A DNA vaccine expressing PB1 protein of influenza A virus protects mice against virus infection

Although influenza DNA vaccine research has focused mainly on viral hemagglutinin and has led to promising results, other virion proteins have also shown some protective potential. In this work, we explored the potential of a DNA vaccine based on the PB1 protein to protect BALB/c mice against lethal influenza A virus infection. The DNA vaccine consisted of pTriEx4 plasmid expressing PB1. As a positive control, a pTriEx4 plasmid expressing influenza A virus HA was used. Two weeks after three subcutaneous doses of DNA vaccine, the mice were challenged intranasally with 1 LD₅₀ of A/Puerto Rico/8/34 (H1N1) virus, and PB1- and HA-specific antibodies, survival rate, body weight change, viral mRNA load, infectious virus titer in the lungs, cytokines IL-2, IL-4 and IL-10, and granzyme-B were measured. The results showed that (i) the PB1-expressing DNA vaccine provided a fair protective immunity in the mouse model and (ii) viral structural proteins such as PB1 represent promising antigens for DNA vaccination against influenza A.     

Monoclonal antibodies protect against respiratory syncytial virus infection in mice.

Twenty-five monoclonal antibodies (Mab) to respiratory syncytial virus (RSV) and two to hepatitis B virus were inoculated intravenously into mice. Twenty-four hours later the mice were challenged intranasally with RSV. Eleven of 14 Mab against fusion protein and four out of six Mab against a larger glycoprotein (GP84) significantly reduced the titre of RSV in the lungs when mice were killed 5 days later. Five Mab against three other RSV proteins and two Mab against hepatitis B virus had no significant effect on RSV infection. These results indicated that serum IgG against one epitope on the fusion protein and another on the larger glycoprotein (GP84) will completely protect mice against challenge. These epitopes are primary candidates for an RSV vaccine produced by techniques of gene cloning and peptide synthesis.     

Reduced activation and proliferation of human lymphocytes exposed to respiratory syncytial virus compared to cells exposed to influenza virus

Both respiratory syncytial virus (RSV) and influenza A virus (IAV) may infect human peripheral blood mononuclear leukocytes (PBMC) during the immune response to viral challenge as the cells are recruited to the respiratory tract. The current studies demonstrated differences in PBMC responses to the two viruses very early after exposure, including reduced fos protein and CD69 expression and IL‐2 production by RSV‐exposed T lymphocytes. Exposure to RSV resulted in reduced lymphocyte proliferation despite evidence of a virus‐specific T lymphocyte frequency equivalent to that for influenza virus. Reduced RSV‐induced proliferation was not due to apoptosis, which was itself reduced relative to that of influenza virus‐exposed T lymphocytes. The data indicate that differential immune responses to RSV and influenza virus are determined early after exposure of human PBMC and support the concept that the anamnestic immune response that might prevent clinically evident reinfection is attenuated very soon after exposure to RSV. Thus, candidate RSV vaccines should be expected to reduce but not prevent clinical illness upon subsequent infection by RSV. Furthermore, effective therapeutic agents for RSV are likely to be needed, especially for high‐risk populations, even after vaccine development.     

Broad humoral and cellular immunity elicited by a bivalent DNA vaccine encoding HA and NP genes from an H5N1 virus

Influenza A virus is highly variable and a major viral respiratory pathogen that can cause severe illness in humans. Therefore it is important to induce a sufficient immune response specific to current strains and to heterosubtypic viruses with vaccines. In this study, we developed a dual-promoter-based bivalent DNA vaccine that encodes both hemagglutinin (HA) and nucleoprotein (NP) proteins from a highly pathogenic A/Chicken/Henan/12/2004 (H5N1) virus. Our results show that the expression levels of HA and NP genes from the dual-promoter plasmid are similar to those seen when they are expressed individually in independent plasmids. When the bivalent DNA vaccine was inoculated via intramuscular injection and in vivo electroporation, high levels of both humoral and cellular immune responses were elicited against homologous H5N1 virus and heterosubtypic H9N2 virus. Furthermore, no obvious antigenic competition was observed between HA and NP proteins in the dual-promoter-based bivalent vaccine compared to monovalent vaccines. Our data suggest that a combination of influenza surface and internal viral genes in a dual-promoter-expressing plasmid may provide a new approach for developing a DNA vaccine that may protect not only specifically against a currently circulating strain, but also may cross-protect broadly against new heterosubtypic viruses.     

