genes from A/Puerto Rico/8/34 (PR8) by genetic reassortment, for evaluation as a potential vaccine candidate in humans. Pathogenicity studies showed that the G9/PR8 reassortant was not highly pathogenic for mice or chickens. Two doses of a formalin-inactivated G9/PR8 virus vaccine induced hemagglutination inhibiting antibodies and conferred complete protection against challenge with G9 and the antigenically distinct H9N2 A/Hong Kong/1073/99 (G1-like) virus in a mouse model. These results indicate that the high growth G9/PR8 reassortant has properties that are desirable in a vaccine seed virus and is suitable for evaluation in humans for use in the event of an H9 pandemic.     

Characterization of a human H9N2 influenza virus isolated in Hong Kong

Two H9N2 viruses were isolated, for the first time, from humans in Hong Kong in 1999. Isolation of influenza viruses with a novel subtype of the hemagglutinin (HA) drew attention of health care authorities worldwide from the view of pandemic preparedness. Sequence analysis of the HA genes reveals that HA of A/Hong Kong/1073/99 (H9N2) is most closely related to that of A/quail/HK/G1/97 (H9N2) that contains the internal genes similar to those of Hong Kong/97 (H5N1) viruses. Phylogenetic and antigenic analyses demonstrated the diversity among H9 HA. A/Hong Kong/1073/99 was shown to cause a respiratory infection in Syrian hamsters, suggesting that the virus can replicate efficiently in mammalian hosts. We developed a whole virion test vaccine with a formalin-inactivated egg-grown HK1073. Intraperitoneal administration of the vaccine twice to hamsters conferred a complete protection against challenge infection by the MDCK cell-grown homologous virus. Receptor specificity of HK1073 appeared different from that of other avian influenza viruses of H9 subtype which recognize preferentially alpha-2,3 linked sialic acid. Hemagglutination of HK1073 with guinea pig erythrocytes was inhibited by both alpha-2,3 and alpha-2,6 linked sialic acid containing polymers. These data suggested that HK1073 had acquired a broader host range, including humans. Together with data so far available, the present study suggested that isolation of the H9 influenza viruses from humans requires precaution against the emergence of a novel human influenza.     

Vaccination with virus-like particles containing H5 antigens from three H5N1 clades protects chickens from H5N1 and H5N8 influenza viruses

Highly pathogenic avian influenza (HPAI) viruses, especially H5N1 strains, represent a public health threat and cause widespread morbidity and mortality in domestic poultry. Recombinant virus-like particles (VLPs) represent a promising novel vaccine approach to control avian influenza including HPAI strains. Influenza VLPs contain viral hemagglutinin (HA), which can be expressed in cell culture within highly immunogenic VLPs that morphologically and antigenically resemble influenza virions, except VLPs are non-infectious. Here we describe a recombinant VLP containing HA proteins derived from three distinct clades of H5N1 viruses as an experimental, broadly protective H5 avian influenza vaccine. A baculovirus vector was configured to co-express the H5 genes from recent H5N1 HPAI isolates A/chicken/Germany/2014 (clade 2.3.4.4), A/chicken/West Java/Subang/29/2007 (clade 2.1.3) and A/chicken/Egypt/121/2012 (clade 2.2.1). Co-expression of these genes in Sf9 cells along with influenza neuraminidase (NA) and retrovirus gag genes resulted in production of triple-clade H555 VLPs that exhibited hemagglutination activity and morphologically resembled influenza virions. Vaccination of chickens with these VLPs resulted in induction of serum antibody responses and efficient protection against experimental challenges with three different viruses including the recent U.S. H5N8 HPAI isolate. We conclude that these novel triple-clade VLPs represent a feasible strategy for simultaneously evoking protective antibodies against multiple variants of H5 influenza virus.     

