A call to cellular & humoral arms: enlisting cognate T cell help to develop broad-spectrum vaccines against influenza A

Influenza A is an important cause of morbidity and mortality worldwide. In the United States alone influenza kills 30,000 to 50,000 people in a non-epidemic year and significantly more in an acute epidemic.(1) An emerging pandemic influenza virus, such as H5N1, could have a devastating economic and social impact. The Surgeon General estimates that at least 43 million Americans, especially those younger than 1 and older than 60, are at risk of death from influenza. Antigenically distinct influenza virus strains emerge regularly, mandating changes in influenza vaccine antigenic composition. Consequently, the immunity engendered by the conventional influenza vaccines is relevant only for a short time. However, by incorporating conserved influenza T cell epitopes, it may be possible to develop more immunogenic, broader-spectrum vaccines that may be efficacious over a longer period. This review summarizes the critical components of effective influenza vaccines, a rational vaccine design approach, and the pertinent influenza immunology.     

Towards a universal influenza vaccine: volunteer virus challenge studies in quarantine to speed the development and subsequent licensing

There are now more than 5 experimental vaccine formulations which induce T and B cell immunity towards the internally situated virus proteins matrix (M1 and M2e) and nucleoprotein (NP), and towards stem and stalk regions of the HA which have a shared antigenic structure amongst many of the 17 influenza A virus sub types. Such ‘universal vaccines’ could be used, at least in theory, as a prophylactic stockpile vaccine for newly emerged epidemic and novel pandemic influenza A viruses or as a supplement to conventional HA/NA vaccines. My own laboratory has approached the problem from the clinical viewpoint by identifying CD4⁺ cells which are present in influenza infected volunteers who resist influenza infection. We have established precisely which peptides in M and NP proteins react with these immune CD4 cells. These experimental vaccines induce immunity in animal models but with a single exception no data have been published on protection against influenza virus infection in humans. The efficacy of the latter vaccine is based on vaccinia virus (MVA) as a carrier and was analyzed in a quarantine unit. Given the absence of induced HI antibody in the new universal vaccines a possible licensing strategy is a virus challenge model in quarantine whereby healthy volunteers can be immunized with the new vaccine and thereafter deliberately infected and clinical signs recorded alongside quantities of virus excreted and compared with unvaccinated controls.     

Vaxfectin-formulated influenza DNA vaccines encoding NP and M2 viral proteins protect mice against lethal viral challenge

Next generation influenza vaccines containing conserved antigens may enhance immunity against seasonal or pandemic influenza virus strains. Using a plasmid DNA (pDNA)-based vaccine approach, we systematically tested combinations of NP, M1, and M2 antigens derived from consensus sequences for protection against lethal influenza challenge and compared formulations for adjuvanting low pDNA vaccine doses. The highest level of protection at the lowest pDNA doses was provided by Vaxfectin-formulated NP + M2. Vaxfectin adjuvanticity was confirmed with a low dose of HA pDNA. These promising proof-of-concept data support the clinical development of Vaxfectin-formulated pDNA encoding NP + M2 consensus proteins.     

基于流感病毒保守抗原的广谱流感疫苗研究进展

流感是由流感病毒感染引起的严重急性呼吸道传染病,接种流感疫苗是预防流感最经济、安全且有效的措施。目前广泛应用的传统流感疫苗的保护效果受到疫苗株与流行株表面抗原匹配程度的明显影响,难以有效应对因流感病毒发生抗原漂移或抗原转换而产生的无法预料的流行或大流行。因而开发能够诱导广谱和持久免疫的广谱流感疫苗是新型流感疫苗研发的重要方向。流感病毒基质、核蛋白和血凝素茎部结构域作为流感病毒的保守抗原,是当前广谱流感疫苗的主要靶抗原。此文就近年来基于流感病毒保守抗原的广谱流感疫苗的抗原选择、免疫保护制剂、临床研究进展做一综述。     

Liposomal adjuvants for human vaccines

ABSTRACT

Introduction: Liposomes are well-known as drug carriers, and are now critical components of two of six types of adjuvants present in licensed vaccines. The liposomal vaccine adjuvant field has long been dynamic and innovative, and research in this area is further examined as new commercial products appear in parallel with new vaccines. In an arena where successful products exist the potential for new types of vaccines with liposomal adjuvants, and alternative liposomal adjuvants that could emerge for new types of vaccines, are discussed.

