缺失ORF73或ORF214基因对重组鸡痘病毒免疫应答的影响

【目的】旨在研究鸡痘病毒ORF73和ORF214编码蛋白是否具有IL-18结合蛋白的功能,以及ORF73或ORF214基因缺失后对重组病毒诱导免疫应答的影响。【方法】以缺失ORF73或ORF214基因并表达H5亚型AIVHA基因的重组鸡痘病毒(rFPVLP-△73LRH5A、rFPVLP-△214LRH5A)作为研究对象,以未缺失ORF73或ORF214基因而表达H5亚型AIVHA基因的重组鸡痘病毒(rFPVLP-12LSH5A)作为对照,检测重组病毒体外诱导SPF鸡脾细胞和外周血淋巴细胞产生IFN情况,同时检测重组病毒免疫SPF鸡后诱导的体液免疫、CD4+/CD8+比值、外周血淋巴细胞的增殖能力和H5亚型AIV强毒攻击后的免疫保护效力。【结果】rFPVLP-△73LRH5A和rFPVLP-△214LRH5A体外诱导脾细胞产生的IFN量显著高于rFPVLP-12LSH5A,而免疫10d后的CD4+/CD8+比值显著低于rFPVLP-12LSH5A;3种重组鸡痘病毒诱导外周血淋巴细胞增殖的能力没有明显差异;3种重组病毒在SPF鸡均产生针对H5亚型AIV的HI抗体,免疫14d后rFPVLP-△214LRH5A组诱生的HI抗体水平显著低于rFPVLP-12LSH5A组的抗体,但3组在免疫21d后HPAIV的致死性攻击时,均100%被保护。【结论】鸡痘病毒ORF73和ORF214编码蛋白具有IL-18结合蛋白抑制IFN产生的功能,虽然缺失株和亲本株重组鸡痘病毒在细胞和体液免疫应答存在一定差异,但在SPF鸡均能诱导产生良好的免疫保护。     

Robust intrapulmonary CD8 T cell responses and protection with an attenuated N1L deleted vaccinia virus

Background

Vaccinia viruses have been used as a model for viral disease and as a protective live vaccine.

Methodology and Principal Findings

We investigated the immunogenicity of an attenuated strain of vaccinia virus engineered to inactivate the N1L gene (vGK5). Using the intranasal route, this recombinant virus was 2 logs less virulent compared to the wildtype VACV-WR. Infection by the intranasal, intraperitoneal, and tail scarification routes resulted in the robust induction of cytolytic virus-specific CD8 T cells in the spleens and the lungs. VACV-specific antibodies were also detected in the sera of mice infected 3–5 months prior with the attenuated vGK5 virus. Finally, mice immunized with vGK5 were significantly protected when challenged with a lethal dose of VACV-WR.

Conclusions

These results indicate that the attenuated vGK5 virus protects against subsequent infection and suggest that the N1L protein limits the strength of the early antiviral CD8 T cell response following respiratory infection.     

Large-scale preparation of biologically active measles virus haemagglutinin expressed by attenuated vaccinia virus vectors

A procedure described here allows the efficient and rapid purification of histidine-tagged measles virus haemagglutinin that is synthesized under the control of powerful promoters (PSFJ1-10 and PSFJ2-16) of the highly attenuated vaccinia virus (VV) strain LC16mO. A single affinity chromatography step purifies recombinant haemagglutinin proteins from the lysates of cells infected with the recombinant VVs. The recovery and purity are both very high (a yield of 0.5-2.8 mg/10(8) cells and purity of >94-98%), indicating that this procedure is approximately 400 times more efficient than the conventional methods used to prepare haemagglutinin. The haemagglutinins are correctly transported to the cell surface and have haemadsorption activity. Moreover, the recombinant haemagglutinin proteins cooperate with the measles virus fusion protein to elicit cell fusion activity. In addition, the antibody titres against measles virus, as measured by enzyme-linked immunosorbent assay using the purified haemagglutinin as the capture antigen, correlated closely with neutralization test titres (R(2) = 0.84, p < 0.05), indicating the preservation of immunologically relevant antigenicity. Such recombinant haemagglutinin preparations will be useful in diagnostic tests that measure functional anti-measles immunity and investigate the biological functions and structure of the haemagglutinin.     