Generation and characterization of a cold-adapted influenza A H9N2 reassortant as a live pandemic influenza virus vaccine candidate

H9N2 subtype influenza A viruses have been identified in avian species worldwide and were isolated from humans in 1999, raising concerns about their pandemic potential and prompting the development of candidate vaccines to protect humans against this subtype of influenza A virus. Reassortant H1N1 and H3N2 human influenza A viruses with the internal genes of the influenza A/Ann Arbor/6/60 (H2N2) (AA) cold-adapted (ca) virus have proven to be attenuated and safe as live virus vaccines in humans. Using classical genetic reassortment, we generated a reassortant virus (G9/AA ca) that contains the hemagglutinin and neuraminidase genes from influenza A/chicken/Hong Kong/G9/97 (H9N2) (G9) and six internal gene segments from the AA ca virus. When administered intranasally, the reassortant virus was immunogenic and protected mice from subsequent challenge with wild-type H9N2 viruses, although it was restricted in replication in the respiratory tract of mice. The G9/AA ca virus bears properties that are desirable in a vaccine for humans and is available for clinical evaluation and use, should the need arise.     

Recombinant H1N1 virus-like particle vaccine elicits protective immunity in ferrets against the 2009 pandemic H1N1 influenza virus

The pandemic virus of 2009 (2009 H1N1) continues to cause illness worldwide, especially in younger age groups. The widespread H1N1 virus infection further emphasizes the need for vaccine strategies that are effective against emerging pandemic viruses and are not dependent on the limitations of traditional egg-based technology. This report describes a recombinant influenza virus-like particle (VLP) vaccine consisting of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins of influenza A/California/04/2009 (H1N1) virus. Influenza H1N1 VLPs with a diameter of approximately 120 nm were released into the culture medium from Sf9 insect cells infected with recombinant baculovirus coexpressing HA, NA, and M1 proteins. Purified recombinant H1N1 VLPs morphologically resembled influenza virions and exhibited biological characteristics of influenza virus, including HA and NA activities. In the ferret challenge model, 2009 influenza H1N1 VLPs elicited high-titer serum hemagglutination inhibition (HI) antibodies specific for the 2009 H1N1 virus and inhibited replication of the influenza virus in the upper and lower respiratory tract tissues following A/Mexico/4482/09 (H1N1) virus challenge. Moreover, a single 15 μg dose of H1N1 VLPs resulted in complete virus clearance in the ferret lung. These results provide support for the use of recombinant influenza VLP vaccine as an effective strategy against pandemic H1N1 virus.     

Recombinant Newcastle disease virus expressing H9 HA protects chickens against heterologous avian influenza H9N2 virus challenge

In order to produce an efficient poultry H9 avian influenza vaccine that provides cross-protection against multiple H9 lineages, two Newcastle disease virus (NDV) LaSota vaccine strain recombinant viruses were generated using reverse genetics. The recombinant NDV-H9Con virus expresses a consensus-H9 hemagglutinin (HA) that is designed based on available H9N2 sequences from Chinese and Middle Eastern isolates. The recombinant NDV-H9Chi virus expresses a chimeric-H9 HA in which the H9 ectodomain of A/Guinea Fowl/Hong Kong/WF10/99 was fused with the cytoplasmic and transmembrane domain of the fusion protein (F) of NDV. Both recombinant viruses expressed the inserted HA stably and grew to high titers. An efficacy study in chickens showed that both recombinant viruses were able to provide protection against challenge with a heterologous H9N2 virus. In contrast to the NDV-H9Chi virus, the NDV-H9Con virus induced a higher hemagglutination inhibition titer against both NDV and H9 viruses in immunized birds, and efficiently inhibited virus shedding through the respiratory route. Moreover, sera collected from birds immunized with either NDV-H9Con or NDV-H9Chi were able to cross-neutralize two different lineages of H9N2 viruses, indicating that NDV-H9Con and NDV-H9Chi are promising vaccine candidates that could provide cross-protection among different H9N2 lineage viruses.     

Multi-clade H5N1 virus-like particles: Immunogenicity and protection against H5N1 virus and effects of beta-propiolactone