Areas covered: Major areas include: virosomes, constructed from phospholipids and proteins from influenza virus particles; liposomes containing natural and synthetic neutral or anionic phospholipids, cholesterol, natural or synthetic monophosphoryl lipid A, and QS21 saponin; non-phospholipid cationic liposomes; and combinations and mixtures of liposomes and immunostimulating ingredients as adjuvants for experimental vaccines.

Expert opinion: Liposomes containing monophosphoryl lipid A and QS21 have considerable momentum that will result soon in emergence of prophylactic vaccines to malaria and shingles, and possible novel cancer vaccines. The licensed virosome vaccines to influenza and hepatitis A will be replaced with virosome vaccines to other infectious diseases. Alternative liposomal formulations are likely to emerge for difficult diseases such as tuberculosis or HIV-1 infection.     

Inulin sugar glasses preserve the structural integrity and biological activity of influenza virosomes during freeze-drying and storage.

Influenza virosomes are reconstituted influenza virus envelopes that may be used as vaccines or as carrier systems for cellular delivery of therapeutic molecules. Here we present a procedure to generate influenza virosomes as a stable dry-powder formulation by freeze-drying (lyophilization) using an amorphous inulin matrix as a stabilizer. In the presence of inulin the structural integrity and fusogenic activity of virosomes were fully preserved during freeze-drying. For example, the immunological properties of the virosomes, i.e. the HA potency in vitro and the immunogenic potential in vivo, were maintained during lyophilization in the presence of inulin. In addition, compared to virosomes dispersed in buffer, inulin-formulated virosomes showed substantially prolonged preservation of the HA potency upon storage. Also the capacity of virosomes to mediate cellular delivery of macromolecules was maintained during lyophilization in the presence of inulin and upon subsequent storage. Specifically, when dispersed in buffer, virosomes with encapsulated plasmid DNA lost their transfection activity completely within 6 weeks, whereas their transfection activity was fully preserved for at least 12 weeks after incorporation in an inulin matrix. Thus, in the presence of inulin as a stabilizing agent, the shelf-life of influenza virosomes with and without encapsulated macromolecules was considerably prolonged. Formulation of influenza virosomes as a dry-powder is advantageous for storage and transport and offers the possibility to develop needle-free dosage forms, e.g. for oral, nasal, pulmonal, or dermal delivery.     

The quest for a nanoparticle-based vaccine inducing broad protection to influenza viruses

Influenza virus infections are a significant public threat and the best approach to prevent them is through vaccination. Because of the perpetual changes of circulating influenza strains, the efficacy of influenza vaccines rarely exceeds 50%. To improve the protection efficacy, we have designed a novel vaccine formulation that shows a broad range of protection. The formulation is made of the matrix protein 2 (M2e) and the nucleoprotein (NP) antigens. The multimerization of NP into nanoparticles improved significantly the immune response to NP. The combination of the NP nanoparticles with the PapMV-M2e nanoparticles enhances significantly the immune response directed to NP revealing the adjuvant property of the PapMV platform. The vaccine formulation combining these two types of nanoparticles protects mice from infectious challenges by two different influenza strains (H1N1 and H3N2) and is a promising influenza A vaccine capable to elicit a broad protection.     

