Adaptation of high-growth influenza H5N1 vaccine virus in Vero cells: implications for pandemic preparedness

Current egg-based influenza vaccine production technology can't promptly meet the global demand during an influenza pandemic as shown in the 2009 H1N1 pandemic. Moreover, its manufacturing capacity would be vulnerable during pandemics caused by highly pathogenic avian influenza viruses. Therefore, vaccine production using mammalian cell technology is becoming attractive. Current influenza H5N1 vaccine strain (NIBRG-14), a reassortant virus between A/Vietnam/1194/2004 (H5N1) virus and egg-adapted high-growth A/PR/8/1934 virus, could grow efficiently in eggs and MDCK cells but not Vero cells which is the most popular cell line for manufacturing human vaccines. After serial passages and plaque purifications of the NIBRG-14 vaccine virus in Vero cells, one high-growth virus strain (Vero-15) was generated and can grow over 10(8) TCID(50)/ml. In conclusion, one high-growth H5N1 vaccine virus was generated in Vero cells, which can be used to manufacture influenza H5N1 vaccines and prepare reassortant vaccine viruses for other influenza A subtypes.     

Improved antigen yield in pandemic H1N1 (2009) candidate vaccine viruses with chimeric hemagglutinin molecules

The candidate pandemic H1N1 vaccine virus NIBRG-121 was derived by reverse genetics and comprises the hemagglutinin (HA) and neuraminidase (NA) genes from A/California/7/2009 (CAL) on an A/Puerto Rico/8/34 (PR8) backbone. NIBRG-121 was found to grow poorly in eggs, compared to seasonal H1N1 candidate vaccine viruses. Based on our previous study with H5N1 candidate vaccine viruses, we generated two new viruses with chimeric PR8/CAL HA genes. Here we show that these new viruses have considerably improved growth in eggs and are therefore better candidate vaccine viruses for use in production of pandemic H1N1 (2009) vaccine.     

ATF3 inhibits adipocyte differentiation of 3T3-L1 cells

The global spread of highly pathogenic avian influenza A H5N1 viruses raises concerns about more widespread infection in the human population. Pre-pandemic vaccine for H5N1 clade 1 influenza viruses has been produced from the A/Viet Nam/1194/2004 strain (VN1194), but recent prevalent avian H5N1 viruses have been categorized into the clade 2 strains, which are antigenically distinct from the pre-pandemic vaccine. To understand the antigenicity of H5N1 hemagglutinin (HA), we produced a neutralizing monoclonal antibody (mAb12-1G6) using the pre-pandemic vaccine. Analysis with chimeric and point mutant HAs revealed that mAb12-1G6 bound to the loop (amino acid positions 140-145) corresponding to an antigenic site A in the H3 HA. mAb12-1G6 failed to bind to the mutant VN1194 HA when only 3 residues were substituted with the corresponding residues of the clade 2.1.3.2 A/Indonesia/5/05 strain (amino acid substitutions at positions Q142L, K144S, and S145P), suggesting that these amino acids are critical for binding of mAb12-1G6. Escape mutants of VN1194 selected with mAb12-1G6 carried a S145P mutation. Interestingly, mAb12-1G6 cross-neutralized clade 1 and clade 2.2.1 but not clade 2.1.3.2 or clade 2.3.4 of the H5N1 virus. We discuss the cross-reactivity, based on the amino acid sequence of the epitope.     

Heterologous Prime-Boost Vaccination with MF59-Adjuvanted H5 Vaccines Promotes Antibody Affinity Maturation towards the Hemagglutinin HA1 Domain and Broad H5N1 Cross-Clade Neutralization