During the past decade, H5N1 highly pathogenic avian influenza (HPAI) viruses have diversified genetically and antigenically, suggesting the need for multiple H5N1 vaccines. However, preparation of multiple vaccines from live H5N1 HPAI viruses is difficult and economically not feasible representing a challenge for pandemic preparedness. Here we evaluated a novel multi-clade recombinant H5N1 virus-like particle (VLP) design, in which H5 hemagglutinins (HA) and N1 neuraminidase (NA) derived from four distinct clades of H5N1 virus were co-localized within the VLP structure. The multi-clade H5N1 VLPs were prepared by using a recombinant baculovirus expression system and evaluated for functional hemagglutination and neuraminidase enzyme activities, particle size and morphology, as well as for the presence of baculovirus in the purified VLP preparations. To remove residual baculovirus, VLP preparations were treated with beta-propiolactone (BPL). Immunogenicity and efficacy of multi-clade H5N1 VLPs were determined in an experimental ferret H5N1 HPAI challenge model, to ascertain the effect of BPL on immunogenicity and protective efficacy against lethal challenge. Although treatment with BPL reduced immunogenicity of VLPs, all vaccinated ferrets were protected from lethal challenge with influenza A/VietNam/1203/2004 (H5N1) HPAI virus, indicating that multi-clade VLP preparations treated with BPL represent a potential approach for pandemic preparedness vaccines.     

Antigenic relationships between type a influenza viruses of human, porcine, equine, and avian origin

This paper summarizes the available information on the relationship of two envelope antigens (haemagglutinin and neuraminidase) of influenzaviruses isolated from different hosts. The relationship of the haemagglutinin antigens was based on the results of haemagglutination inhibition tests with postinfection sera and that of the neuraminidase antigens on the results of neuraminidase inhibition and gel precipitation tests with hyperimmune and monospecific sera. On the basis of the antigenic specificity of the haemagglutinin, the influenzaviruses of human origin are divided into several subtypes (H0, H1, H2); viruses of equine origin could be divided into two subtypes (Heq1, Heq2). Porcine influenza strains are regarded as belonging to a single subtype, all of them being related to the prototype swine influenzavirus A (swine/Iowa/15/30). Within the avian influenzaviruses, 6 antigenic subtypes were described in earlier studies. Antigenic relationships between the haemagglutinin of strains from different hosts were infrequent but were demonstrated and confirmed between human A/Hong Kong/68 and equine viruses and between A/Hong Kong/68 and swine/Taiwan/69. The swine/Taiwan/69 virus also shared the related neuraminidase with A/Hong Kong/68 virus, and represents the only isolation from nonhuman sources of an influenzavirus identical with a human pandemic strain. The studies on the antigenic specificity of the neuraminidases demonstrated 8 antigenic varieties of neuraminidase among avian influenzaviruses and also that the neuraminidase grouping did not correspond with the antigenic grouping with regard to haemagglutinin. The relationships between human and nonhuman influenzaviruses are emphasized because of their significance to studies on the origin of influenza pandemics in man.     

Influenza A and B virus-like particles produced in mammalian cells are highly immunogenic and induce functional antibodies

Influenza virus-like particles (VLPs) represent an attractive alternative to traditional influenza vaccine formulations. Influenza VLPs mimic the natural virus while lacking the genetic material, are easily recognized by the immune system, and are considered safe. The use of a mammalian cell platform offers many advantages for VLP production, such as flexibility and the same glycosylation patterns as a human virus. In this study, the influenza VLPs containing hemagglutinin (HA), neuraminidase (NA) and matrix M1 proteins were expressed in CHO-K1, Vero or 293 T cell lines using transient transfection. After production in 3L bioreactor and purification, extensive characterization was performed on two batches of VLPs produced in 293 T, the best cell line for VLP expression; one batch expressed the HA and NA genes from A/Hong Kong/4801/2014 (H3N2) strain and the other, HA and NA genes from B/Phuket/3073/2013. Characterizations provided evidence that mammalian VLPs closely emulate the exterior of authentic virus particles in terms of both antigen presentation and biological properties. The two VLPs produced contained more NA proteins on their surface with a HA:NA ratio around 1:1 than influenza viruses which present a HA:NA ratio of around 4:1. Immunogenicity studies in BALB/c mice demonstrated that the VLPs, administered intra-muscularly, were highly immunogenic at low doses, with the induction of functional antibodies against HA and NA. Immunogenicity was also shown in a human in vitro model (MIMIC® system). In conclusion, we believe that influenza vaccines made of VLPs produced in mammalian cell lines, constitute a potential alternative to the classical influenza vaccines.     