Immunization by influenza virus-like particles protects aged mice against lethal influenza virus challenge

Influenza virus-like particles (VLPs) were produced in Sf9 insect cells by co-expressing the matrix protein M1 and the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) using the recombinant baculovirus expression system. The VLPs were morphologically similar to influenza virions. Both HA and NA proteins were incorporated into VLPs and these proteins retained their functional activities. Further, influenza VLPs but not inactivated influenza viruses (IIV) stimulated secretion of inflammatory cytokines from mouse bone marrow-derived dendritic cells (BMDC). Immunogenicity of influenza VLPs and their protective efficacies against lethal influenza virus challenge were evaluated in young and aged mice. Immunization with influenza VLPs induced strong antibody responses against HA that inhibited hemagglutination by influenza virus, similar to IIV vaccines. Compared to young mice, antibody responses in aged mice immunized with a low dose of either influenza VLPs or IIV vaccines exhibited markedly reduced avidity for HA. However, immunization of aged mice with a high dose of influenza VLPs induced antibody responses with high avidity similar to those in young mice. Furthermore, all vaccinated animals survived a lethal challenge by a mouse-adapted influenza virus (A/PR/8/34), indicating that influenza VLPs are highly efficacious for protection against influenza virus infection in both young and aged mice.     

流感病毒基质蛋白2胞外域作为广谱流感疫苗靶点研究现状

流感是由流感病毒引起的呼吸系统疾病,接种流感疫苗是预防流感的重要手段。疫苗的主要抗原是流感病毒表面蛋白血凝素和神经氨酸酶,但这两者都存在抗原漂移的现象,给疫苗设计带来巨大困难。广谱流感疫苗的候选抗原之一是高度保守的流感病毒基质蛋白2胞外域（matrix protein 2 ectodomain,M2e）。M2e在天然感染的情况下免疫原性较弱,但是可通过相关的佐剂、包裹材料和蛋白疫苗提高免疫原性。此文介绍了加强M2e免疫效果方式的研究以及M2e疫苗的临床研究进展。     

Modulation of immune response induced by co-administration of DNA vaccine encoding HBV surface antigen and HCV envelope antigen in BALB/c mice

Plasmid DNA vaccines encoding the hepatitis B virus (HBV) surface and hepatitis C virus (HCV) envelope antigens, respectively, were constructed, and attempt were made to find the possibility of a divalent vaccine against HBV and HCV. The expression of each plasmid in Cos-1 cells was confirmed using immunocytochemistry. To measure the induced immune response by these plasmids in vivo, female BALB/c mice were immunized intramuscularly with 100 microg of either both or just one of the plasmids. Anti-HBV and HCV-specific antibodies and related cytokines were evaluated to investigate the generation of both humoral and cellular immune responses. As a result, specific anti-HBV and anti-HCV serum antibodies from mice immunized with these plasmids were observed using immunoblot. The levels of IL-2 and RANTES showing a Th1 immune response were significantly increased, but there was no change in the level of IL-4 (Th2 immune response) in any of the immunized groups. Compared with each plasmid DNA vaccine, the combined vaccine elicited similar immune responses in both humoral and cell-mediated immunities. These results suggest that the combined DNA vaccine can induce not only comparable immunity experimentally without antigenic interference, but also humoral and Th1 dominant cellular immune responses. Therefore, they could serve as candidates for a simultaneous bivalent vaccine against HBV and HCV infections.     