Chimeric subgroup A respiratory syncytial virus with the glycoproteins substituted by those of subgroup B and RSV without the M2-2 gene are attenuated in African green monkeys

Using the existing reverse genetics system developed for the subgroup A respiratory syncytial virus (RSV), a chimeric virus (designated rA-G(B)F(B)) that expresses subgroup B-specific antigens was constructed by replacing the G and F genes of the A2 strain with those of the 9320 strain of subgroup B RSV. rA-G(B)F(B) grew well in tissue culture, but it was attenuated in the respiratory tracts of cotton rats and African green monkeys. To further attenuate this chimeric RSV, the M2-2 open reading frame was removed from rA-G(B)F(B). rA-G(B)F(B)DeltaM2-2 was highly attenuated in replication in the respiratory tracts of the infected monkeys, but it provided complete protection against wild-type subgroup B RSV challenge following two doses of infection. In this study, rA2DeltaM2-2 (a recombinant A2 RSV that lacks the M2-2 gene) was also evaluated in African green monkeys. The replication of rA2DeltaM2-2 was highly restricted in both the upper and lower respiratory tracts of the infected monkeys and it induced titers of serum anti-RSV neutralizing antibody that were slightly lower than those induced by wild-type rA2. When rA2DeltaM2-2-infected monkeys were challenged with wild-type A2 virus, the replication of the challenge virus was reduced by approximately 100-fold in the upper respiratory tract and 45,000-fold in the lower respiratory tracts. rA2DeltaM2-2 and rA-G(B)F(B)DeltaM2-2 could represent a bivalent RSV vaccine composition for protection against multiple strains from the two RSV subgroups.     

Plasmid DNA encoding the respiratory syncytial virus G protein is a promising vaccine candidate

Respiratory syncytial virus (RSV) remains a major cause of severe respiratory diseases in infants, young children, and the elderly. However, development of a RSV vaccine has been hampered by the outcome of the infant trials in the 1960s with a formalin-inactivated RSV preparation. Enhanced lung disease was induced by the vaccination post-RSV exposure. Previous studies in mice primed with RSV G protein either formulated in adjuvants or delivered by recombinant vaccinia viruses have indicated that enhanced lung pathology resulted from a Th2-type host immune response against the viral G protein. However, in the present report, we have demonstrated that vaccination with plasmid vectors encoding either a full-length or a secreted G protein (DNA-G) clearly elicited balanced systemic and pulmonary Th1/Th2 cytokine responses in mice and did not induce an atypical pulmonary inflammatory reaction post-RSV challenge in cotton rats. DNA-G immunization also induced marked virus neutralizing antibody responses and protection against RSV infection of the lower respiratory tract of both mice and cotton rats. So far, only genetic immunization has been able to induce a balanced Th1/Th2 response with the RSV G protein, reminiscent of that induced by live RSV. Therefore, DNA-G is a promising immunogen for inclusion in a nucleic acid RSV vaccine.     

Respiratory syncytial virus, pneumonia virus of mice, and influenza A virus differently affect respiratory allergy in mice

BACKGROUND: Respiratory viral infections in early childhood may interact with the immune system and modify allergen sensitization and/or allergic manifestations. In mice, respiratory syncytial virus (RSV) infection during allergic provocation aggravates the allergic T helper (Th) 2 immune response, characterized by the production of IL-4, IL-5, and IL-13, and inflammatory infiltrates. However, it is unclear whether the RSV-enhanced respiratory allergic response is a result of non-specific virus-induced damage of the lung, or virus-specific immune responses. OBJECTIVE: In the present study we investigated whether RSV, pneumonia virus of mice (PVM) and influenza A virus similarly affect the allergic response. METHODS: BALB/c mice were sensitized and challenged with ovalbumin (OVA), and inoculated with virus during the challenge period. Pulmonary inflammation, lung cytokine mRNA responses, and IgE production in serum were assessed after the last OVA-challenge. RESULTS: Like RSV, PVM enhanced the OVA-induced pulmonary IL-4, IL-5, and IL-13 mRNA expression, which was associated with enhanced perivascular inflammation. In addition, PVM increased the influx of eosinophils in lung tissue. In contrast, influenza virus decreased the Th2 cytokine mRNA expression in the lungs. However, like PVM, influenza virus enhanced the pulmonary eosinophilic infiltration in OVA-allergic mice. CONCLUSION: The Paramyxoviruses RSV and PVM both are able to enhance the allergic Th2 cytokine response and perivascular inflammation in BALB/c mice, while the Orthomyxovirus influenza A is not.     