Studies on relationships between human and porcine influenza. 2. Immunological comparisons of human A-Hong Kong-68 virus with influenza A viruses of porcine origin

Antigenic comparisons were made between the human A/Hong Kong/68 (H3N2) virus and a collection of influenza A viruses of swine origin. Haemagglutination-inhibition and neuraminidase-inhibition tests were used in addition to immunoprecipitin tests with monospecific antisera prepared against purified haemagglutinin and neuraminidase preparations. The antigenic relationships revealed by the studies are summarized as follows: (1) swine/Taiwan/7310/70 virus contained envelope antigens that were antigenically indistinguishable from those of A/Hong Kong/68 virus, (2) “classical” strains of swine influenzavirus related to A/swine/Iowa/15/30 (Hsw1N1) isolated between 1930 and 1967 contained neuraminidase that was antigenically distinct from that of A/Hong Kong/68 virus but related to that of human A0 and A1 viruses, and (3) the haemagglutinins of certain strains of “classical” influenza A virus appeared to show a minor antigenic relationship with the haemagglutinin of A/Hong Kong/68 virus. Immunoprecipitin tests suggested that this relationship was confined to only one of the two antigenic components of the haemagglutinin subunit. The antigenic relationships are discussed in respect of possible epidemiological relationships between human and swine influenza A viruses. It is proposed that the swine/Taiwan isolates be designated A/swine/Taiwan/70 (H3N2), indicating their antigenic identity with the human A/Hong Kong/1/68 virus.     

长沙市首例人感染H9N2禽流感病毒的分离及基因特征分析

2015年9月从来源于国家级流感样病例监测哨点医院的标本中分离出一株H9N2亚型禽流感病毒。为了了解病毒的来源、重组、变异及耐药等分子特征,本研究对这株病毒的神经氨酸酶(Neuraminidase,NA)和基质蛋白(Matrix protein,M)基因进行测序及序列特征分析。结果显示长沙市人感染H9N2毒株的NA基因与A/duck/jiangxi/20147/2013(H9N2)禽源关系最近,遗传进化分支属于欧亚系中A/Duck/Hong Kong/Y280/97(H9N2)分支。M基因与A/duck/wenzhou/YHQL64/2014 (H9N2)禽源关系最近,属于A/Quail/Hong Kong/G1/97(H9N2)分支。该病毒分离株的NA和M基因与2014年长沙市活禽环境中的H9N2病毒高度同源。影响NA蛋白功能的关键氨基酸位点相对保守,未产生与抗神经氨酸酶抑制剂相关的氨基酸突变;M2蛋白的第31位氨基酸由S突变为N导致病毒对金刚烷胺类药物耐药。因此,长沙市的人感染H9N2禽流感病毒有可能是来源于长沙市活禽环境中的H9N2病毒,它们是通过家禽贸易传播并发生病毒重配而产生。     

Infection of mice with a human influenza A/H3N2 virus induces protective immunity against lethal infection with influenza A/H5N1 virus

The transmission of highly pathogenic avian influenza (HPAI) A viruses of the H5N1 subtype from poultry to man and the high case fatality rate fuels the fear for a pandemic outbreak caused by these viruses. However, prior infections with seasonal influenza A/H1N1 and A/H3N2 viruses induce heterosubtypic immunity that could afford a certain degree of protection against infection with the HPAI A/H5N1 viruses, which are distantly related to the human influenza A viruses. To assess the protective efficacy of such heterosubtypic immunity mice were infected with human influenza virus A/Hong Kong/2/68 (H3N2) 4 weeks prior to a lethal infection with HPAI virus A/Indonesia/5/05 (H5N1).     

Production and characterization of mammalian virus-like particles from modified vaccinia virus Ankara vectors expressing influenza H5N1 hemagglutinin and neuraminidase