Human Genetics and Responses to Influenza Vaccination

Influenza A and B viruses are negative-strand RNA viruses that cause regular outbreaks of respiratory disease and substantially impact on morbidity and mortality. Our primary defense against the influenza virus infection is provided by neutralizing antibodies that inhibit the function of the virus surface coat proteins hemagglutinin and neuraminidase. Production of these antibodies by B lymphocytes requires help from CD4+ T cells. The most commonly used vaccines against the influenza virus comprise purified preparations of hemagglutinin and neuraminidase, and are designed to induce a protective neutralizing antibody response. Because of regular antigenic change in these proteins (drift and shift mutation), the vaccines have to be administered on an annual basis. Current defense strategies center on prophylactic vaccination of those individuals who are considered to be most at risk from the serious complications of infection (principally individuals aged >65 years and those with chronic respiratory, cardiac, or metabolic disease).The clinical effectiveness of influenza virus vaccination is dependent on several vaccine-related factors, including the quantity of hemagglutinin within the vaccine, the number of doses administered, and the route of immunization. In addition, the immunocompetence of the recipient, their previous exposure to influenza virus and influenza virus vaccines, and the closeness of the match between the vaccine and circulating influenza virus strains, all influence the serologic response to vaccination.However, even when these vaccines are administered to young fit adults a proportion of individuals do not mount a significant serologic response to the vaccine. It is not clear whether these nonresponding individuals are genetically pre-programmed to be nonresponders or whether failure to respond to the vaccine is a random event. There is good evidence that nonresponsiveness to hepatitis B vaccine, another purified protein vaccine, is at least partially modulated by an individual's human leucocyte antigen (HLA) alleles. Because CD4+ T cells, which control the neutralizing antibody response to influenza virus, recognize antigens in association with HLA class II molecules, we recently conducted a small study to investigate whether there was any association between HLA class II molecules and nonresponsiveness to influenza virus vaccination. This work revealed that the HLA-DRB1*0701 allele was over represented among persons who fail to mount a neutralizing antibody response. This preliminary finding is important because it potentially identifies a group who may not be protected by current vaccination strategies. Further investigation into the role of HLA polymorphisms and nonresponse to influenza virus vaccination, and vaccination against viruses in general, is clearly required.     

Several hemagglutinins of same serotype for induction of broad immunity against influenza A virus antigenic drift variants: WO2008048984

The respiratory disease influenza gives rise to severe public health concerns. During inter-pandemic periods, the constant problem of the annually recurring seasonal influenza is perpetuated by the ability of influenza viruses to alter their surface antigens continuously (antigenic drift). Therefore, vaccines eliciting broad immunity against drift variants still remain a major objective in vaccine development. The patent WO2008048984 evaluated in this article claims an approach which aims to elicit homosubtypic protection against drift variants by simultaneous vaccination with several hemagglutinins (HAs) of the same serotype. The proposed multivalent vaccine based on simultaneous administration of several HAs, the results obtained from mice immunization studies and the implications of this concept are discussed in light of their relevance to application in humans. This proof-of-principle study suggests that a multivalent HA vaccine could elicit broad protection against drifted virus variants of one HA subtype. In the future, the dependence of broad efficacy on large antigenic distances among the HAs used for immunization as well as the antigenic distance between the HAs administered to that of the challenge virus, the immunological correlates of broad efficacy, and the suitability of this concept for domestic animals and humans remain to be investigated.     

流感病毒血凝素在多种系统中表达的研究进展

流感疫苗是预防流感病毒感染最有效的方法。传统灭活流感疫苗通过在鸡胚中培养病毒后经纯化获得。流感每年都会发生季节性流行,流感病毒高度多变的特性使生产有效疫苗成为一项挑战。为了克服流感疫苗生产对鸡胚的依赖,需要开发新的流感疫苗生产策略。由于血凝素是流感病毒主要表面抗原之一,重组血凝素亚单位疫苗为流感疫苗的生产提供了一个方案。本文将对流感病毒血凝素在大肠埃希菌、毕赤酵母、昆虫细胞、哺乳动物细胞多种系统中表达的研究进行综述。     

A conserved matrix epitope based DNA vaccine protects mice against influenza A virus challenge

DNA vaccination represents a unique strategy to overcome the limitations of immunization with conventional vaccines which is restricted by the high variability of influenza viruses. We evaluated the protective efficacy of a plasmid DNA (pDNA), encoding an evolutionarily conserved epitope of viral matrix protein, against the influenza A virus infection. It was found that the mice immunized via the intra-muscular route purely elicited cell mediated immune response to the pDNA, with enhanced level of Th1 cytokines viz. IL-12 and IFNγ production in the stimulated splenocyte supernatant. The cytotoxic T lymphocytes in the spleen of immunized mice significantly lysed the virus-infected MDCK cells. A significant decrease in virus replication was also observed in the lungs of immunized mice and 83% of the mice were protected against the lethal challenge of influenza A viruses. These findings suggest that the plasmid DNA expressing a single matrix epitope may serve as a promising vaccine candidate to provide effective immunity in the susceptible (mouse) population.     