Induction of potent immune responses by cationic microparticles with adsorbed human immunodeficiency virus DNA vaccines

The effectiveness of cationic microparticles with adsorbed DNA at inducing immune responses was investigated in mice, guinea pigs, and rhesus macaques. Plasmid DNA vaccines encoding human immunodeficiency virus (HIV) Gag and Env adsorbed onto the surface of cationic poly(lactide-coglycolide) (PLG) microparticles were shown to be substantially more potent than corresponding naked DNA vaccines. In mice immunized with HIV gag DNA, adsorption onto PLG increased CD8(+) T-cell and antibody responses by approximately 100- and approximately 1,000-fold, respectively. In guinea pigs immunized with HIV env DNA adsorbed onto PLG, antibody responses showed a more rapid onset and achieved markedly higher enzyme-linked immunosorbent assay and neutralizing titers than in animals immunized with naked DNA. Further enhancement of antibody responses was observed in animals vaccinated with PLG/DNA microparticles formulated with aluminum phosphate. The magnitude of anti-Env antibody responses induced by PLG/DNA particles was equivalent to that induced by recombinant gp120 protein formulated with a strong adjuvant, MF-59. In guinea pigs immunized with a combination vaccine containing HIV env and HIV gag DNA plasmids on PLG microparticles, substantially superior antibody responses were induced against both components, as measured by onset, duration, and titer. Furthermore, PLG formulation overcame an apparent hyporesponsiveness of the env DNA component in the combination vaccine. Finally, preliminary data in rhesus macaques demonstrated a substantial enhancement of immune responses afforded by PLG/DNA. Therefore, formulation of DNA vaccines by adsorption onto PLG microparticles is a powerful means of increasing vaccine potency.     

Induction of cellular immune responses to simian immunodeficiency virus gag by two recombinant negative-strand RNA virus vectors

A recombinant Newcastle disease virus (rNDV) expressing simian immunodeficiency virus (SIV) Gag protein (rNDV/SIVgag) was generated. The rNDV/SIVgag virus induced Gag-specific cellular immune responses in mice, leading to a specific anti-Gag antiviral immunity. This was evidenced by the inhibition of growth of recombinant vaccinia virus expressing an identical Gag antigen (rVac/SIVgag) but not of wild-type vaccinia virus in rNDV/SIVgag-immunized mice. Among intravenous, intraperitoneal, or intranasal immunization routes, intranasal administration induced the strongest protective response against challenge with rVac/SIVgag. We further demonstrated that these immune responses were greatly enhanced after booster immunization with recombinant influenza viruses expressing immunogenic portions of SIV Gag. The magnitude of the protective immune response correlated with the levels of cellular immune responses to Gag, which were still evident 9 weeks after immunization. These results suggest that rNDV and influenza virus vectors are suitable candidate vaccines against AIDS as well as against other infectious diseases.     

Induction of anti-HIV-1 immune responses by retroviral vectors.

Retroviral vectors encoding HIV-1 proteins, in particular, the envelope from HIV-1 IIIB, have been constructed and used to generate infectious vector particles. Murine cells transduced with these vectors express HIV proteins. Vector-transduced cells, when injected into syngeneic BALB/c mice, induce potent CD8+, class I MHC-restricted cytotoxic T-lymphocyte responses and elicit the production of neutralizing antibody specific for HIV-1. The induction of similar responses in primates may provide the basis for considering the use of these vectors as immunostimulants in humans. The retroviral vectors or vector-transduced cells would probably be first employed as an immunotherapeutic for HIV-infected individuals.     

Induction of CD8+ T-lymphocyte responses to a secreted antigen of Mycobacterium tuberculosis by an attenuated vaccinia virus

Protective immunity against Mycobacterium tuberculosis infection requires the activation of mycobacterium-specific CD8+ T cells, as well as CD4+ T cells. Therefore, optimizing strategies that stimulate CD8+ T cells recognizing dominant mycobacterial antigens, including secreted proteins, may lead to the development of more effective vaccines against tuberculosis. To generate a viral vaccine that is safe in humans, the early secreted protein, MPT64, was expressed in the attenuated vaccinia virus (VV) strain, modified vaccinia virus Ankara (MVA-64). The immunogenicity of MVA-64 was compared with that of the Western Reserve strain of VV (VVWR-64). The replication-defective MVA-64 was as efficient as VVWR-64 in inducing specific antibodies and cytolytic T-cell responses to a defined H-2-Db-restricted epitope on MTP-64. In addition, priming with MPT64-expressing plasmid DNA (DNA-64), and boosting with either MVA-64 or VVWR-64, markedly enhanced MPT64-specific cytolytic and IFN-gamma-producing CD8+ T-cell responses. These findings suggest that MVA may be a suitable vaccine carrier for stimulating mycobacterium-specific CD8+ T-cell responses and may be particularly relevant for developing vaccines for use in regions endemic for tuberculosis and HIV infection.     