Several studies have described the production of influenza virus-like particles (VLP) using a variety of platform systems. These VLPs are non-replicating particles that spontaneously self-assemble from expressed influenza virus proteins and have been proposed as vaccine candidates for both seasonal and pandemic influenza. Although still in the early stages of development and evaluation as influenza vaccines, influenza VLPs have a variety of other valuable uses such as examining and understanding correlates of protection against influenza and investigating virus-cell interactions. The most common production system for influenza VLPs is the baculovirus-insect cell expression which has several attractive features including the ease in which new gene combinations can be constructed, the immunogenicity elicited and protection afforded by the produced VLPs, and the scalability offered by the system. However, there are differences between the influenza VLPs produced by baculovirus expression systems in insect cells and the influenza viruses produced for use as current vaccines or the virus produced during a productive clinical infection. We describe here the development of a modified vaccinia virus Ankara (MVA) system to generate mammalian influenza VLPs containing influenza H5N1 proteins. The MVA vector system is flexible for manipulating and generating various VLP constructs, expresses high level of influenza hemagglutinin (HA), neuraminidase (NA), and matrix (M) proteins, and can be scaled up to produce VLPs in quantities sufficient for in vivo studies. We show that mammalian VLPs are generated from recombinant MVA vectors expressing H5N1 HA alone, but that increased VLP production can be achieved if NA is co-expressed. These mammalian H5N1 influenza VLPs have properties in common with live virus, as shown by electron microscopy analysis, their ability to hemagglutinate red blood cells, express neuraminidase activity, and to bind influenza specific antibodies. Importantly, these VLPs are able to elicit a protective immune response in a mouse challenge model, suggesting their utility in dissecting the correlates of immunity in such models. Mammalian derived VLPs may also provide a useful tool for studying virus-cell interactions and may have potential for development as pandemic vaccines.     

Diversity of influenza A virus subtypes isolated from domestic poultry in Hong Kong.

The second phase of a 2-year influenza virus surveillance programme of domestic avian species in Hong Kong (up to October 1977) yielded influenza A virus, Newcastle disease virus, and Hong Kong paramyxovirus, as well as unidentified haemagglutinating agents. These viruses were isolated from the trachea or cloaca of apparently healthy domestic ducks, geese, and chickens originating from China and Hong Kong. Twenty-five combinations of haemagglutinin and neuraminidase surface antigens were identified from the 136 influenza A viruses isolated. Eight of the combinations do not appear to have been previously reported — Hav3Nav2, Hav4Nav2, Hav4Nav4, Hav4Nav5, Hav4Neq1, Hav6Nav4, Hav6Nav6, and Hav9Nav1. The existence of such a diverse pool of influenza virus genetic information may play a role in the emergence of new human pandemic strains.     

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     

Influenza activity in China: 1998-1999

During 1989-1999, influenza A H3N2 and H1N1 subtypes and B type viruses were still co-circulating in human population in China, while influenza A (H3N2) virus was predominant strain. The two antigenically and genetically distinguishable strains of influenza B virus were also still co-circulating in men in southern China. The antigenic analysis indicated that most of the H3N2 viruses were A/Panama/2007/99 (H3N2)-like strain, the most of the H1N1 viruses were antigenically similar to A/Beijing/262/95 (H1N1) virus. However, most of the influenza B viruses were B/Beijing/184/93-like strain, but few of them were antigenically similar to B/Shandong/7/97 virus. In the summer of 1998, the influenza outbreaks caused by H3N2 subtype of influenza A virus occurred widely in southern China. Afterwards, during 1998-1999 influenza season, a severe influenza epidemic caused by H3N2 virus emerged in northern China. The morbidity was reached as high as 10% in Beijing area. It was interesting that during influenza, surveillance from 1998 to 1999, five strains of avian influenza A (H9N2) virus were isolated from outpatients with influenza-like illness in July-August of 1998, and another one was repeatedly isolated from a child suffering from influenza-like disease in November of 1999 in Guangdong province. The genetic analysis revealed that the five strains isolated in 1998 were genetically closely related to H9N2 viruses being isolated from chickens (G9 lineage virus), whereas, A/Guangzhou/333/99 (H9N2) virus was a reassortant derived from reassortment between G9 and G1 lineage of avian influenza A (H9N2) viruses due to its genes encoding the HA, NA, NP and NS proteins, closely related to G9 lineage virus, the rest of the genes encoding the M and three polymerase (PB2, PB1 and PA) were closely related to G1 lineage strain of H9N2 virus. However, no avian influenza A (H5N1) virus has so far been isolated neither from in or outpatients with influenza-like disease in mainland China. Unfortunately, where did the reassortment occur and how did the reassortant transmit to men? These questions are still unknown.     