流感DNA疫苗免疫BALB/c小鼠克服母源性抗体干扰的研究

目的 为了克服母源性抗体对子代的免疫抑制作用,寻找避免母源性抗体干扰的流感疫苗免疫策略.方法 以小鼠为动物模型,接种流感灭活疫苗或DNA疫苗,并用致死量流感病毒感染.感染后检测小鼠的存活率、肺部病毒滴度、体内抗体滴度等指标,对疫苗的保护效果进行评价.结果 母代与子代免疫相同的疫苗,不论是灭活疫苗还是DNA疫苗,子代体内的母源性抗体都抑制了子代免疫后的自动免疫应答,表现为子鼠接种疫苗后不能抵御致死量流感病毒感染;母代免疫流感灭活疫苗,子代免疫神经氨酸酶DNA疫苗,子鼠能够克服母源性抗体干扰,抵御致死量流感病毒感染;母代和子代免疫不同的DNA疫苗,即母代免疫血凝素或神经氨酸酶DNA疫苗,子代免疫神经氨酸酶或血凝素DNA疫苗,也能达到克服母源性抗体干扰的目的 .结论 流感DNA疫苗免疫BALB/c小鼠能克服母源性抗体的干扰,这为临床新生儿抗母源性抗体干扰的研究提供了实验参考.     

流感DNA疫苗免疫BALB/c小鼠克服母源性抗体干扰的研究

目的 为了克服母源性抗体对子代的免疫抑制作用,寻找避免母源性抗体干扰的流感疫苗免疫策略.方法 以小鼠为动物模型,接种流感灭活疫苗或DNA疫苗,并用致死量流感病毒感染.感染后检测小鼠的存活率、肺部病毒滴度、体内抗体滴度等指标,对疫苗的保护效果进行评价.结果 母代与子代免疫相同的疫苗,不论是灭活疫苗还是DNA疫苗,子代体内的母源性抗体都抑制了子代免疫后的自动免疫应答,表现为子鼠接种疫苗后不能抵御致死量流感病毒感染;母代免疫流感灭活疫苗,子代免疫神经氨酸酶DNA疫苗,子鼠能够克服母源性抗体干扰,抵御致死量流感病毒感染;母代和子代免疫不同的DNA疫苗,即母代免疫血凝素或神经氨酸酶DNA疫苗,子代免疫神经氨酸酶或血凝素DNA疫苗,也能达到克服母源性抗体干扰的目的 .结论 流感DNA疫苗免疫BALB/c小鼠能克服母源性抗体的干扰,这为临床新生儿抗母源性抗体干扰的研究提供了实验参考.     

流感DNA疫苗免疫BALB/c小鼠克服母源性抗体干扰的研究

目的 为了克服母源性抗体对子代的免疫抑制作用,寻找避免母源性抗体干扰的流感疫苗免疫策略.方法 以小鼠为动物模型,接种流感灭活疫苗或DNA疫苗,并用致死量流感病毒感染.感染后检测小鼠的存活率、肺部病毒滴度、体内抗体滴度等指标,对疫苗的保护效果进行评价.结果 母代与子代免疫相同的疫苗,不论是灭活疫苗还是DNA疫苗,子代体内的母源性抗体都抑制了子代免疫后的自动免疫应答,表现为子鼠接种疫苗后不能抵御致死量流感病毒感染;母代免疫流感灭活疫苗,子代免疫神经氨酸酶DNA疫苗,子鼠能够克服母源性抗体干扰,抵御致死量流感病毒感染;母代和子代免疫不同的DNA疫苗,即母代免疫血凝素或神经氨酸酶DNA疫苗,子代免疫神经氨酸酶或血凝素DNA疫苗,也能达到克服母源性抗体干扰的目的 .结论 流感DNA疫苗免疫BALB/c小鼠能克服母源性抗体的干扰,这为临床新生儿抗母源性抗体干扰的研究提供了实验参考.     

