Protection of Mice against Lethal Challenge with 2009 H1N1 Influenza A Virus by 1918-Like and Classical Swine H1N1 Based Vaccines

The recent 2009 pandemic H1N1 virus infection in humans has resulted in nearly 5,000 deaths worldwide. Early epidemiological findings indicated a low level of infection in the older population (>65 years) with the pandemic virus, and a greater susceptibility in people younger than 35 years of age, a phenomenon correlated with the presence of cross-reactive immunity in the older population. It is unclear what virus(es) might be responsible for this apparent cross-protection against the 2009 pandemic H1N1 virus. We describe a mouse lethal challenge model for the 2009 pandemic H1N1 strain, used together with a panel of inactivated H1N1 virus vaccines and hemagglutinin (HA) monoclonal antibodies to dissect the possible humoral antigenic determinants of pre-existing immunity against this virus in the human population. By hemagglutinination inhibition (HI) assays and vaccination/challenge studies, we demonstrate that the 2009 pandemic H1N1 virus is antigenically similar to human H1N1 viruses that circulated from 1918–1943 and to classical swine H1N1 viruses. Antibodies elicited against 1918-like or classical swine H1N1 vaccines completely protect C57B/6 mice from lethal challenge with the influenza A/Netherlands/602/2009 virus isolate. In contrast, contemporary H1N1 vaccines afforded only partial protection. Passive immunization with cross-reactive monoclonal antibodies (mAbs) raised against either 1918 or A/California/04/2009 HA proteins offered full protection from death. Analysis of mAb antibody escape mutants, generated by selection of 2009 H1N1 virus with these mAbs, indicate that antigenic site Sa is one of the conserved cross-protective epitopes. Our findings in mice agree with serological data showing high prevalence of 2009 H1N1 cross-reactive antibodies only in the older population, indicating that prior infection with 1918-like viruses or vaccination against the 1976 swine H1N1 virus in the USA are likely to provide protection against the 2009 pandemic H1N1 virus. This data provides a mechanistic basis for the protection seen in the older population, and emphasizes a rationale for including vaccination of the younger, naïve population. Our results also support the notion that pigs can act as an animal reservoir where influenza virus HAs become antigenically frozen for long periods of time, facilitating the generation of human pandemic viruses.     

Modifications in the polymerase genes of a swine-like triple-reassortant influenza virus to generate live attenuated vaccines against 2009 pandemic H1N1 viruses

On 11 June 2009, the World Health Organization (WHO) declared that the outbreaks caused by novel swine-origin influenza A (H1N1) virus had reached pandemic proportions. The pandemic H1N1 (H1N1pdm) virus is the predominant influenza virus strain in the human population. It has also crossed the species barriers and infected turkeys and swine in several countries. Thus, the development of a vaccine that is effective in multiple animal species is urgently needed. We have previously demonstrated that the introduction of temperature-sensitive mutations into the PB2 and PB1 genes of an avian H9N2 virus, combined with the insertion of a hemagglutinin (HA) tag in PB1, resulted in an attenuated (att) vaccine backbone for both chickens and mice. Because the new pandemic strain is a triple-reassortant (TR) virus, we chose to introduce the double attenuating modifications into a swine-like TR virus isolate, A/turkey/OH/313053/04 (H3N2) (ty/04), with the goal of producing live attenuated influenza vaccines (LAIV). This genetically modified backbone had impaired polymerase activity and restricted virus growth at elevated temperatures. In vivo characterization of two H1N1 vaccine candidates generated using the ty/04 att backbone demonstrated that this vaccine is highly attenuated in mice, as indicated by the absence of signs of disease, limited replication, and minimum histopathological alterations in the respiratory tract. A single immunization with the ty/04 att-based vaccines conferred complete protection against a lethal H1N1pdm virus infection in mice. More importantly, vaccination of pigs with a ty/04 att-H1N1 vaccine candidate resulted in sterilizing immunity upon an aggressive intratracheal challenge with the 2009 H1N1 pandemic virus. Our studies highlight the safety of the ty/04 att vaccine platform and its potential as a master donor strain for the generation of live attenuated vaccines for humans and livestock.     