In an open label clinical study (2007), MF59-adjuvanted hemagglutinin (HA) vaccine from H5N1-A/Vietnam/1194/2004 (clade 1) was administered to subjects previously vaccinated (primed) with clade 0 H5N3 (A/duck/Singapore/97) vaccine at least 6 years earlier (in 1999 or 2001). The primed individuals responded rapidly and generated high neutralizing antibody titers against the H5N1-Vietnam strain within 7 days of a single booster vaccination. Furthermore, significant cross-neutralization titers were measured against H5N1 clade 0, 1, and 2 viruses. In the current study, the impact of MF59 adjuvant during heterologous priming on the quality of humoral polyclonal immune response in different vaccine arms were further evaluated using real time kinetics assay by surface plasmon resonance (SPR). Total anti-H5N1 HA1 polyclonal sera antibody binding from the heterologous prime-boost groups after a single MF59-H5N1 boost was significantly higher compared with sera from unprimed individuals that received two MF59-H5N1 vaccinations. The antigen-antibody complex dissociation rates (surrogate for antibody affinity) of the polyclonal sera against HA1 of H5N1-A/Vietnam/1194/2004 from the MF59-H5N3 primed groups were significantly higher compared to sera from unadjuvanted primed groups or unprimed individuals that received two MF59-H5N1 vaccines. Furthermore, strong inverse correlations were observed between the antibody dissociation off-rates of the immune sera against HA1 (but not HA2) and the virus neutralization titers against H5 vaccine strains and heterologous H5N1 strains. These findings supports the use of oil-in-water-adjuvanted pandemic influenza vaccines to elicit long term memory B cells with high affinity BCR capable of responding to potential variant pandemic viruses likely to emerge and adapt to human transmissions.     

Influenza H5 hemagglutinin DNA primes the antibody response elicited by the live attenuated influenza A/Vietnam/1203/2004 vaccine in ferrets

Priming immunization plays a key role in protecting individuals or populations to influenza viruses that are novel to humans. To identify the most promising vaccine priming strategy, we have evaluated different prime-boost regimens using inactivated, DNA and live attenuated vaccines in ferrets. Live attenuated influenza A/Vietnam/1203/2004 (H5N1) candidate vaccine (LAIV, VN04 ca) primed ferrets efficiently while inactivated H5N1 vaccine could not prime the immune response in seronegative ferrets unless an adjuvant was used. However, the H5 HA DNA vaccine alone was as successful as an adjuvanted inactivated VN04 vaccine in priming the immune response to VN04 ca virus. The serum antibody titers of ferrets primed with H5 HA DNA followed by intranasal vaccination of VN04 ca virus were comparable to that induced by two doses of VN04 ca virus. Both LAIV-LAIV and DNA-LAIV vaccine regimens could induce antibody responses that cross-neutralized antigenically distinct H5N1 virus isolates including A/HongKong/213/2003 (HK03) and prevented nasal infection of HK03 vaccine virus. Thus, H5 HA DNA vaccination may offer an alternative option for pandemic preparedness.     

Emulsified Nanoparticles Containing Inactivated Influenza Virus and CpG Oligodeoxynucleotides Critically Influences the Host Immune Responses in Mice

Background

Antigen sparing and cross-protective immunity are regarded as crucial in pandemic influenza vaccine development. Both targets can be achieved by adjuvantation strategy to elicit a robust and broadened immune response. We assessed the immunogenicity of an inactivated H5N1 whole-virion vaccine (A/Vietnam/1194/2004 NIBRG-14, clade 1) formulated with emulsified nanoparticles and investigated whether it can induce cross-clade protecting immunity.

Methodology/Principal Findings

After formulation with PELC, a proprietary water-in-oil-in-water nanoemulsion comprising of bioresorbable polymer/Span®85/squalene, inactivated virus was intramuscularly administered to mice in either one-dose or two-dose schedule. We found that the antigen-specific serum antibody responses elicited after two doses of non-adjuvanted vaccine were lower than those observed after a single dose of adjuvanted vaccine, PELC and the conventional alum adjuvant as well. Moreover, 5 µg HA of PELC-formulated inactivated virus were capable of inducing higher antibodies than those obtained from alum-adjuvanted vaccine. In single-dose study, we found that encapsulating inactivated virus into emulsified PELC nanoparticles could induce better antibody responses than those formulated with PELC-adsorbed vaccine. However, the potency was rather reduced when the inactivated virus and CpG (an immunostimulatory oligodeoxynucleotide containing unmethylated cytosine-guanosine motifs) were co-encapsulated within the emulsion. Finally, the mice who received PELC/CpG(adsorption)-vaccine could easily and quickly reach 100% of seroprotection against a homologous virus strain and effective cross-protection against a heterologous virus strain (A/Whooper swan/Mongolia/244/2005, clade 2.2).