Induction of positive cellular and humoral immune responses by a prime-boost vaccine encoded with simian immunodeficiency virus gag/pol

It is believed likely that immune responses are responsible for controlling viral load and infection. In this study, when macaques were primed with plasmid DNA encoding SIV gag and pol genes (SIVgag/pol DNA) and then boosted with replication-deficient vaccinia virus DIs recombinant expressing the same genes (rDIsSIVgag/pol), this prime-boost regimen generated higher levels of Gag-specific CD4+ and CD8+ T cell responses than did either SIVgag/pol DNA or rDIsSIVgag/pol alone. When the macaques were i.v. challenged with pathogenic simian/HIV, the prime-boost group maintained high CD4+ T cell counts and reduced plasma viral loads up to 30 wk after viral challenge, whereas the rDIsSIVgag/pol group showed only a partial attenuation of the viral infection, and the group immunized with SIVgag/pol DNA alone showed none at all. The protection levels were better correlated with the levels of virus-specific T cell responses than the levels of neutralization Ab responses. These results demonstrate that a vaccine regimen that primes with DNA and then boosts with a replication-defective vaccinia virus DIs generates anti-SIV immunity, suggesting that it will be a promising vaccine regimen for HIV-1 vaccine development.     

Dirofilaria immitis: cell-mediated and humoral immune responses in experimentally-infected dogs.

Four dogs were experimentally infected with 30 Dirofilaria immitis infective larvae, four dogs received two such infections and four dogs served as uninfected controls. A partially-purified D. immitis antigen was used in an indirect hemagglutination assay to determine anti-D. immitis antibody titers. Anti-D. immitis antibody was first detected in infected dogs 4 weeks after infection. Titers were highest 2 weeks after the appearance of microfilariae and diminished to low levels thereafter in the single infection group. Antibody levels in the double infection group decreased similarly but were demonstrable throughout the study. Antibody titers were significantly higher in the infected dogs, but there were no differences in titers between single and double infection groups.The responses of peripheral lymphocytes to phytohemagglutinin P and pokeweed mitogen were significantly depressed in infected dogs. Peripheral blood lymphocyte transformation could not be induced with D. immitis antigens. Differences between groups in T-cell function were not demonstrated by total hemagglutinating antibody or 2-mercaptoethanol labile hemagglutinating antibody following immunization with sheep erythrocytes.     

Induction of potent antitumor CTL responses by recombinant vaccinia encoding a melan-A peptide analogue

There is considerable interest in the development of vaccination strategies that would elicit strong tumor-specific CTL responses in cancer patients. One strategy consists of using recombinant viruses encoding amino acid sequences corresponding to natural CTL-defined peptide from tumor Ags as immunogens. However, studies with synthetic tumor antigenic peptides have demonstrated that introduction of single amino acid substitutions may dramatically increase their immunogenicity. In this study we have used a well-defined human melanoma tumor Ag system to test the possibility of translating the immunological potency of synthetic tumor antigenic peptide analogues into recombinant vaccinia viruses carrying constructs with the appropriate nucleotide substitutions. Our results indicate that the use of a mutated minigene construct directing the expression of a modified melanoma tumor Ag leads to improved Ag recognition and, more importantly, to enhanced immunogenicity. Thus, recombinant vaccinia viruses containing mutated minigene sequences may lead to new strategies for the induction of strong tumor-specific CTL responses in cancer patients.     

