Immunogenicity analysis following human immunodeficiency virus recombinant DNA and recombinant vaccinia virus Tian Tan prime-boost immunization

This study assessed and compared the immunogenicity of various immunization strategies in mice using combinations of recombinant DNA(pCCMp24) and recombinant attenuated vaccinia virus Tian Tan(rddVTT-CCMp24).Intramuscular immunization was performed on days 0(prime) and 21(boost).The immunogenicity of the vaccine schedules was determined by measuring human immunodeficiency virus(HIV)-specific binding antibody levels and cytokine(interleukin-2 and interleukin-4) concentrations in peripheral blood,analyzing lymphocyte proliferation capacity against HIV epitopes and CD4 + /CD8 + cell ratio,and monitoring interferon-gamma levels at different times post-immunization.The results showed that pCCMp24,rddVTT-CCMp24 and their prime-boost immunization induced humoral and cellular immune responses.The pCCMp24/rddVTT-CCMp24 immunization strategy increased CD8 + T cells and induced more IFN-γ-secreting cells compared with single-shot rDNA.The prime-boost immunization strategy also induced the generation of cellular immunological memory to HIV epitope peptides.These results demonstrated that prime-boost immunization with rDNA and rddVTT-CCMp24 had a tendency to induce greater cellular immune response than single-shot vaccinations,especially IFN-γ response,providing a basis for further studies.     

含H5N1多个结构抗原的DNA疫苗与重组痘苗病毒疫苗联合免疫小鼠可明显增强免疫原性

本研究旨在研发经济、高效的人高致病性禽流感病毒H5N1实验疫苗并优化免疫方案。利用本实验室前期构建的含有H5N1(安徽株)结构基因的多个单顺反子(HAop和NAop)和双顺反子(HAop/M2和NAop/M1)DNA疫苗及重组痘苗病毒(天坛株)疫苗,采用不同方案(单独或联合)免疫BALB/c小鼠,初步分析了抗原特异性体液免疫(HA血凝抑制抗体,NA特异性抗体,中和抗体及M1与M2特异性抗体)和细胞免疫应答(IFN-γELIS-pot)的特点。结果表明:DNA疫苗与重组痘苗病毒(天坛株)疫苗联合免疫可以激发较强的多个抗原特异的免疫应答,尤其是体液免疫应答,明显优于DNA疫苗或重组痘苗病毒(天坛株)疫苗单独免疫;联合免疫方案中DNA疫苗初免所激发的HA与NA特异的体液免疫应答强于重组痘苗病毒(天坛株)疫苗初免,然而M1与M2特异的体液免疫应答则反之。本研究为新型H5N1疫苗的研发及免疫方案的优化奠定了基础。     

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

Background  

Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol.     

Identification and characterization of novel recombinant vaccine antigens for immunization against genital Chlamydia trachomatis

Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection worldwide, with over 91 million cases estimated annually. An effective subunit vaccine against Chlamydia may require a multivalent subunit cocktail of antigens in a single formulation for broad coverage of a heterogeneous major histocompatibility complex population. Herein, we describe the identification of novel C. trachomatis antigens by CD4+ and CD8+ T-cell expression cloning, serological expression cloning, and an in silico analysis of the C. trachomatis genome. These antigens elicited human CD4+ T-cell responses, and a subset proved to be immunogenic and protective when administered as immunoprophylactic vaccines against C. trachomatis challenge. Candidate vaccines consisting of the prioritized C. trachomatis antigens adjuvanted in a GlaxoSmithKline proprietary AS01B adjuvant were prioritized based on induction of solid protection against challenge in C57BL/6 and BALB/c mice with C. trachomatis . Some of the vaccines prevented bacterial shedding and colonization of the upper genital tract to varying degrees by mechanisms that may include CD4+ T cells.     

Immunogenicity and growth inhibitory efficacy of the prime-boost immunization regime with DNA followed by recombinant vaccinia virus carrying the P29 gene of Babesia gibsoni in dogs

In recent studies, heterologous prime-boost approaches, employing plasmid DNA and viral vector pathogen-delivering sequences, have been considered an effective protection strategy for intracellular parasite infections. Here, we evaluated the efficacy of such a strategy against the canine Babesia gibsoni infection. The DNA (pCAGGS-P29) and recombinant vaccinia virus (vvP29) both encoding the P29 of B. gibsoni were used in this study. The dogs were immunized 3 times with priming DNA and boosted once with recombinant virus. The dogs immunized with P29 developed a significant level of IgG2 antibody against P29. The response was strongly boosted by the inoculation of vvP29. The peripheral IFN-gamma responses of the dogs immunized with P29 were significantly higher than those of controls after the parasite inoculation. Moreover, the P29 immunized group showed a significantly low level of parasitemia. In conclusion, this study supports the efficacy of a prime-boost strategy for dogs against canine B. gibsoni infection.     