流感病毒RNA聚合酶蛋白PB1在小鼠中可诱导亚型间交叉免疫保护

目的探索流感病毒RNA聚合酶PB2和PB1亚基作为实验性流感疫苗候选抗原的可能性.方法以复制型质粒（pSCA）为载体分别构建表达甲1型和甲3型流感PB2和PB1的复制型DNA疫苗,免疫小鼠后分别用甲1型流感病毒（A/PR/8/34）进行鼻腔攻击,观察针对不同亚型流感病毒的复制型DNA疫苗的免疫保护效果.结果本实验所构建的复制型DNA疫苗在真核细胞中均可表达外源基因;本实验采用的复制型质粒载体（pSCA）与传统质粒载体（pcDNA3）在诱导小鼠产生抗体方面无差异,并且都诱导了偏向TH1类的免疫反应;表达甲1型和甲3型流感PB1基因的复制型DNA疫苗均可保护小鼠抵御甲1型流感病毒（A/PR/8/34）的攻击.结论表达甲型流感病毒PB1的复制型DNA疫苗能保护小鼠抵御同型和异型流感病毒的攻击,本实验为流感疫苗研究提供新的候选抗原.     

Interferon production by human mononuclear leukocytes: differences between respiratory syncytial virus and influenza viruses.

The ability of respiratory syncytial virus (RSV) to induce interferon production by human mononuclear leukocytes was compared with that of influenza viruses. Cell culture fluids were assayed for interferon activity 1, 3 and 7 days after exposure to RSV or to one of two subtypes of influenza A virus (H0N1 and H3N2). RSV induced interferon production inconsistently and in low titers. Varying the multiplicity of infection did not improve the ability of RSV to induce interferon production. In contrast, influenza viruses were effective inducers of interferon production. Seropositivity to the influenza virus strains was not associated with increased interferon titers. Interferon produced after exposure to RSV or to the influenza viruses was resistant to low pH treatment. The data suggest that interferon production may not be a major component of human immunological defense against RSV infection.     

Mutations in the genes coding for hemagglutinin and neuraminidase in cold-adapted variants of the influenza virus A/Leningrad/134/57 (H2N2)

An analysis of ts-mutations in the genomes of native and cold-adapted variants of influenza A/Leningrad/134/57 (H2N2) virus based on the use of fowl plague virus ts mutants was carried out. The recombination test was done by the conventional method in chick embryo fibroblast culture (genes PB2, PB1, PA, NP, NA, M and NS) or cell systems permissive for reproduction of human influenza virus (gene HA). The cold-adapted strain A/Len/17 used for preparation of live influenza vaccine (LIV) for adults was shown to have 4 ts mutations: three in "internal" genes (PB2, NP, and M) and one in gene 4 coding for hemagglutinin (HA). The more attenuated cold-adapted donor A/Len/47 for preparation of similar LIV for children acquired three additional ts mutations: two (PB1 and NS) in "internal" genes and one in gene 6 coding for neuraminidase (NA). The accumulation of ts mutations in the genome of cold-adapter strains was found to be accompanied by a decrease in their pneumotropicity for mice as well as their detectability in different organs of these animals.     

In vitro growth profiles of respiratory syncytial virus in the presence of influenza virus

To elucidate epidemiological interference between respiratory syncytial (RSV) and influenza viruses, the influence of influenza A (HlN1) virus on the growth of RSV was examined. Although RSV grew in MDCK cells, coinfection with influenza A virus led to a reduction of progeny RSV. The degree of growth interference depended on the time of infection with influenza A virus post infection (p.i.) with RSV. In fact, infection with influenza A virus 12 hrs p.i. with RSV did not influence growth of the latter virus. On the contrary, growth suppression of influenza A virus by RSV was observed when the coinfection began at the later stages of RSV infection. Suppression of the growth of RSV by influenza A infection was further demonstrated at the level of viral protein synthesis. An indirect immunofluorescence (IF) test revealed that a large proportion of infected cells synthesized both RSV and influenza A virus antigens. Scanning electron microscopic (SEM) examination demonstrated that influenza A and RSV virions possessing surface antigens specific for each virus were selectively released from dually infected cells. In the present study, we proved for the first time that the growth of RSV is blocked by competitive infection with influenza A virus in a susceptible cell population, competitive protein synthesis and selective budding of RSV and influenza viruses from the same infected cells.     