Conclusions/Significance

Encapsulating inactivated H5N1 influenza virus and CpG into emulsified nanoparticles critically influences the humoral responses against pandemic influenza. These results demonstrated that the use of PELC could be as antigen-sparing in preparation for a potential shortage of prophylactic vaccines against local infectious diseases, in particular pandemic influenza. Moreover, the cross-clade neutralizing antibody responses data verify the potential of such adjuvanted H5N1 candidate vaccine as an effective tool in pre-pandemic preparedness.     

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.     

Vesicular stomatitis virus-based H5N1 avian influenza vaccines induce potent cross-clade neutralizing antibodies in rhesus macaques

We analyzed the ability of a vaccine vector based on vesicular stomatitis virus (VSV) to induce a neutralizing antibody (NAb) response to avian influenza viruses (AIVs) in rhesus macaques. Animals vaccinated with vectors expressing either strain A/Hong Kong/156/1997 or strain A/Vietnam/1203/2004 H5 hemagglutinin (HA) were able to generate robust NAb responses. The ability of the vectors to induce NAbs against homologous and heterologous AIVs after a single dose was dependent upon the HA antigen incorporated into the VSV vaccine. The vectors expressing strain A/Vietnam/1203/2004 H5 HA were superior to those expressing strain A/Hong Kong/156/1997 HA at inducing cross-clade NAbs.     

Evaluation of the efficacy of a pre-pandemic H5N1 vaccine (MG1109) in mouse and ferret models

The threat of a highly pathogenic avian influenza (HPAI) H5N1 virus causing the next pandemic remains a major concern. In this study, we evaluated the immunogenicity and efficacy of an inactivated whole-virus H5N1 pre-pandemic vaccine (MG1109) formulated by Green Cross Co., Ltd containing the hemagglutinin (HA) and neuraminidase (NA) genes of the clade 1 A/Vietnam/1194/04 virus in the backbone of A/Puerto Rico/8/34 (RgVietNam/04xPR8/34). Administration of the MG1109 vaccine (2-doses) in mice and ferrets elicited high HI and SN titers in a dose-dependent manner against the homologous (RgVietNam/04xPR8/34) and various heterologous H5N1 strains, (RgKor/W149/06xPR8/34, RgCambodia/04xPR8/34, RgGuangxi/05xPR8/34), including a heterosubtypic H5N2 (A/Aquatic bird/orea/W81/05) virus. However, efficient cross-reactivity was not observed against heterosubtypic H9N2 (A/Ck/Korea/H0802/08) and H1N1 (PR/8/34) viruses. Mice immunized with 1.9 μg HA/dose of MG1109 were completely protected from lethal challenge with heterologous wild-type HPAI H5N1 A/EM/Korea/W149/06 (clade 2.2) and mouse-adapted H5N2 viruses. Furthermore, ferrets administered at least 3.8 μg HA/dose efficiently suppressed virus growth in the upper respiratory tract and lungs. Vaccinated mice and ferrets also demonstrated attenuation of clinical disease signs and limited virus spread to other organs. Thus, this vaccine provided immunogenic responses in mouse and ferret models even against challenge with heterologous HPAI H5N1 and H5N2 viruses. Since the specific strain of HPAI H5N1 virus that would potentially cause the next outbreak is unknown, pre-pandemic vaccine preparation that could provide cross-protection against various H5 strains could be a useful approach in the selection of promising candidate vaccines in the future.     

