Induction of antibody response by DNA immunization of newborn baboons against influenza virus.

Previous studies showed that DNA immunization of newborn mice with plasmids expressing influenza virus antigens induced protective immunity. We have now extended the study of neonatal responsiveness to DNA vaccines to nonhuman primates. Baboons immunized as neonates with plasmids expressing type A influenza virus hemagglutinin (HA) and nucleoprotein (NP) in doses ranging from 40 microg to 1 mg per plasmid per dose developed virus-specific humoral responses. The titer and kinetics of appearance of virus-specific IgG antibodies were dose dependent. Specific antibodies were detected by enzyme-linked immunosorbent assay (ELISA) as early as 1 month after birth in baboons immunized with the highest and intermediate doses of vaccine. Virus-neutralizing antibodies were detected in the group of baboons immunized with the highest dose. The specificity of virus-neutralizing antibodies was found to be directed against homologous determinants of HA; however, the IgG antibodies also cross-reacted with HA of a drift variant. Thus, DNA vaccination of newborn baboons with a prototype vaccine against influenza virus resulted in induction of specific humoral immunity.     

Long-term humoral and cellular immunity induced by a single immunization with replication-defective adenovirus recombinant vector

This study examines the suitability of replication-defective adenovirus vectors for engineering recombinant vaccines. The immunological abilities and limitations of E1-deleted adenoviruses containing the lac Z gene (Ad-β-gal) were investigated by examining the humoral and cellular immune responses to the β-galactosidase protein. BALB/c mice (H-2^d) were given in a single injection of recombinant adenovirus. The cytotoxic T lymphocyte (CTL) response of spleen cells was evaluated. Recognized target cells were H-2^d-derived tumor cells transfected by the lac Z gene, or incubated with the 876–884 β-galactosidase peptide known to be restricted by the L^d molecule of the major histocompatibility complex. A long-lasting β-galactosidase-specific cytotoxic T cell response was obtained. By contrast, CTL from mice immunized with the L^d-restricted peptide were less specific for the endogenous epitope presented by the transfectants expressing β-galactosidase. Ad-β-gal-immunized mice were also protected against an intra-cerebral challenge with a recombinant vaccinia virus expressing the lac-Z gene. These results suggest that Ad-β-gal-induced CTL have protective abilities in vivo. The induction of β-galactosidase-specific T helper lymphocytes and humoral IgG responses were also examined. A proliferative response occurred only late after immunization and the primed T lymphocytes produced interleukin-2, but no interleukin-4. A humoral IgG response to the β-galactosidase protein was detected 15–30 days after a single immunization and remained stable for 6 months without boosting. Lastly, we followed the evolution of the immune response over the course of successive immunizations. The magnitude and kinetics of the cellular and humoral responses were similar to those obtained after a single immunization. Consistent with these observations, an adenovirus-specific neutralizing antibody response was detected as early as the second immunization. Thus, a single immunization with a replication-defective adenovirus recombinant vector induces long-lasting humoral and cellular immune responses specific to the transgene product.     

Mucosal immunization with attenuated Shigella flexneri harboring an influenza hemagglutinin DNA vaccine protects mice against a lethal influenza challenge

Mucosal surfaces are important for the induction of immunity against influenza virus. In a murine intranasal immunization model, we demonstrated that the attenuated Shigella flexneri Deltaasd strain 15D, carrying a DNA construct encoding the influenza virus hemagglutinin (HA), induces protective immunity against a lethal respiratory challenge with influenza A/WSN/33. Influenza virus-specific IFN-gamma T cells were detected among splenocytes, and anti-HA IgG was detected in serum post-immunization, albeit at low levels. Following influenza virus challenge, an accelerated anti-HA IgA antibody response was detected in bronchoalveolar lavage (BAL) washings from mice vaccinated with attenuated shigella containing the HA construct. These results suggest that S. flexneri Deltaasd strain 15D is a promising vector for mucosal DNA vaccine immunization against influenza virus and other mucosal pathogens.     

Induction of herpes simplex virus immunity in newborn mice.

It was demonstrated that newborn mice, surviving an infection with attenuated herpes simplex virus, become resistant to challenge with a pathogenic strain of herpes simplex virus. This resistance did not seem to be mediated by antibody.     

Induction of humoral immunity and pulmonary mast cells in mice and rats after immunization with aerosolized antigen.