Characterization of cross protection of Swine-Origin Influenza Virus (S-OIV) H1N1 and reassortant H5N1 influenza vaccine in BALB/c mice given a single-dose vaccination

Background

Influenza virus has antigen drift and antigen shift effect, vaccination with some influenza vaccine might not induce sufficient immunity for host to the threat of other influenza virus strains. S-OIV H1N1 and H5N1 influenza vaccines in single-dose immunization were evaluated in mice for cross protection to the challenge of A/California/7/2009 H1N1 or NIBRG-14 H5N1 virus.

Results

Both H1N1 and H5N1 induced significant homologous IgG, HAI, and microneutralization antibody responses in the mice, while only vaccines plus adjuvant produced significant heterogeneous IgG and HAI antibody responses. Both alum and MPLA adjuvants significantly reduced the S-OIV H1N1 vaccine dose required to elicit protective HAI antibody titers from 0.05 μg to 0.001 μg. Vaccines alone did not protect mice from challenge with heterogeneous influenza virus, while H5N1 vaccine plus alum and MPLA adjuvants did. Mouse body weight loss was also less significant in the presence of adjuvant than in the vaccine without adjuvant. Furthermore, both H1N1 and H5N1 lung viral titers of immunized mice were significantly reduced post challenge with homologous viruses.

Conclusion

Only in the presence of MPLA adjuvant could the H5N1 vaccine significantly reduce mouse lung viral titers post H1N1 virus challenge, and not vice versa. MPLA adjuvant induced cross protection with a single dose vaccination to the challenge of heterogeneous influenza virus in mice. Lung viral titer seemed to be a better indicator compared to IgG, neutralization antibody, and HAI titer to predict survival of mice infected with influenza virus.     

Live Attenuated Influenza Viruses Containing NS1 Truncations as Vaccine Candidates against H5N1 Highly Pathogenic Avian Influenza

Due to the high mortality associated with recent, widely circulating strains of H5N1 influenza virus in poultry, the recurring introduction of H5N1 viruses from birds to humans, and the difficulties in H5N1 eradication by elimination of affected flocks, an effective vaccine against HPAI (highly pathogenic avian influenza) is highly desirable. Using reverse genetics, a set of experimental live attenuated vaccine strains based on recombinant H5N1 influenza virus A/Viet Nam/1203/04 was generated. Each virus was attenuated through expression of a hemagglutinin protein in which the polybasic cleavage site had been removed. Viruses were generated which possessed a full-length NS1 or a C-terminally truncated NS1 protein of 73, 99, or 126 amino acids. Viruses with each NS genotype were combined with a PB2 polymerase gene which carried either a lysine or a glutamic acid at position 627. We predicted that glutamic acid at position 627 of PB2 would attenuate the virus in mammalian hosts, thus increasing the safety of the vaccine. All recombinant viruses grew to high titers in 10-day-old embryonated chicken eggs but were attenuated in mammalian cell culture. Induction of high levels of beta interferon by all viruses possessing truncations in the NS1 protein was demonstrated by interferon bioassay. The viruses were each found to be highly attenuated in a mouse model. Vaccination with a single dose of any virus conferred complete protection from death upon challenge with a mouse lethal virus expressing H5N1 hemagglutinin and neuraminidase proteins. In a chicken model, vaccination with a single dose of a selected virus encoding the NS1 1-99 protein completely protected chickens from lethal challenge with homologous HPAI virus A/Viet Nam/1203/04 (H5N1) and provided a high level of protection from a heterologous virus, A/egret/Egypt/01/06 (H5N1). Thus, recombinant influenza A/Viet Nam/1203/04 viruses attenuated through the introduction of mutations in the hemagglutinin, NS1, and PB2 coding regions display characteristics desirable for live attenuated vaccines and hold potential as vaccine candidates in poultry as well as in mammalian hosts.     