H5N1 VLP vaccine induced protection in ferrets against lethal challenge with highly pathogenic H5N1 influenza viruses

In this study, recombinant virus-like particles (VLPs) were evaluated as a candidate vaccine against emerging influenza viruses with pandemic potential. The VLPs are composed of the hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1) proteins of the H5N1 A/Indonesia/05/2005 (clade 2.1; [Indo/05]) virus, which were expressed using baculovirus in Spodoptera frugiperda (Sf9) cells. Ferrets received either 2 injections of the VLP vaccine at escalating doses (based on HA content), recombinant HA, or were mock vaccinated. Vaccinated ferrets were then challenged with either H5N1 Indo/05 or H5N1 A/Viet Nam 1203/2004 (VN/04) wild-type viruses. All ferrets that received the VLP vaccine survived regardless of the VLP dose or challenge strain, whereas seven of eight mock vaccinated ferrets died. The VLP vaccine induced HAI antibodies against the homologous H5N1 clade 2.1 strain, as well as heterologous strains from H5N1 clades 1, 2.2, and 2.3. The magnitude of the HAI titers correlated with VLP dose. Neutralizing antibody responses against the Indo/05 and VN/04 strains showed a similar pattern. Affinity of the anti-HA antibodies raised by the H5N1 Indo/05 VLPs had a higher association rate to the homologous clade 2.1 HA than to the clade 1 (VN/04) HA; however, once bound, antibodies had similar slow disassociation rates. These results provide support for continued development of the H5N1 VLPs as a candidate vaccine against pandemic influenza. Exploration of immunologic correlates of protection for H5N1 vaccines beyond HAI and neutralizing antibody responses is warranted.     

Lack of evidence for human-to-human transmission of avian influenza A (H9N2) viruses in Hong Kong, China 1999

In April 1999, isolation of avian influenza A (H9N2) viruses from humans was confirmed for the first time. H9N2 viruses were isolated from nasopharyngeal aspirate specimens collected from two children who were hospitalized with uncomplicated, febrile, upper respiratory tract illnesses in Hong Kong during March 1999. Novel influenza viruses have the potential to initiate global pandemics if they are sufficiently transmissible among humans. We conducted four retrospective cohort studies of persons exposed to these two H9N2 patients to assess whether human-to-human transmission of avian H9N2 viruses had occurred. No serologic evidence of H9N2 infection was found in family members or health-care workers who had close contact with the H9N2-infected children, suggesting that these H9N2 viruses were not easily transmitted from person to person.     

The 2001/2002 influenza season and the vaccine composition for the 2002/2003 season

The epidemic in the influenza season 2001/2002 was of moderate activity just like in 2000/2001. The influenza epidemic started in week 2 of 2002 when the clinical influenza activity reported by the general practitioner network of the Netherlands Institute of Primary Health Care (NIVEL) increased. This was caused by influenza A viruses of the H3N2 subtype in particular. All influenza A viruses of this subtype were closely related to the vaccine strain for this subtype, A/Moscow/10/99. Influenza B viruses and influenza A/H1 viruses isolated this season had surprising features. The influenza B viruses originated from two lineages. Viruses of the B/Yamagata/16/88 lineage have been circulating for more than twelve years. The vaccine reference strain B/Sichuan/379/99 belongs to this lineage. The B/Victoria/2/87 lineage reappeared again after an absence in Europe of more than ten years and accounted for 50% of the influenza B viruses that were isolated in the Netherlands. Therefore the vaccine will have provided only partial protection against influenza B. The only influenza A/H1 virus that was isolated appeared to be of a new subtype H1N2. The H1 hemagglutinin of this virus was closely related to that of the vaccine strain A/New Caledonia/20/99. The N2 neuraminidase originated from recent human influenza A/H3N2 viruses. Therefore the vaccine probably provided good protection against the new H1N2 subtype. Based in part on these data, the World Health Organization has advised that the vaccines for the season 2002/2003 should contain the following or comparable influenza-virus strains: A/Moscow/10/99 (H3N2), A/New Caledonia/20/99 (H1N1) and B/Hong Kong/330/01, the latter being an influenza B virus of the B/Victoria/2/87 lineage.