Bacterial HA1 vaccine against pandemic H5N1 influenza virus: evidence of oligomerization, hemagglutination, and cross-protective immunity in ferrets

The impending influenza virus pandemic requires global vaccination to prevent large-scale mortality and morbidity, but traditional influenza virus vaccine production is too slow for rapid responses. We have developed bacterial systems for expression and purification of properly folded functional hemagglutinin as a rapid response to emerging pandemic strains. A recombinant H5N1 (A/Vietnam/1203/2004) hemagglutinin globular domain (HA1) was produced in Escherichia coli under controlled redox refolding conditions. Importantly, the properly folded HA1(1-320), i.e., HA1 lacking amino acids 321 to 330, contained ≥75% functional oligomers without addition of foreign oligomerization sequence. Site-directed mutagenesis mapped the oligomerization signal to the HA1 N-terminal Ile-Cys-Ile residues at positions 3 to 5. The purified HA1 oligomers (but not monomers) bound fetuin and agglutinated red blood cells. Upon immunization of rabbits, the oligomeric HA1(1-320) elicited potent neutralizing antibodies against homologous and heterologous H5N1 viruses more rapidly than HA1(28-320) containing only monomers. Ferrets vaccinated with oligomeric HA1 (but not monomeric HA1 with the N terminus deleted) at 15 and 3 μg/dose were fully protected from lethality and weight loss after challenge with homologous H5N1 (A/Vietnam/1203/2004, clade 1) virus, as well as heterologous clade 2.2 H5N1 (A/WooperSwan/Mongolia/244/2005) virus. Protection was associated with a significant reduction in viral loads in the nasal washes of homologous and heterologous virus challenged ferrets. This is the first study that describes the presence of an N-terminal oligomerization sequence in the globular domain of influenza virus hemagglutinin. Our findings suggest that functional oligomeric rHA1-based vaccines can be produced efficiently in bacterial systems and can be easily upscaled in response to a pandemic influenza virus threat.     

Oral Priming with Replicating Adenovirus Serotype 4 Followed by Subunit H5N1 Vaccine Boost Promotes Antibody Affinity Maturation and Expands H5N1 Cross-Clade Neutralization

A Phase I trial conducted in 2009–2010 demonstrated that oral vaccination with a replication competent Ad4-H5 (A/Vietnam) vector with dosages ranging from 10⁷-10¹¹ viral particles was well tolerated. HA-specific T-cell responses were efficiently induced, but very limited hemagglutination-inhibiting (HI) humoral responses were measured. However, a single boost of Ad4-H5-Vtn vaccinated individuals with a unadjuvanted licensed H5N1 (A/Vietnam) subunit vaccine resulted in superior HI titers compared with unprimed subjects. In the current study, the impact of Ad4-H5 priming on the quality of the polyclonal humoral immune response was evaluated using a real-time kinetics assay by surface plasmon resonance (SPR). Total binding of serum polyclonal antibodies from the Ad4-H5-Vtn primed groups against both homologous H5N1-A/Vietnam/1194/2004 (clade 1) and heterologous A/Indonesia-5/2005 (clade 2.1) HA1 head domain was significantly higher compared with sera from individuals that received subunit H5N1 vaccination alone. SPR measurements also demonstrated that the antigen-antibody complex dissociation rates (a surrogate for antibody affinity) of serum antibodies against the HA1 of H5N1-A/Vietnam were significantly higher in the Ad4-H5 primed groups compared with those from the unprimed group. Furthermore, strong correlations were observed between the antibody affinities for HA1 (but not HA2) and the virus neutralization titers against the homologous strain and a panel of heterologous clade 2 H5N1 strains. These findings support the concept of oral prime-boost vaccine approaches against pandemic influenza to elicit long-term memory B cells with high affinity capable of rapid response to variant pandemic viruses likely to emerge and adapt to human transmissions.     

Detection and Characterization of Clade 1 Reassortant H5N1 Viruses Isolated from Human Cases in Vietnam during 2013