Single-dose mucosal immunization with a candidate universal influenza vaccine provides rapid protection from virulent H5N1, H3N2 and H1N1 viruses

Background

The sudden emergence of novel influenza viruses is a global public health concern. Conventional influenza vaccines targeting the highly variable surface glycoproteins hemagglutinin and neuraminidase must antigenically match the emerging strain to be effective. In contrast, “universal” vaccines targeting conserved viral components could be used regardless of viral strain or subtype. Previous approaches to universal vaccination have required protracted multi-dose immunizations. Here we evaluate a single dose universal vaccine strategy using recombinant adenoviruses (rAd) expressing the conserved influenza virus antigens matrix 2 and nucleoprotein.

Methodology/Principal Findings

In BALB/c mice, administration of rAd via the intranasal route was superior to intramuscular immunization for induction of mucosal responses and for protection against highly virulent H1N1, H3N2, or H5N1 influenza virus challenge. Mucosally vaccinated mice not only survived, but had little morbidity and reduced lung virus titers. Protection was observed as early as 2 weeks post-immunization, and lasted at least 10 months, as did antibodies and lung T cells with activated phenotypes. Virus-specific IgA correlated with but was not essential for protection, as demonstrated in studies with IgA-deficient animals.

Conclusion/Significance

Mucosal administration of NP and M2-expressing rAd vectors provided rapid and lasting protection from influenza viruses in a subtype-independent manner. Such vaccines could be used in the interval between emergence of a new virus strain and availability of strain-matched vaccines against it. This strikingly effective single-dose vaccination thus represents a candidate off-the-shelf vaccine for emergency use during an influenza pandemic.     

Molecular Basis of Live-Attenuated Influenza Virus

Human influenza is a seasonal disease associated with significant morbidity and mortality. The most effective means for controlling infection and thereby reducing morbidity and mortality is vaccination with a three inactivated influenza virus strains mixture, or by intranasal administration of a group of three different live attenuated influenza vaccine strains. Comparing to the inactivated vaccine, the attenuated live viruses allow better elicitation of a long-lasting and broader immune (humoral and cellular) response that represents a naturally occurring transient infection. The cold-adapted (ca) influenza A/AA/6/60 (H2N2) (AA ca) virus is the backbone for the live attenuated trivalent seasonal influenza vaccine licensed in the United States. Similarly, the influenza A components of live-attenuated vaccines used in Russia have been prepared as reassortants of the cold-adapted (ca) H2N2 viruses, A/Leningrad/134/17/57-ca (Len/17) and A/Leningrad/134/47/57-ca (Len/47) along with virulent epidemic strains. However, the mechanism of temperature-sensitive attenuation is largely elusive. To understand how modification at genetic level of influenza virus would result in attenuation of human influenza virus A/PR/8/34 (H1N1,A/PR8), we investigated the involvement of key mutations in the PB1 and/or PB2 genes in attenuation of influenza virus in vitro and in vivo. We have demonstrated that a few of residues in PB1 and PB2 are critical for the phenotypes of live attenuated, temperature sensitive influenza viruses by minigenome assay and real-time PCR. The information of these mutation loci could be used for elucidation of mechanism of temperature-sensitive attenuation and as a new strategy for influenza vaccine development.     

Reassortant H9N2 influenza viruses containing H5N1-like PB1 genes isolated from black-billed magpies in Southern China