Rats (BN X Wistar) and mice (CBA/Ca) were immunized by exposure in 10-day periods to an aerosol of ovalbumin (OA). In rats this immunization resulted in IgE antibodies detectable at very low levels in bronchial washings, whereas IgG, IgA and IgM antibodies were recorded both in serum and in bronchial washings. In mice, exposure to aerosolized antigen resulted in specific IgE and IgG antibodies in serum. The levels of IgM antibodies were low and no IgA antibodies could be recorded with the enzyme-linked immunosorbent assay (ELISA). Histological examination of lung tissue from immunized rats and mice revealed increased numbers of cells with characteristics of both immature and mature mast cells. In addition, in the rats these cells were more closely located to the bronchi in immunized than in control animals. In the latter animals the mast cells were located around the blood vessels. Immature mast cells were located in the bronchiole-associated lymphatic tissue (BALT) which showed a marked proliferation in immunized animals. The findings indicate that sensitization via the airways provides possibilities to develop a model in rodents for studies of IgE-mediated allergy in the lung.     

Suppression of humoral and cellular immunity in normal mice by diazepam

Diazepam, a benzodiazepine derivative useful in the treatment of anxiety disorders, was found to depress both primary antibody to sheep red blood cells and delayed-type hypersensitivity responses in normal mice. This immunodepressant activity warrants further investigation owing to its potential consequences on human health and the putative involvement of specific receptors on immunocompetent cells.     

Protection against influenza A virus infection in mice by oral immunization with a polyvalent bacterial lysate.

A study is presented which investigated whether oral immunization with a polyvalent bacterial lysate (Paspat oral) can sufficiently enhance cell-mediated defense mechanisms to protect mice against influenza A virus infection. It was found that oral immunization reduced mortality due to influenza A infection with 15-70%, depending on the quantity of virus administered and and the moment of infection. Cyclosporin A severely reduced the protective effect of oral immunization, suggesting that a major effect of oral immunization in these studies is T-cell activation. The effect of oral immunization on macrophageal activity was evaluated by measuring cyclic-AMP in alveolar macrophages (AMs) obtained by bronchoalveolar lavage. Before infection, basal activity levels of AMs in immunized mice were significantly lower than in controls. Five days after infection, however, basal activity level of AMs in immunized mice was significantly higher than AM activity in controls. Stimulation of AMs with PGE2 significantly reduced cellular activity in both groups, before and after infection. However, cellular activity of AMs from immunized animals was less reduced than cellular activity of control macrophages. Activity of AMs of immunized animals was significantly more reduced by histamine than activity of control macrophages. It is concluded that oral immunization with Paspat oral stimulates T-cell-dependent immune mechanisms, resulting in protection against influenza A virus infection in mice.     

Pathological aspects of immunization of mice against influenza virus infection.

Groups of mice were immunized against influenza Ao/NWS virus by a single intranasal administration of inactivated homologous virus, by 2 intranasal doses of vaccine separated by an interval of 2 weeks, or by 2 intraperitoneal doses of the same vaccine. When subjected 2 weeks later to a standard challenge of 6 x 10(5) egg infecting units Ao/NWS virus instilled intranasally, mortality fell significantly from 64% in unimmunized mice to 39% in mice given a single intranasal dose of vaccine and to 29% in animals which received double intranasal vaccine. The best protection was conferred by double intraperitoneal immunization, after which mortality was 10%. Immunity waned with time, since the mortality of mice doubly immunized by the respiratory route and challenged 30 weeks later was 49%. Intrapulmonary lymphoid tissue developed in large amounts in a proportion of mice immunized by all methods and challenged after an interval of 2 weeks. Attention is drawn to this reaction as a possible unfavourable consequence of vaccination. There were no lesions in the lungs or central nervous system after immunization without subsequent challenge. The importance of histopathology in vaccine trials in experimental animals is emphasized by the consistently higher detection rate of lesions in lungs by histological examination than by visual inspection alone.     

Role of maternal immunity in the protection of newborn ferrets against infection with a virulent influenza virus

Intranasal infection of newborn ferrets with a virulent strain of influenza virus invariably resulted in their deaths following virus replication to high titre in both lung and nasal turbinates (Collie et al., 1980). However, a similar challenge of newborn ferrets born to mothers immunized by infection with virulent or attenuated viruses resulted in complete protection; no virus replicated in their lungs and little or no virus was isolated from their nasal turbinates. Protection appeared to be antibody-mediated since it was sub-type-specific and milk-derived since newborn ferrets born to non-immune mothers but fostered onto immune mothers exhibited a similar level of protection to neonates born to and suckled by immune mothers.     