Highly pathogenic avian influenza (HPAI) H5N1 is endemic in Vietnamese poultry and has caused sporadic human infection in Vietnam since 2003. Human infections with HPAI H5N1 are of concern due to a high mortality rate and the potential for the emergence of pandemic viruses with sustained human-to-human transmission. Viruses isolated from humans in southern Vietnam have been classified as clade 1 with a single genome constellation (VN3) since their earliest detection in 2003. This is consistent with detection of this clade/genotype in poultry viruses endemic to the Mekong River Delta and surrounding regions. Comparison of H5N1 viruses detected in humans from southern Vietnamese provinces during 2012 and 2013 revealed the emergence of a 2013 reassortant virus with clade 1.1.2 hemagglutinin (HA) and neuraminidase (NA) surface protein genes but internal genes derived from clade 2.3.2.1a viruses (A/Hubei/1/2010-like; VN12). Closer analysis revealed mutations in multiple genes of this novel genotype (referred to as VN49) previously associated with increased virulence in animal models and other markers of adaptation to mammalian hosts. Despite the changes identified between the 2012 and 2013 genotypes analyzed, their virulence in a ferret model was similar. Antigenically, the 2013 viruses were less cross-reactive with ferret antiserum produced to the clade 1 progenitor virus, A/Vietnam/1203/2004, but reacted with antiserum produced against a new clade 1.1.2 WHO candidate vaccine virus (A/Cambodia/W0526301/2012) with comparable hemagglutination inhibition titers as the homologous antigen. Together, these results indicate changes to both surface and internal protein genes of H5N1 viruses circulating in southern Vietnam compared to 2012 and earlier viruses.     

Comparative clinical trial of vaccines against avian influenza

Scientic-production association "Microgen" has finished 1st phase of clinical trials of candidate vaccines against avian influenza in order to assess their reactogenicity, safety, and immunogenicity. Two vaccines constructed from NIBRG-14 vaccine strain [A/Vietnam/1 194/2004 (H5N1)], obtained from World Health Organization, were studied: "OrniFlu" (inactivated subunit influenza vaccine adsorbed on aluminium hydroxide) and inactivated polymer-subunit influenza vaccine with polyoxydonium (IPSIV). Clinical trial of the vaccines with different quantity of antigen (15, 30, and 45 mcg of H5N1 virus hemagglutinin) was carried out in Influenza Research Institute (St. Petersburg) and in Mechnikov Research Institute of Vaccines and Sera (Moscow). Analysis of results allowed to conclude that both vaccines were safe, well tolerated and characterized by low reactogenicity. Two-doses vaccination schedule was needed to meet required seroconversion and seroprotection rates (> or =1:40 in > or =70% of vaccinated volunteers). "Orni-Flu" vaccine containing 15 mcg of hemagglutinin and optimal quantity of aluminium hydroxide (0.5 mg) in one dose as well as IPSIV containing 45 mcg of hemagglutinin and 0.75 mg of polyoxydonium in one dose were most immunogenic after 2 doses - seroprotection rates in microneutralization assay were 72.2% and 77.0% respectively. Marked influence of aluminium hydroxide content on immunogenicity of the "OrniFlu" vaccine was confirmed in the study. Optimal quantity of adjuvant was 0.5 mg per dose. According to basic concept of vaccine development, preference is given to vaccine that under minimal quantity of antigen induces sufficient specific immune response and is safe in volunteers. "OrniFlu" vaccine containing 15 mcg of H5N1 virus hemagglutinin and optimal quantity of aluminium hydroxide (0.5 mg) corresponded to these requirements that allowed researchers to recommend it for clinical trials of 2nd phase.     

枸杞多糖对H5N1流感全病毒灭活疫苗的体液免疫增强作用

探讨枸杞多糖(Lycium barbarum polysaccharide,LBP)作为佐剂对H5亚型流感病毒全病毒灭活疫苗的体液免疫增强效果。将流感病毒A/Vietnam/1194/2004(H5N1)灭活疫苗与不同剂量的枸杞多糖混合后以腹腔注射的方式共同免疫小鼠,免疫后三周收集血清用于特异性抗体检测。实验中设立氢氧化铝佐剂组作对照共同评价LBP作为佐剂的免疫增强效果。结果显示,小鼠血清中针对H5灭活疫苗的特异性抗体水平在一定范围内随着LBP剂量的增加而提高。LBP在800μg剂量时血清特异性抗体水平较无佐剂组显著增强,并与氢氧化铝佐剂组大致相当。因而,LBP有可能成为一种有效的流感灭活疫苗免疫佐剂。     

A Replicating Modified Vaccinia Tiantan Strain Expressing an Avian-Derived Influenza H5N1 Hemagglutinin Induce Broadly Neutralizing Antibodies and Cross-Clade Protective Immunity in Mice