H9N2 influenza A viruses have become endemic in different types of terrestrial poultry and wild birds in Asia, and are occasionally transmitted to humans and pigs. To evaluate the role of black-billed magpies (Pica pica) in the evolution of influenza A virus, we conducted two epidemic surveys on avian influenza viruses in wild black-billed magpies in Guangxi, China in 2005 and characterized three isolated black-billed magpie H9N2 viruses (BbM viruses). Phylogenetic analysis indicated that three BbM viruses were almost identical with 99.7 to 100% nucleotide homology in their whole genomes, and were reassortants containing BJ94-like (Ck/BJ/1/94) HA, NA, M, and NS genes, SH/F/98-like (Ck/SH/F/98) PB2, PA, and NP genes, and H5N1-like (Ck/YN/1252/03, clade 1) PB1 genes. Genetic analysis showed that BbM viruses were most likely the result of multiple reassortments between co-circulating H9N2-like and H5N1-like viruses, and were genetically different from other H9N2 viruses because of the existence of H5N1-like PB1 genes. Genotypical analysis revealed that BbM viruses evolved from diverse sources and belonged to a novel genotype (B46) discovered in our recent study. Molecular analysis suggested that BbM viruses were likely low pathogenic reassortants. However, results of our pathogenicity study demonstrated that BbM viruses replicated efficiently in chickens and a mammalian mouse model but were not lethal for infected chickens and mice. Antigenic analysis showed that BbM viruses were antigenic heterologous with the H9N2 vaccine strain. Our study is probably the first report to document and characterize H9N2 influenza viruses isolated from black-billed magpies in southern China. Our results suggest that black-billed magpies were susceptible to H9N2 influenza viruses, which raise concerns over possible transmissions of reassortant H9N2 viruses among poultry and wild birds.     

Immunity to influenza A H9N2 viruses induced by infection and vaccination

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

H5N1亚型禽流感病毒基因重排后毒力的变化

[目的]为了探讨高致病性禽流感病毒对水禽致病性差异的分子致病机理.[方法]我们对从野鸭分离到的H5N1亚型禽流感病毒的生物学特性进行鉴定,其中A/mallard/Huadong/Y/2003(Y)是对麻鸭无致病性病毒,而 A/mallard/Huadong/S/2005(S)是对麻鸭高致病性病毒.利用反向遗传技术构建一系列单个和多个基因组合替换基因重排病毒,并验证重排病毒在麻鸭上的致病力.[结果]研究表明,PB2, PB1, PA(3P), HA单基因以及3P基因组合替换的使S病毒对麻鸭的毒力完全致弱,但相应的基因替换后仅使Y病毒对麻鸭的毒力略有上升.两病毒的其它基因对毒力影响较小.[结论]H5N1亚型禽流感病毒对麻鸭的致病力受多基因调控,且这种调控作用在不同病毒骨架上的影响不一致,强毒受影响程度远比弱毒的大.     

Isolation and genetic characterization of avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China

As pigs are susceptible to both human and avian influenza viruses, they have been proposed to be intermediate hosts or mixing vessels for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we reported avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China. Homology and phylogenetic analyses showed that the H1N1 virus (A/swine/Zhejiang/1/07) was closely to avian-like H1N1 viruses and seemed to be derived from the European swine H1N1 viruses, which was for the first time reported in China; and the two H1N2 viruses (A/swine/Shanghai/1/07 and A/swine/Guangxi/13/06) were novel ressortant H1N2 influenza viruses containing genes from the classical swine (HA, NP, M and NS), human (NA and PB1) and avian (PB2 and PA) lineages, which indicted that the reassortment among human, avian, and swine influenza viruses had taken place in pigs in China and resulted in the generation of new viruses. The isolation of avian-like H1N1 influenza virus originated from the European swine H1N1 viruses, especially the emergence of two novel ressortant H1N2 influenza viruses provides further evidence that pigs serve as intermediate hosts or “mixing vessels”, and swine influenza virus surveillance in China should be given a high priority.     

MVA-Based H5N1 Vaccine Affords Cross-Clade Protection in Mice against Influenza A/H5N1 Viruses at Low Doses and after Single Immunization