Induction of humoral and cellular immune responses against hepatitis C virus by vaccination with replicon particles derived from Sindbis-like virus XJ-160

A replication-defective, recombinant Sindbis virus vector was utilized in a novel immunization strategy to induce humoral and cellular responses against hepatitis C virus (HCV). The recombinant vector, pVaXJ-E1E2, expressing the gene for HCV glycoproteins E2 and E1, was constructed by inserting the E1E2 gene into the replicon pVaXJ, a DNA vector derived from Sindbis-like virus XJ-160. The defective replicon particles, XJ-E1E2, were produced by transfecting BHK-21^E+Capsid cells, the packaging cell lines for the vector from XJ-160 virus, with pVaXJ-E1E2. Both glycoproteins, E2 and E1, were stably expressed, as indicated by immunofluorescence assay (IFA) and Western blotting. Mice were vaccinated using a prime-boost strategy with XJ-E1E2 particles combined with Freund’s incomplete adjuvant via intramuscular injection at 0 and 2 weeks. HCV-specific IgG antibody levels and cellular immune responses were evaluated by IFA and IFN-γ ELISPOT, respectively. The results showed that the defective XJ-E1E2 particles in combination with Freund’s incomplete adjuvant induced effective humoral and cellular immune responses against HCV glycoprotein E1 or E2, suggesting that a defective Sindbis particle vaccine is capable of eliciting an effective immune response. These findings have important implications for the development of HCV vaccine candidates.     

Microneedle vaccination with stabilized recombinant influenza virus hemagglutinin induces improved protective immunity

The emergence of the swine-origin 2009 influenza pandemic illustrates the need for improved vaccine production and delivery strategies. Skin-based immunization represents an attractive alternative to traditional hypodermic needle vaccination routes. Microneedles (MNs) can deliver vaccine to the epidermis and dermis, which are rich in antigen-presenting cells (APC) such as Langerhans cells and dermal dendritic cells. Previous studies using coated or dissolvable microneedles emphasized the use of inactivated influenza virus or virus-like particles as skin-based vaccines. However, most currently available influenza vaccines consist of solubilized viral protein antigens. Here we test the hypothesis that a recombinant subunit influenza vaccine can be delivered to the skin by coated microneedles and can induce protective immunity. We found that mice vaccinated via MN delivery with a stabilized recombinant trimeric soluble hemagglutinin (sHA) derived from A/Aichi/2/68 (H3) virus had significantly higher immune responses than did mice vaccinated with unmodified sHA. These mice were fully protected against a lethal challenge with influenza virus. Analysis of postchallenge lung titers showed that MN-immunized mice had completely cleared the virus from their lungs, in contrast to mice given the same vaccine by a standard subcutaneous route. In addition, we observed a higher ratio of antigen-specific Th1 cells in trimeric sHA-vaccinated mice and a greater mucosal antibody response. Our data therefore demonstrate the improved efficacy of a skin-based recombinant subunit influenza vaccine and emphasize the advantage of this route of vaccination for a protein subunit vaccine.     

Recombinant parainfluenza virus 5 (PIV5) expressing the influenza A virus hemagglutinin provides immunity in mice to influenza A virus challenge

Parainfluenza virus type 5 (PIV5), formerly known as simian virus 5 (SV5), is a non-segmented negative strand RNA virus that offers several advantages as a vaccine vector. PIV5 infects many cell types causing little cytopathic effect, it replicates in the cytoplasm of infected cells, and does not have a DNA phase in its life cycle thus avoiding the possibility of introducing foreign genes into the host DNA genome. Importantly, PIV5 can infect humans but it is not associated with any known human illness. PIV5 grows well in tissue culture cells, including Vero cells, which have been approved for vaccine production, and the virus can be obtained easily from the media. To test the feasibility of using PIV5 as a live vaccine vector, the hemagglutinin (HA) gene from influenza A virus strain A/Udorn/72 (H3N2) was inserted into the PIV5 genome as an extra gene between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Recombinant PIV5 containing the HA gene of Udorn (rPIV5-H3) was recovered and it replicated similarly to wild type PIV5, both in vitro and in vivo. The HA protein expressed by rPIV5-H3-infected cells was incorporated into the virions and addition of the HA gene did not increase virus virulence in mice. The efficacy of rPIV5-H3 as a live vaccine was examined in 6-week-old BALB/c mice. The results show that a single dose inoculation provides broad and considerable immunity against influenza A virus infection.     