To combat the possibility of a zoonotic H5N1 pandemic in a timely fashion, it is necessary to develop a vaccine that would confer protection against homologous and heterologous human H5N1 influenza viruses. Using a replicating modified vaccinia virus Tian Tan strain (MVTT) as a vaccine vector, we constructed MVTT_HA-QH and MVTT_HA-AH, which expresses the H5 gene of a goose-derived Qinghai strain A/Bar-headed Goose/Qinghai/1/2005 or human-derived Anhui Strain A/Anhui/1/2005. The immunogenicity profiles of both vaccine candidates were evaluated. Vaccination with MVTT_HA-QH induced a significant level of neutralizing antibodies (Nabs) against a homologous strain and a wide range of H5N1 pseudoviruses (clades 1, 2.1, 2.2, 2.3.2, and 2.3.4). Neutralization tests (NT) and Haemagglutination inhibition (HI) antibodies inhibit the live autologous virus as well as a homologous A/Xingjiang/1/2006 and a heterologous A/Vietnam/1194/2004, representing two human isolates from clade 2.2 and clade 1, respectively. Importantly, mice vaccinated with intranasal MVTT_HA-QH were completely protected from challenge with lethal dosages of A/Bar-headed Goose/Qinghai/1/2005 and the A/Viet Nam/1194/2004, respectively, but not control mice that received a mock MVTT_S vaccine. However, MVTT_HA-AH induced much lower levels of NT against its autologous strain. Our results suggest that it is feasible to use the H5 gene from A/Bar-headed Goose/Qinghai/1/2005 to construct an effective vaccine, when using MVTT as a vector, to prevent infections against homologous and genetically divergent human H5N1 influenza viruses.     

长沙A(H1N1)亚型季节性流感暴发疫情的实验室诊断及其HA基因特性分析

对2009 年长沙麓山国际学校流感暴发疫情进行实验室诊断, 并探索新分离的A(H1N1)亚型流感病毒血凝素(HA)的基因特性。对流感暴发疫情的25 份鼻/咽拭子标本进行RT-PCR检测和流感病毒分离, 然后利用CEQ?8000 Genetic Analysis System对病毒分离株(A/Yuelu/314/2009)进行测序, 测序结果提交至GenBank(登录号: FJ912843)并用ClustalX和Mega4.1软件进行序列分析。结果显示, 分离出A(H1N1)亚型流感毒株18株, 检出21份A(H1N1)亚型流感病毒核酸阳性; A/Yuelu/314/2009(H1N1) HA基因序列与2008~2009 年疫苗株(A/Brisbane/59/2007)比较显示: 核苷酸和氨基酸同源性均为99%, 有6个位点的氨基酸发生了变异(V148A、S158N、G202A、I203D、A206T、W435R), 其中一个S158N氨基酸变异位于B抗原表位, HA基因序列上共有潜在糖基化位点9 个(27、28、40、71、151、176、303、497、536), 与A/Brisbane/59/2007相同且氨基酸序列保守。本实验诊断出此次流感暴发疫情的病原体为A(H1N1)型季节性流感病毒, 研究还发现A/Yuelu/314/2009(H1N1)长沙分离株与A/Brisbane/59/2007 疫苗株基因序列比较显示并未形成一个新的变种, 推测是由于分离株与疫苗株之间基因特性的改变和人群对A(H1N1)亚型流感病毒免疫力降低导致了此次长沙麓山国际学校A(H1N1)亚型流感的暴发。     

Intranasal immunization with H5N1 vaccine plus Poly I:Poly C12U, a Toll-like receptor agonist, protects mice against homologous and heterologous virus challenge

The avian H5N1 influenza virus has the potential to cause a new pandemic. Since it is difficult to predict which strain of influenza will cause a pandemic, it is advantageous to produce vaccines that confer cross-protective immunity. Mucosal vaccine administration was reported to induce cross-protective immunity by inducing secretion of IgA at the mucosal surface. Adjuvants can also enhance the development of fully protective mucosal immunity. Here we show that a new mucosal adjuvant, poly I:poly C12U (Ampligen), a Toll-like receptor 3 agonist proven to be safe in a Phase III human trial, is an effective adjuvant for H5N1 influenza vaccination. Intranasal administration of a candidate influenza vaccine with Ampligen resulted in secretion of IgA, and protected mice that were subsequently challenged with homologous A/Vietnam/1194/2004 and heterologous A/HK/483/97 and A/Indonesia/6/2005 virus.     