流感DNA疫苗免疫BALB/c小鼠克服母源性抗体干扰的研究

目的 为了克服母源性抗体对子代的免疫抑制作用,寻找避免母源性抗体干扰的流感疫苗免疫策略.方法 以小鼠为动物模型,接种流感灭活疫苗或DNA疫苗,并用致死量流感病毒感染.感染后检测小鼠的存活率、肺部病毒滴度、体内抗体滴度等指标,对疫苗的保护效果进行评价.结果 母代与子代免疫相同的疫苗,不论是灭活疫苗还是DNA疫苗,子代体内的母源性抗体都抑制了子代免疫后的自动免疫应答,表现为子鼠接种疫苗后不能抵御致死量流感病毒感染;母代免疫流感灭活疫苗,子代免疫神经氨酸酶DNA疫苗,子鼠能够克服母源性抗体干扰,抵御致死量流感病毒感染;母代和子代免疫不同的DNA疫苗,即母代免疫血凝素或神经氨酸酶DNA疫苗,子代免疫神经氨酸酶或血凝素DNA疫苗,也能达到克服母源性抗体干扰的目的 .结论 流感DNA疫苗免疫BALB/c小鼠能克服母源性抗体的干扰,这为临床新生儿抗母源性抗体干扰的研究提供了实验参考.     

流感DNA疫苗免疫BALB/c小鼠克服母源性抗体干扰的研究

目的 为了克服母源性抗体对子代的免疫抑制作用,寻找避免母源性抗体干扰的流感疫苗免疫策略.方法 以小鼠为动物模型,接种流感灭活疫苗或DNA疫苗,并用致死量流感病毒感染.感染后检测小鼠的存活率、肺部病毒滴度、体内抗体滴度等指标,对疫苗的保护效果进行评价.结果 母代与子代免疫相同的疫苗,不论是灭活疫苗还是DNA疫苗,子代体内的母源性抗体都抑制了子代免疫后的自动免疫应答,表现为子鼠接种疫苗后不能抵御致死量流感病毒感染;母代免疫流感灭活疫苗,子代免疫神经氨酸酶DNA疫苗,子鼠能够克服母源性抗体干扰,抵御致死量流感病毒感染;母代和子代免疫不同的DNA疫苗,即母代免疫血凝素或神经氨酸酶DNA疫苗,子代免疫神经氨酸酶或血凝素DNA疫苗,也能达到克服母源性抗体干扰的目的 .结论 流感DNA疫苗免疫BALB/c小鼠能克服母源性抗体的干扰,这为临床新生儿抗母源性抗体干扰的研究提供了实验参考.     

流感DNA疫苗免疫BALB/c小鼠克服母源性抗体干扰的研究

目的 为了克服母源性抗体对子代的免疫抑制作用,寻找避免母源性抗体干扰的流感疫苗免疫策略.方法 以小鼠为动物模型,接种流感灭活疫苗或DNA疫苗,并用致死量流感病毒感染.感染后检测小鼠的存活率、肺部病毒滴度、体内抗体滴度等指标,对疫苗的保护效果进行评价.结果 母代与子代免疫相同的疫苗,不论是灭活疫苗还是DNA疫苗,子代体内的母源性抗体都抑制了子代免疫后的自动免疫应答,表现为子鼠接种疫苗后不能抵御致死量流感病毒感染;母代免疫流感灭活疫苗,子代免疫神经氨酸酶DNA疫苗,子鼠能够克服母源性抗体干扰,抵御致死量流感病毒感染;母代和子代免疫不同的DNA疫苗,即母代免疫血凝素或神经氨酸酶DNA疫苗,子代免疫神经氨酸酶或血凝素DNA疫苗,也能达到克服母源性抗体干扰的目的 .结论 流感DNA疫苗免疫BALB/c小鼠能克服母源性抗体的干扰,这为临床新生儿抗母源性抗体干扰的研究提供了实验参考.