Role of genes that modulate host immune responses in the immunogenicity and pathogenicity of vaccinia virus

Poxvirus vaccine vectors, although capable of eliciting potent immune responses, pose serious health risks in immunosuppressed individuals. We therefore constructed five novel recombinant vaccinia virus vectors which contained overlapping deletions of coding regions for the B5R, B8R, B12R, B13R, B14R, B16R, B18R, and B19R immunomodulatory gene products and assessed them for both immunogenicity and pathogenicity. All five of these novel vectors elicited both cellular and humoral immunity to the inserted HIV-BH10 env comparable to that induced by the parental Wyeth strain vaccinia virus. However, deletion of these immunomodulatory genes did not increase the immunogenicity of these vectors compared with the parental vaccinia virus. Furthermore, four of these vectors were slightly less virulent and one was slightly more virulent than the Wyeth strain virus in neonatal mice. Attenuated poxviruses have potential use as safer alternatives to current replication-competent vaccinia virus. Improved vaccinia virus vectors can be generated by deleting additional genes to achieve a more significant viral attenuation.     

Differential activation of innate immune responses by adenovirus and adeno-associated virus vectors

Adenovirus vectors induce acute inflammation of infected tissues due to activation of the innate immune system and expression of numerous chemokines and cytokines in transduced target cells. In contrast, adeno-associated virus (AAV) vectors are not associated with significant inflammation experimentally or clinically. We tested the ability of AAV vectors to induce the expression of chemokines in vitro and to activate the innate immune system in vivo. In human HeLa cells and murine renal epithelium-derived cells (REC cells) the adenovirus vector AdlacZ induced the expression of multiple inflammatory chemokines including RANTES, interferon-inducible protein 10 (IP-10), interleukin-8 (IL-8), MIP-1beta, and MIP-2 in a dose-dependent manner. The use of AAVlacZ did not induce the expression of these chemokines above baseline levels despite 40-fold-greater titers than AdlacZ and greater amounts of intracellular AAVlacZ genomes according to Southern and slot blot analysis. This finding confirmed that the lack of AAVlacZ induction of chemokine was not due to reduced transduction. In DBA/2 mice, the intravenous administration of 2.5 x 10(11) particles of AAVlacZ resulted in the rapid induction of liver tumor necrosis factor alpha (TNF-alpha), RANTES, IP-10, MIP-1beta, MCP-1, and MIP-2 mRNAs. However, 6 h following injection, chemokine mRNA levels returned to baseline. As expected, administration of 10-fold less AdlacZ caused an induction of liver TNF-alpha and chemokine mRNAs that persisted for more than 24 h posttransduction. Whereas intravenous administration of 2.5 x 10(11) particles of AAVlacZ triggered a transient infiltration of neutrophils and CD11b(+) cells into liver, this response stood in contrast to widespread inflammation and toxicity induced by AdlacZ. Kupffer cell depletion abolished AAVlacZ but not AdlacZ-induced chemokine expression and neutrophil infiltration. In summary, these results show that AAV vectors activate the innate immune system to a lesser extent than do adenovirus vectors and offer a possible explanation for the reduced inflammatory properties of AAV compared to adenovirus vectors.     

Induction of humoral immune responses following vaccination with envelope-containing, formaldehyde-treated, thermally inactivated human immunodeficiency virus type 1

The lack of success of subunit human immunodeficiency virus type 1 (HIV-1) vaccines to date suggests that multiple components or a complex virion structure may be required. We previously demonstrated retention of the major conformational epitopes of HIV-1 envelope following thermal treatment of virions. Moreover, antibody binding to some of these epitopes was significantly enhanced following thermal treatment. These included the neutralizing epitopes identified by monoclonal antibodies 1b12, 2G12, and 17b, some of which have been postulated to be partially occluded or cryptic in native virions. Based upon this finding, we hypothesized that a killed HIV vaccine could be derived to elicit protective humoral immune responses. Shedding of HIV-1 envelope has been described for some strains of HIV-1 and has been cited as one of the major impediments to developing an inactivated HIV-1 vaccine. In the present study, we demonstrate that treatment of virions with low-dose formaldehyde prior to thermal inactivation retains the association of viral envelope with virions. Moreover, mice and nonhuman primates vaccinated with formaldehyde-treated, thermally inactivated virions produce antibodies capable of neutralizing heterologous strains of HIV in peripheral blood mononuclear cell-, MAGI cell-, and U87-based infectivity assays. These data indicate that it is possible to create an immunogen by using formaldehyde-treated, thermally inactivated HIV-1 virions to induce neutralizing antibodies. These findings have broad implications for vaccine development.     

Biochemical relationship of thymocyte mitogenic factor and factors enhancing humoral and cell-mediated immune responses.