Generation and Characterization of Live Attenuated Influenza A(H7N9) Candidate Vaccine Virus Based on Russian Donor of Attenuation

Background

Avian influenza A (H7N9) virus has emerged recently and continues to cause severe disease with a high mortality rate in humans prompting the development of candidate vaccine viruses. Live attenuated influenza vaccines (LAIV) are 6:2 reassortant viruses containing the HA and NA gene segments from wild type influenza viruses to induce protective immune responses and the six internal genes from Master Donor Viruses (MDV) to provide temperature sensitive, cold-adapted and attenuated phenotypes.

Methodology/Principal Findings

LAIV candidate A/Anhui/1/2013(H7N9)-CDC-LV7A (abbreviated as CDC-LV7A), based on the Russian MDV, A/Leningrad/134/17/57 (H2N2), was generated by classical reassortment in eggs and retained MDV temperature-sensitive and cold-adapted phenotypes. CDC-LV7A had two amino acid substitutions N123D and N149D (H7 numbering) in HA and one substitution T10I in NA. To evaluate the role of these mutations on the replication capacity of the reassortants in eggs, the recombinant viruses A(H7N9)RG-LV1 and A(H7N9)RG-LV2 were generated by reverse genetics. These changes did not alter virus antigenicity as ferret antiserum to CDC-LV7A vaccine candidate inhibited hemagglutination by homologous A(H7N9) virus efficiently. Safety studies in ferrets confirmed that CDC-LV7A was attenuated compared to wild-type A/Anhui/1/2013. In addition, the genetic stability of this vaccine candidate was examined in eggs and ferrets by monitoring sequence changes acquired during virus replication in the two host models. No changes in the viral genome were detected after five passages in eggs. However, after ten passages additional mutations were detected in the HA gene. The vaccine candidate was shown to be stable in the ferret model; post-vaccination sequence data analysis showed no changes in viruses collected in nasal washes present at day 5 or day 7.

Conclusions/Significance

Our data indicate that the A/Anhui/1/2013(H7N9)-CDC-LV7A reassortant virus is a safe and genetically stable candidate vaccine virus that is now available for distribution by WHO to vaccine manufacturers.     

一种含不同流感病毒血凝素抗原表位的核酸疫苗

流感病毒表面抗原血凝素( hemagglutinin,HA)是流感核酸疫苗重要的靶抗原,针对HA的保护性中和抗体主要由HA上的五个抗原表位诱导产生.在本文中,我们构建了一种以新甲型H1N1流感病毒HA1为骨架的含2个A/PR/8( H1N1)流感病毒HA抗原表位和3个新甲型H1N1流感病毒HA抗原表位的核酸疫苗,并在B...     

A Novel Bivalent Vaccine Based on a PB2-Knockout Influenza Virus Protects Mice from Pandemic H1N1 and Highly Pathogenic H5N1 Virus Challenges

Vaccination is an effective means to protect against influenza virus. Although inactivated and live-attenuated vaccines are currently available, each vaccine has disadvantages (e.g., immunogenicity and safety issues). To overcome these problems, we previously developed a replication-incompetent PB2-knockout (PB2-KO) influenza virus that replicates only in PB2 protein-expressing cells. Here, we generated two PB2-KO viruses whose PB2-coding regions were replaced with the HA genes of either A/California/04/2009 (H1N1pdm09) or A/Vietnam/1203/2004 (H5N1). The resultant viruses comparably, or in some cases more efficiently, induced virus-specific antibodies in the serum, nasal wash, and bronchoalveolar lavage fluid of mice relative to a conventional formalin-inactivated vaccine. Furthermore, mice immunized with these PB2-KO viruses were protected from lethal challenges with not only the backbone virus strain but also strains from which their foreign HAs originated, indicating that PB2-KO viruses with antigenically different HAs could serve as bivalent influenza vaccines.