Parenteral and mucosal prime-boost immunization strategies in mice with hepatitis B surface antigen and CpG DNA

Synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG ODN) are potent adjuvants to protein antigens administered by parenteral or mucosal routes to BALB/c mice. To date, there have been no studies using combined parenteral/mucosal approaches with CpG DNA as adjuvant. In this study we evaluated different parenteral prime-mucosal boost and mucosal prime-parenteral boost strategies using hepatitis B surface antigen (HBsAg) alone or with different adjuvants: aluminum hydroxide (alum), cholera toxin (CT), CpG ODN. In addition, since CpG ODN has previously been shown to act synergistically with other adjuvants after parenteral or mucosal delivery, we also evaluated adjuvant combinations: alum+CpG ODN and CT+CpG ODN. The effects of adjuvant and administration strategy on systemic and mucosal humoral responses were measured, as well as cell-mediated immune responses (cytotoxic T lymphocyte activity). These results were compared to parenteral only or mucosal only strategies. Our findings demonstrate that parenteral immunization can prime for mucosal responses even when different lymph nodes were being targeted. HBsAg-specific immune responses (IgG in plasma, cytotoxic T lymphocytes) induced by parenteral prime could all be significantly enhanced by mucosal boosting and despite the fact that intramuscular immunization alone could not induce mucosal IgA, it could prime for a subsequent mucosal boost. In addition, the presence of adjuvant at time of boosting could influence the nature of subsequent immune responses (Th1 vs. Th2). Mice primed intranasally could have their systemic immune responses boosted with a parenteral administration and it was also possible to enhance mucosal responses induced by intranasal prime with an intramuscular boost.     

A novel particulate influenza vaccine induces long-term and broad-based immunity in mice after oral immunization.

The immunogenicity of a novel particulate oral influenza vaccine was examined in terms of antibody response and protection in mice. Oral immunization with chicken erythrocytes (CRBC) adsorbed with gamma-irradiated influenza A virus induced high levels of immunoglobulin G antibodies and protection in the lung compared with gamma-irradiated virus administered alone or CRBC. Immunoglobulin A antibodies were the predominant antibodies in nasal washings, and their presence did not correlate with protection as well as immunoglobulin G antibodies. Immunity was not specific for the immunizing virus subtype, as antibodies and enhanced lung clearance of virus were demonstrated with different virus subtypes. However, mice were not protected when challenged with live influenza B virus. The antibody response and the degree of protection were dependent on both the concentration of virus adsorbed to CRBC and number of CRBC adsorbed to virus. Virus-adsorbed CRBC given subcutaneously failed to induce antibodies or protection. Oral immunization with A/Qld/6/72 (H3N2) virus gave a high level of protection over 12 weeks, which could be demonstrated with different subtypes. Protection correlated with antibody levels in the lung determined by both enzyme-linked immunosorbent and hemagglutination inhibition assays, although the levels detected by the latter declined over time.     

Class II restriction in mice to the malaria candidate vaccine ring infected erythrocyte surface antigen (RESA) as synthetic peptides or as expressed in recombinant vaccinia

The immune response to three peptides corresponding to the repeat regions of the malaria candidate vaccine ring infected E surface Ag (RESA) were studied. Both antibody responses and lymphocyte stimulation in mice injected with these peptides without carrier were found to be restricted to certain MHC class II haplotypes. Mice bearing IAk were strong responders to all three peptides. Mice bearing IAd were strong responders only to the 3' repeat peptides, the octamer and tetramer. Mice bearing Is or Iq did not respond to any repeat peptides. Remarkably, the pattern of genetic restriction of the antibody response to the entire RESA as expressed in vaccinia indicated that there were no other epitopes besides the three repeats. Because only one class II haplotype (i.e., k) out of five tested responded strongly to this peptide and only two out of five (i.e., k and d) responded to the octamer or tetramer, it may be difficult to achieve a good immune response against RESA in most or all humans.     