用反向遗传技术产生冷适应致弱的重组A型人流感病毒的研究

目的 以鸡胚高度适应株A/PR/8/34株为重组流感病毒骨架,利用反向遗传技术拯救冷适应致弱的重组A型人流感病毒.方法 对冷适应、温度敏感、减毒的A/Ann Arbor/6/60(H2N2)流感病毒株的PB2基因片段,进行了全基因序列合成,同时人工引入PB2265(N265S)氨基酸的突变.PB2基因片段通过与改造后的转录/表达载体pAD3000连接,构建PB2基因的拯救载体.该重组质粒与PR8进行"7 1"组合的病毒拯救,共转染COS-1细胞.结果 经测序获得序列准确的拯救质粒pMDV-A-PB2,利用反向遗传技术成功拯救出了具有血凝性(1×25)的冷适应的重组A型流感病毒.结论 利用反向遗传技术成功拯救冷适应致弱的重组A型人流感病毒,该系统为深入研究甲型人流感病毒的基因功能和新型疫苗研发奠定了基础.     

Influenza and respiratory syncytial virus in the elderly

Respiratory infections are one of the leading causes of morbidity and mortality worldwide: influenza and respiratory syncytial virus (RSV) are the predominant pathogens responsible. Annual vaccination and the use of antiviral drugs provides both protection and treatment against influenza, particularly protecting those patients most at risk, including the elderly and individuals with chronic comorbidities. Currently, there are extremely limited options in the protection against RSV infection, making those at-risk patients vulnerable to serious disease, complications and death. This review focuses on recent trends in respiratory illness in the elderly, particularly focusing on the burden of influenza and RSV on hospitalizations and mortality. We discuss the potential benefit of influenza vaccination on these outcomes, including the recent controversial debate over the effectiveness of influenza vaccination.     

Influenza A virus with defective M2 ion channel activity as a live vaccine

We propose a rational approach to the design of live virus vaccines against influenza infection by alteration of the influenza A virus M2 protein, which is responsible for ion channel activity. Previously we demonstrated that a mutant A/WSN/33 (H1N1) influenza virus with defective M2 ion channel activity did not show appreciable growth defects in cell culture, although its growth was attenuated in mice (T. Watanabe, S. Watanabe, H. Ito, H. Kida, and Y. Kawaoka, 2001, J. Virol. 75, 5656-5662). Here, we show that this M2 ion channel defective mutant virus, the M2del29-31, protected mice against challenge with lethal doses of influenza virus, indicating the potential of incorporating this M2 alteration in a live influenza vaccine as one of the attenuating mutations.     

含H5N1-HA基因重组腺病毒疫苗的构建及其诱导免疫应答的初步探讨

目的 构建包含H5N1-HA基因的重组腺病毒疫苗并探讨其免疫效果.方法 用Admax系统构建包含H5N1-HA基因的重组腺病毒疫苗,并用PCR、Western-Blot等方法对重组病毒疫苗进行鉴定;疫苗免疫小鼠后,通过HI实验和ELISPOT实验检测其体液免疫和细胞免疫反应,评价其免疫效果.结果 成功得到了含有H5N1-HA基因的重组腺病毒疫苗;基因表达鉴定表明,HA基因能够在细胞中进行表达;血凝抑制实验结果显示小鼠产生的针对HA抗体滴度在1:320和1:640之间;ELISPOT结果显示实验组和对照组(PBS)相比斑点数量差异有统计学意义(P<0.05),以上免疫结果表明重组腺病毒载体疫苗可以诱导小鼠产生良好的特异性体液和细胞免疫反应.结论 含H5NA-HA的重组腺病毒疫苗可以诱导小鼠产生良好的免疫反应,为研制人禽流感疫苗打下基础.     