Human infections with highly pathogenic avian influenza viruses of the H5N1 subtype, frequently reported since 2003, result in high morbidity and mortality. It is feared that these viruses become pandemic, therefore the development of safe and effective vaccines is desirable. MVA-based H5N1 vaccines already proved to be effective when two immunizations with high doses were used. Dose-sparing strategies would increase the number of people that can be vaccinated when the amount of vaccine preparations that can be produced is limited. Furthermore, protective immunity is induced ideally after a single immunization. Therefore the minimal requirements for induction of protective immunity with a MVA-based H5N1 vaccine were assessed in mice. To this end, mice were vaccinated once or twice with descending doses of a recombinant MVA expressing the HA gene of influenza virus A/Vietnam/1194/04. The protective efficacy was determined after challenge infection with the homologous clade 1 virus and a heterologous virus derived from clade 2.1, A/Indonesia/5/05 by assessing weight loss, virus replication and histopathological changes. It was concluded that MVA-based vaccines allowed significant dose-sparing and afford cross-clade protection, also after a single immunization, which are favorable properties for an H5N1 vaccine candidate.     

DNA Vaccine Encoding Hemagglutinin Provides Protective Immunity against H5N1 Influenza Virus Infection in Mice

In Hong Kong in 1997, a highly lethal H5N1 avian influenza virus was apparently transmitted directly from chickens to humans with no intermediate mammalian host and caused 18 confirmed infections and six deaths. Strategies must be developed to deal with this virus if it should reappear, and prospective vaccines must be developed to anticipate a future pandemic. We have determined that unadapted H5N1 viruses are pathogenic in mice, which provides a well-defined mammalian system for immunological studies of lethal avian influenza virus infection. We report that a DNA vaccine encoding hemagglutinin from the index human influenza isolate A/HK/156/97 provides immunity against H5N1 infection of mice. This immunity was induced against both the homologous A/HK/156/97 (H5N1) virus, which has no glycosylation site at residue 154, and chicken isolate A/Ck/HK/258/97 (H5N1), which does have a glycosylation site at residue 154. The mouse model system should allow rapid evaluation of the vaccine’s protective efficacy in a mammalian host. In our previous study using an avian model, DNA encoding hemagglutinin conferred protection against challenge with antigenic variants that differed from the primary antigen by 11 to 13% in the HA1 region. However, in our current study we found that a DNA vaccine encoding the hemagglutinin from A/Ty/Ir/1/83 (H5N8), which differs from A/HK/156/97 (H5N1) by 12% in HA1, prevented death but not H5N1 infection in mice. Therefore, a DNA vaccine made with a heterologous H5 strain did not prevent infection by H5N1 avian influenza viruses in mice but was useful in preventing death.     

Development and Pre-Clinical Evaluation of Two LAIV Strains against Potentially Pandemic H2N2 Influenza Virus

H2N2 Influenza A caused the Asian flu pandemic in 1957, circulated for more than 10 years and disappeared from the human population after 1968. Given that people born after 1968 are naïve to H2N2, that the virus still circulates in wild birds and that this influenza subtype has a proven pandemic track record, H2N2 is regarded as a potential pandemic threat. To prepare for an H2N2 pandemic, here we developed and tested in mice and ferrets two live attenuated influenza vaccines based on the haemagglutinins of the two different H2N2 lineages that circulated at the end of the cycle, using the well characterized A/Leningrad/134/17/57 (H2N2) master donor virus as the backbone. The vaccine strains containing the HA and NA of A/California/1/66 (clade 1) or A/Tokyo/3/67 (clade 2) showed a temperature sensitive and cold adapted phenotype and a reduced reproduction that was limited to the respiratory tract of mice, suggesting that the vaccines may be safe for use in humans. Both vaccine strains induced haemagglutination inhibition titers in mice. Vaccination abolished virus replication in the nose and lung and protected mice from weight loss after homologous and heterologous challenge with the respective donor wild type strains. In ferrets, the live attenuated vaccines induced high virus neutralizing, haemagglutination and neuraminidase inhibition titers, however; the vaccine based on the A/California/1/66 wt virus induced higher homologous and better cross-reactive antibody responses than the A/Tokyo/3/67 based vaccine. In line with this observation, was the higher virus reduction observed in the throat and nose of ferrets vaccinated with this vaccine after challenge with either of the wild type donor viruses. Moreover, both vaccines clearly reduced the infection-induced rhinitis observed in placebo-vaccinated ferrets. The results favor the vaccine based on the A/California/1/66 isolate, which will be evaluated in a clinical study.     