Protection against pulmonary blastomycosis: correlation with cellular and humoral immunity in mice after subcutaneous nonlethal infection.

A model of pulmonary blastomycosis in the mouse, in which the portal of entry is the same as natural human infection, was used to study resistance to challenge after subcutaneous infection. One week after subcutaneous infection, mice were partially resistant to pulmonary challenge, and mice challenged two weeks after infection were resistant. Measurement of cellular and humoral immune responses to Blastomyces dermatitidis antigens after subcutaneous infection showed the following. (i) Delayed-type hypersensitivity appeared 1 week after infection, and responses increased for 3 weeks thereafter. (ii) Proliferative responses in vitro appeared in spleen cells at 1 week and in contralateral lymph node cells at 3 weeks, (iii) Serum antibody, detected by an enzyme-linked immunosorbent assay, appeared 1 week after infection and then increased in titer. (iv) Peritoneal macrophages were activated to inhibit replication of B. dermatitidis in vitro by the first week after infection. Prior subcutaneous infection also resulted in rapid clearing of a second subcutaneous challenge, as well as resistance to a lethal intraperitoneal challenge. This resistance was associated with the development of cell-mediated and humoral immune responses. These data provide a chronological framework for selective transfer experiments.     

Subcutaneous immunization with baculovirus surface-displayed hemagglutinin of pandemic H1N1 Influenza A virus induces protective immunity in mice

The protective immunity of baculovirus displaying influenza virus hemagglutinin (BacHA) against influenza 2009 H1N1 virus infection in a murine model was investigated. The results showed that mice vaccinated with live BacHA or an inactive form of adjuvanted BacHA had enhanced specific antibody responses and induced protective immunity against 2009 H1N1 virus infection, suggesting the potential of baculovirus as a live or inactivated vaccine.     

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.     

猪瘟基因疫苗免疫小鼠体液及细胞免疫动态变化研究

目的：探讨猪瘟基因疫苗免疫小鼠后机体产生体液和细胞免疫发生规律，为猪瘟基因疫苗推广应用提供科学依据。方法：应用FACS、MTT法及间接ELISA试验对猪瘟基因疫苗免疫小鼠脾脏及外周血中CD4^ 和CD8^ 淋巴细胞数、淋巴细胞的转化功能及特异的抗猪瘟病毒血清IgG抗体水平等动态变化进行了观察。结果：猪瘟基因疫苗免疫小鼠后脾细胞及外周血淋巴细胞对ConA和LPS均有明显的反应性。CD4^ 和CD8^ 细胞数量在免疫后20天开始反应，32天达到高峰后开始下降。鼠血清特异性猪瘟病毒血清抗体IgG随免疫时间延长而增加。结论：猪瘟基因疫苗免疫动物可诱导机体体液及细胞免疫。     

Induction of xanthine oxidase by virus infections in newborn mice

In the liver tissue of newborn mice, xanthine oxidase activity is very low during the first 7 to 14 days of life. Infection of mice with several different viruses prematurely induced xanthine oxidase activity 2- to 10-fold in the liver tissue. Generally, overt signs of illness appeared after xanthine oxidase induction; however, some viruses induced the enzyme activity without causing morbidity or deaths. The elevated enzyme activity could not be correlated with alteration of either lactate dehydrogenase or glutamate-pyruvate transaminase. Likewise, there were no histological changes in the livers of infected animals when xanthine oxidase levels were abnormally elevated. These observations suggest that measurement of xanthine oxidase may be an effective method for the detection of subclinical or inapparent viral infections in either naturally infected newborn mice or in newborn mice inoculated with suspected virus-containing materials.     

Mucosal immunity against influenza induced by attenuated recombinant Sendai virus

Live-attenuated influenza vaccines (LAIVs) have been shown to be more immunogenic and capable of inducing a broader immune response than inactivated vaccine. However, use of LAIVs is still limited owing to the safety concerns. Le et al. generated an attenuated recombinant Sendai virus - GP42-H1 expressing the hemagglutinin (HA) gene of influenza A virus. The HA protein was expressed on the cell surface of CV-1 cells infected with GP42-H1. Intranasal immunization of mice with GP42-H1 induced HA-specific IgG and IgA antibodies in sera and mucosal sites without causing any disease symptoms. Immunized mice were also protected from lethal dose challenge of influenza A virus.