Antiviral activity of arbidol against influenza A virus, respiratory syncytial virus, rhinovirus, coxsackie virus and adenovirus in vitro and in vivo

Summary Arbidol, ethyl-6-bromo-4-[(dimethylamino)-methyl]-5-hydroxy-1-methyl-2-[(phenylthio)methyl]-in dole-3-carboxylate hydrochloride monohydrate, is an antiviral chemical agent. In this report, we studied the antiviral activity of arbidol against a panel of human respiratory viruses, namely influenza A virus (FLU-A, A/PR/8/34 H1N1), respiratory syncytial virus (RSV), human rhinovirus type 14 (HRV 14), coxsackie virus B3 (CVB3) and adenovirus type 7 (AdV-7) in vitro in cell culture. Arbidol was found to present potent inhibitory activity against enveloped and non-enveloped RNA viruses, including FLU-A, RSV, HRV 14 and CVB3 when added before, during, or after viral infection, with 50% inhibitory concentration (IC₅₀) ranging from 2.7 to 13.8 μg/ml. However, arbidol showed selective antiviral activity against AdV-7, a DNA virus, only when added after infection (therapeutic index (TI) = 5.5). Orally administered arbidol at 50 or 100 mg/kg/day beginning 24 h pre-virus exposure for 6 days significantly reduced mean pulmonary virus yields and the rate of mortality in mice infected with FLU-A (A/PR/8/34 H1N1). Our results suggest that arbidol has the ability to elicit protective broad-spectrum antiviral activity against a number of human pathogenic respiratory viruses.     

Immunogenic activity of the influenza virus dependent on ne uraminidase (author's transl)]

The immunity of mice to challenge with influenza virus was studied after immunization with killed influenza vaccine. The results showed the resistance of the animals developing early after one and two intranasal vaccinations to depend not only upon the hemagglutinating properties of the vaccine but also upon its neuraminidase activity. When mice were immunized with a vaccine the neuraminidase of which was blocked with specific antibody or inactivated by heating and formalin, the protective effect against challenge with influenza virus decreased 2-3-fold as compared to that of the vaccine possessing active neuraminidase.     

The live attenuated subgroup B respiratory syncytial virus vaccine candidate RSV 2B33F is attenuated and immunogenic in chimpanzees, but exhibits partial loss of the ts phenotype following replication in vivo

The cold-adapted (ca), temperature-sensitive (ts) respiratory syncytial virus (RSV) subgroup B vaccine candidate, designated RSV 2B33F, was found previously to be restricted in replication, immunogenic, and protective against wild-type (wt) virus challenge in rodents and African green monkeys. We sought to investigate the level of attenuation, immunogenicity and genetic stability of this vaccine candidate in seronegative chimpanzees. The 2B33F vaccine candidate was attenuated in chimpanzees and manifested a ten- and 1000-fold restriction in replication in the upper and lower respiratory tracts respectively, compared with its wt RSV 2B parent virus. Despite this attenuation, chimpanzees immunized with RSV 2B33F were completely resistant to respiratory tract disease and virus replication upon challenge with wt virus. The ts phenotype of the RSV 2B33F mutant exhibited some alteration during replication in vivo in three of four chimpanzees tested. Virus present in nasopharyngeal swab or tracheal lavage secretions of these three chimpanzees was biologically cloned by plaque passage in Vero cells at permissive temperature. The plaque progeny retained the ts phenotype, but uniformly produced plaques at 39 and 40 degrees C to a level intermediate between that of the 2B33F input virus and the 2B wt parent virus, indicating that partial loss of the level of temperature sensitivity occurred following replication in vivo. The implications of these findings for RSV vaccine development are discussed.     

Protective Efficacy of the Conserved NP,PB1, and M1 Proteins as Immunogens in DNA- and Vaccinia Virus-Based Universal Influenza A Virus Vaccines in Mice

The conventional hemagglutinin (HA)- and neuraminidase (NA)-based influenza vaccines need to be updated most years and are ineffective if the glycoprotein HA of the vaccine strains is a mismatch with that of the epidemic strain. Universal vaccines targeting conserved viral components might provide cross-protection and thus complement and improve conventional vaccines. In this study, we generated DNA plasmids and recombinant vaccinia viruses expressing the conserved proteins nucleoprotein (NP), polymerase basic 1 (PB1), and matrix 1 (M1) from influenza virus strain A/Beijing/30/95 (H3N2). BALB/c mice were immunized intramuscularly with a single vaccine based on NP, PB1, or M1 alone or a combination vaccine based on all three antigens and were then challenged with lethal doses of the heterologous influenza virus strain A/PR/8/34 (H1N1). Vaccines based on NP, PB1, and M1 provided complete or partial protection against challenge with 1.7 50% lethal dose (LD₅₀) of PR8 in mice. Of the three antigens, NP-based vaccines induced protection against 5 LD₅₀ and 10 LD₅₀ and thus exhibited the greatest protective effect. Universal influenza vaccines based on the combination of NP, PB1, and M1 induced a strong immune response and thus might be an alternative approach to addressing future influenza virus pandemics.