Virulence and genetic compatibility of polymerase reassortant viruses derived from the pandemic (H1N1) 2009 influenza virus and circulating influenza A viruses

Gene mutations and reassortment are key mechanisms by which influenza A virus acquires virulence factors. To evaluate the role of the viral polymerase replication machinery in producing virulent pandemic (H1N1) 2009 influenza viruses, we generated various polymerase point mutants (PB2, 627K/701N; PB1, expression of PB1-F2 protein; and PA, 97I) and reassortant viruses with various sources of influenza viruses by reverse genetics. Although the point mutations produced no significant change in pathogenicity, reassortment between the pandemic A/California/04/09 (CA04, H1N1) and current human and animal influenza viruses produced variants possessing a broad spectrum of pathogenicity in the mouse model. Although most polymerase reassortants had attenuated pathogenicity (including those containing seasonal human H3N2 and high-pathogenicity H5N1 virus segments) compared to that of the parental CA04 (H1N1) virus, some recombinants had significantly enhanced virulence. Unexpectedly, one of the five highly virulent reassortants contained a A/Swine/Korea/JNS06/04(H3N2)-like PB2 gene with no known virulence factors; the other four had mammalian-passaged avian-like genes encoding PB2 featuring 627K, PA featuring 97I, or both. Overall, the reassorted polymerase complexes were only moderately compatible for virus rescue, probably because of disrupted molecular interactions involving viral or host proteins. Although we observed close cooperation between PB2 and PB1 from similar virus origins, we found that PA appears to be crucial in maintaining viral gene functions in the context of the CA04 (H1N1) virus. These observations provide helpful insights into the pathogenic potential of reassortant influenza viruses composed of the pandemic (H1N1) 2009 influenza virus and prevailing human or animal influenza viruses that could emerge in the future.     

Protection against divergent influenza H1N1 virus by a centralized influenza hemagglutinin

Influenza poses a persistent worldwide threat to the human population. As evidenced by the 2009 H1N1 pandemic, current vaccine technologies are unable to respond rapidly to this constantly diverging pathogen. We tested the utility of adenovirus (Ad) vaccines expressing centralized consensus influenza antigens. Ad vaccines were produced within 2 months and protected against influenza in mice within 3 days of vaccination. Ad vaccines were able to protect at doses as low as 10(7) virus particles/kg indicating that approximately 1,000 human doses could be rapidly generated from standard Ad preparations. To generate broadly cross-reactive immune responses, centralized consensus antigens were constructed against H1 influenza and against H1 through H5 influenza. Twenty full-length H1 HA sequences representing the main branches of the H1 HA phylogenetic tree were used to create a synthetic centralized gene, HA1-con. HA1-con minimizes the degree of sequence dissimilarity between the vaccine and existing circulating viruses. The centralized H1 gene, HA1-con, induced stronger immune responses and better protection against mismatched virus challenges as compared to two wildtype H1 genes. HA1-con protected against three genetically diverse lethal influenza challenges. When mice were challenged with 1934 influenza A/PR/8/34, HA1-con protected 100% of mice while vaccine generated from 2009 A/TX/05/09 only protected 40%. Vaccination with 1934 A/PR/8/34 and 2009 A/TX/05/09 protected 60% and 20% against 1947 influenza A/FM/1/47, respectively, whereas 80% of mice vaccinated with HA1-con were protected. Notably, 80% of mice challenged with 2009 swine flu isolate A/California/4/09 were protected by HA1-con vaccination. These data show that HA1-con in Ad has potential as a rapid and universal vaccine for H1N1 influenza viruses.     