Supernatants from concanavalin A-induced mouse spleen cells contain soluble factors which: 1) augment the anti-sheep erythrocyte hemolytic plaque-forming cell response of nude mouse spleen cells, 2) facilitate the generation of antigen-specific cytotoxic cells by alloantigen-stimulated thymocytes, and 3) enhance the mitogenic response of thymocytes in the presence or absence of phytohemagglutinin. The uninduced control spleen cell supernatants contained variable but significantly less biologic activity within the difference assay systems. The principle enhancing activity (apparent m.w. 35,000 to 38,000) in each of the assay systems was found to be inseparable after a multiple step purification scheme involving ammonium sulfate precipitation, two-cycle gel filtration, hydroxylapatite chromatography, and hydrophobic chromatography on Phenyl Sepharose. These results indicate that either a single factor (designated thymocyte mitogenic factor) augments both humoral and cell-mediated immune responses or, alternatively, the different biologic activities are mediated by different factors that share a common core structure.     

Induction of humoral and cellular immune responses in mice by a recombinant fowlpox virus expressing the E2 protein of bovine viral diarrhea virus

A recombinant fowlpox virus (rFPV/E2) expressing the E2 protein of bovine viral diarrhea virus (BVDV) was constructed and characterized. Mice were immunized with recombinant virus and both humoral and cellular immune responses were studied. rFPV/E2 induced BVDV-specific antibodies which were detected by ELISA. In addition, mouse sera were shown to neutralize BVDV. A cytokine ELISA assay revealed that mice vaccinated with rFPV/E2 induced 7-fold more interferon-gamma than parental fowlpox virus.     

Vaccination with a feline immunodeficiency virus multiepitopic peptide induces cell-mediated and humoral immune responses in cats, but does not confer protection.

Cats were immunized with a 46-residue multiepitopic synthetic peptide of feline immunodeficiency virus (FIV) comprising immunodominant epitopes present in the third variable domain of the envelope glycoprotein, transmembrane glycoprotein (TM), and p24 Gag core protein, using Quil A as an adjuvant. All vaccinated cats developed a humoral response which recognized the synthetic peptide immunogen and the intact viral core and envelope proteins. A FIV Gag- and Env-specific effector cytotoxic T-lymphocyte response was also detected in the peripheral blood of vaccinated cats, which peaked at week 30. This response appeared to be major histocompatibility complex restricted. Epitope mapping studies revealed that both the cellular and humoral immune responses were directed principally to a peptide within the TM glycoprotein, CNQNQFFCK. However, vaccination did not confer protection when cats were challenged with the Petaluma isolate of FIV at week 35.     

Cross-protective immune responses induced in rhesus macaques by immunization with attenuated macrophage-tropic simian immunodeficiency virus.

The simian immunodeficiency virus (SIV) macaque model of AIDS has provided a valuable system with which to investigate vaccine approaches for protection against human immunodeficiency virus type 1 (HIV-1) infection. In particular, the ability of macaques persistently infected with attenuated infectious molecular clones of SIV to resist challenge with the pathogenic parental swarm has conclusively demonstrated that protective immunity can be achieved by immunization prior to exposure. The breadth of these protective responses and the immunological correlates of protection, however, have not been identified. In addition, vaccine studies have mainly employed lymphocyte-tropic strains of HIV-1 and SIV. Recent studies have implicated macrophage-tropic strains in the transmission of HIV-1 and have suggested that these virus strains should be examined in vaccine strategies. Macrophage-tropic viruses may confer additional advantages in the induction of protective immunity by replication in antigen-presenting cells. In this study, the immune response of rhesus macaques inoculated with an attenuated macrophage-tropic recombinant of SIVmac239 (SIV/17E-Cl) was evaluated with respect to protective immunity by heterologous challenge at various times after infection. Vigorous type-specific neutralizing-antibody responses restricted to SIV/17E-Cl were evident by 2 weeks postinfection. By 7 months, however, cross-reactive neutralizing antibodies emerged which neutralized not only SIV/17E-Cl but also the heterologous primary isolate SIV/DeltaB670. Challenge of SIV/17E-Cl-infected monkeys with SIV/DeltaB670 at various times postinfection demonstrated that protective responses were associated with the appearance of cross-reactive neutralizing antibodies. Furthermore, passive transfer of sera from SIV/17E-Cl-infected animals passively protected two of four naive recipients.