Generation of Live Attenuated Novel Influenza Virus A/California/7/09 (H1N1) Vaccines with High Yield in Embryonated Chicken Eggs

Several live attenuated influenza virus A/California/7/09 (H1N1) (CA09) candidate vaccine variants that possess the hemagglutinin (HA) and neuraminidase (NA) gene segments from the CA09 virus and six internal protein gene segments from the cold-adapted influenza virus A/Ann Arbor/6/60 (H2N2) virus were generated by reverse genetics. The reassortant viruses replicated relatively poorly in embryonated chicken eggs. To improve virus growth in eggs, reassortants expressing the HA and NA of CA09 were passaged in MDCK cells and variants exhibiting large-plaque morphology were isolated. These variants replicated at levels approximately 10-fold higher than the rate of replication of the parental strains in embryonated chicken eggs. Sequence analysis indicated that single amino acid changes at positions 119, 153, 154, and 186 were responsible for the improved growth properties in MDCK cells and eggs. In addition, the introduction of a mutation at residue 155 that was previously shown to enhance the replication of a 1976 swine influenza virus also significantly improved the replication of the CA09 virus in eggs. Each variant was further evaluated for receptor binding preference, antigenicity, attenuation phenotype, and immunogenicity. Mutations at residues 153, 154, and 155 drastically reduced viral antigenicity, which made these mutants unsuitable as vaccine candidates. However, changes at residues 119 and 186 did not affect virus antigenicity or immunogenicity, justifying their inclusion in live attenuated vaccine candidates to protect against the currently circulating 2009 swine origin H1N1 viruses.Human infections with the swine origin influenza virus A (H1N1) were first detected in April 2009 and spread across the globe, resulting in WHO declaring a pandemic on 12 June 2009 for the first time in the past 41 years. More than 296,471 people have had confirmed infections with this novel H1N1 virus, and there have been at least 3,486 deaths as of September 18, 2009. In the last century, an influenza H1N1 virus caused the devastating 1918-1919 pandemic; this pandemic was characterized by a mild outbreak in the spring of 1918, followed by a lethal wave globally in the fall of that year which killed as many as 50 million people worldwide (20, 29). The 2009 H1N1 viruses circulating globally since April 2009 have not caused a significant rise in mortality related to influenza. Nucleotide sequence analysis suggested that E627 in PB2, a deletion of the PDZ ligand domain in NS1, and the lack of the PB1-F2 open reading frame in the 2009 H1N1 viruses may contribute to the relatively mild virulence (20, 26, 27). Recent animal studies have shown that the 2009 H1N1 influenza viruses did not replicate in tissues beyond the respiratory tract and did not cause significant mortality in the ferret model; however, the 2009 H1N1 viruses are capable of infecting deep in the lung tissues and caused more significant lesions in the lung tissues of animals, including nonhuman primates, than typical seasonal strains (13, 17, 19). Children and young adults are particularly susceptible to the 2009 H1N1 virus infection because they have no or low immunity to the novel 2009 H1N1 strains (11, 13). The widespread and rapid distribution of the 2009 H1N1 viruses in humans raises a concern about the evolution of more virulent strains during passage in the population. One fear is that mutant forms of the 2009 H1N1 viruses may exhibit significantly increased virulence (2, 19). Therefore, there is an urgent need to develop an effective vaccine to control the influenza pandemic caused by the swine origin H1N1 viruses.Live attenuated influenza vaccine (LAIV) has been licensed in the United States annually since 2003. The seasonal vaccine protects against influenza illness and elicits both systemic and mucosal immune responses, including serum hemagglutination inhibition (HAI) antibodies that react to antigenically drifted strains (3, 4). A critical attribute of an effective pandemic vaccine is its capability to elicit an immune response in immunonaive individuals; LAIV has been shown to offer protection following a single dose in young children. However, two doses of vaccines are recommended for children younger than 9 years of age who have never been immunized with influenza vaccines. In order to produce LAIV to protect against the newly emerged swine origin H1N1 influenza virus, we have produced several 6:2 reassortant candidate vaccine strains that express the hemagglutinin (HA) and neuraminidase (NA) gene segments from influenza virus A/California/4/09 (A/CA/4/09) (H1N1) or A/CA/7/09 (H1N1), as well as the six internal protein gene segments (PB1, PB2, PA, NP, M, and NS) from cold-adapted A/Ann Arbor/6/60 (H2N2) (AA60) virus, which is the master donor virus for all influenza virus A strains in trivalent seasonal LAIV. Initial evaluation of these candidate vaccine strains indicated that they did not replicate as efficiently as seasonal H1N1 influenza vaccine strains in embryonated chicken eggs. In this report, we describe directed modifications of the HA gene segment that improved vaccine yields in eggs, resulting in a number of vaccine candidates that are available for human use.     

用电穿孔方法研究 H5N1 禽流感病毒DNA 疫苗对动物的免疫保护作用

为了研究 H5N1 DNA 疫苗对小鼠和鸡的保护效率,用 H5N1 禽流感病毒 HA DNA 疫苗免疫 BALB/c 小鼠和 SPF 鸡 . 小鼠和鸡分别经电穿孔和肌肉注射免疫两次,间隔为 3 周 . 二次免疫后,用致死量的同源病毒进行攻毒实验 . 空白对照组在攻毒后全部死亡,而经电穿孔免疫的小鼠和鸡均获得了完全的保护,并能有效地抑制病毒在小鼠肺脏和鸡泄殖腔的繁殖 . 同时,电穿孔免疫的小鼠和鸡均产生了高水平的特异性抗体 . 经电穿孔免疫的小鼠攻毒后 CTL 反应明显加强 . 这些结果表明, HA DNA 疫苗能有效地保护小鼠和鸡对禽流感病毒的感染,同时也表明电穿孔免疫是 DNA 疫苗免疫的有效途径之一 .     

A Mouse Model for the Evaluation of Pathogenesis and Immunity to Influenza A (H5N1) Viruses Isolated from Humans

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

Impact of amino acid mutations in PB2, PB1-F2, and NS1 on the replication and pathogenicity of pandemic (H1N1) 2009 influenza viruses

Here, we assessed the effects of PB1-F2 and NS1 mutations known to increase the pathogenicity of influenza viruses on the replication and pathogenicity in mice of pandemic (H1N1) 2009 influenza viruses. We also characterized viruses possessing a PB1-F2 mutation that was recently identified in pandemic (H1N1) 2009 influenza virus isolates, with and without simultaneous mutations in PB2 and NS1. Our results suggest that some NS1 mutations and the newly identified PB1-F2 mutation have the potential to increase the replication and/or pathogenicity of pandemic (H1N1) 2009 influenza viruses.     

Growth determinants for H5N1 influenza vaccine seed viruses in MDCK cells

H5N1 influenza A viruses are exacting a growing human toll, with more than 240 fatal cases to date. In the event of an influenza pandemic caused by these viruses, embryonated chicken eggs, which are the approved substrate for human inactivated-vaccine production, will likely be in short supply because chickens will be killed by these viruses or culled to limit the worldwide spread of the infection. The Madin-Darby canine kidney (MDCK) cell line is a promising alternative candidate substrate because it supports efficient growth of influenza viruses compared to other cell lines. Here, we addressed the molecular determinants for growth of an H5N1 vaccine seed virus in MDCK cells, revealing the critical responsibility of the Tyr residue at position 360 of PB2, the considerable requirement for functional balance between hemagglutinin (HA) and neuraminidase (NA), and the partial responsibility of the Glu residue at position 55 of NS1. Based on these findings, we produced a PR8/H5N1 reassortant, optimized for this cell line, that derives all of its genes for its internal proteins from the PR8(UW) strain except for the NS gene, which derives from the PR8(Cambridge) strain; its N1 NA gene, which has a long stalk and derives from an early H5N1 strain; and its HA gene, which has an avirulent-type cleavage site sequence and is derived from a circulating H5N1 virus. Our findings demonstrate the importance and feasibility of a cell culture-based approach to producing seed viruses for inactivated H5N1 vaccines that grow robustly and in a timely, cost-efficient manner as an alternative to egg-based vaccine production.