Immunocontraception in mice using repeated, multi-antigen peptides: immunization with purified recombinant antigens

Two immunocontraceptive antigens (AgE and AgF) were constructed that included different combinations of highly species-specific peptides from the mouse reproductive antigens SP56, ZP3, ZP2, and ZP1 in the form of multi-antigen peptides (MAPs). Both AgE and AgF contained three tandem repeats each of ZP2 and ZP3 peptide epitopes and a single copy of a ZP1 peptide sequence all of which had previously been demonstrated to individually have immunodominant or contraceptive effects. In addition, AgF contained a single contraceptive peptide derived from SP56, the putative ZP3 receptor protein on sperm. The antigens were expressed and affinity purified as recombinant repeated multi-antigen (polyepitope) peptides using an Escherichia coli maltose binding protein (MBP) expression system. Female BALB/c mice actively immunized with these antigens in Freund's adjuvants produced variable serum antibody responses to the component peptides. Fertility rates for animals immunized with AgE (40%) and AgF (20%) were significantly reduced compared to MBP immunized mice (90%), but the reduction in fertility did not correlate with peptide-specific serum antibody levels. Ovaries from all immunized mice appeared histologically normal with no evidence of oophoritis. These results demonstrate that high levels of immunocontraception can be achieved in mice, without apparent side-effects, using species-specific immunogens that include repeated peptides from proteins involved in fertilization.     

含乙型肝炎病毒S-PreS1 基因的DNA疫苗与蛋白颗粒疫苗联合免疫可增强免疫应答

为研制新型有效的HBV治疗性疫苗,构建了含PreS1与S融合基因的HBV DNA疫苗,即pVRC-HBSS1 (PreS1 21–47 aa融合在S抗原1–223的羧基端),并制备了CHO表达相同结构的蛋白颗粒亚单位疫苗HBSS1。在Balb/C小鼠中采用不同的DNA免疫方式 (即肌肉注射、皮内注射加电转) 初免3次,蛋白颗粒亚单位疫苗 (不同佐剂) 肌肉注射加强免疫1次,然后我们分析比较了各组疫苗所引起的免疫应答特点。抗体检测结果表明：皮内注射结合电转初免组产生的PreS1与 S特异性抗体水平皆高于肌肉直接注射组。进一步还发现DNA疫苗与蛋白颗粒亚单位疫苗两种疫苗联合应用后S抗原特异的细胞免疫应答 (IFN-γ ELISpot分析) 明显高于DNA疫苗或蛋白颗粒亚单位单独应用,其中皮内注射+电转结合蛋白颗粒亚单位疫苗联合免疫组可产生最强的细胞免疫应答。这些研究为新型HBV 治疗性疫苗的优化设计与合理应用提供了依据。     

Immunogenicity of heterologous prime-boost regimens involving recombinant adenovirus serotype 11 (Ad11) and Ad35 vaccine vectors in the presence of anti-ad5 immunity

The high prevalence of preexisting immunity to adenovirus serotype 5 (Ad5) in human populations will likely limit the immunogenicity and clinical utility of recombinant Ad5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 and other pathogens. A potential solution to this problem is to utilize rAd vaccine vectors derived from rare Ad serotypes such as Ad35 and Ad11. We have previously reported that rAd35 vectors were immunogenic in the presence of anti-Ad5 immunity, but the immunogenicity of heterologous rAd prime-boost regimens and the extent that cross-reactive anti-vector immunity may limit this approach have not been fully explored. Here we assess the immunogenicity of heterologous vaccine regimens involving rAd5, rAd35, and novel rAd11 vectors expressing simian immunodeficiency virus Gag in mice both with and without anti-Ad5 immunity. Heterologous rAd prime-boost regimens proved significantly more immunogenic than homologous regimens, as expected. Importantly, all regimens that included rAd5 were markedly suppressed by anti-Ad5 immunity. In contrast, rAd35-rAd11 and rAd11-rAd35 regimens elicited high-frequency immune responses both in the presence and in the absence of anti-Ad5 immunity, although we also detected clear cross-reactive Ad35/Ad11-specific humoral and cellular immune responses. Nevertheless, these data suggest the potential utility of heterologous rAd prime-boost vaccine regimens using vectors derived from rare human Ad serotypes.     

Suppression of hyperglycemia in NOD mice after inoculation with recombinant vaccinia viruses

In autoimmune (type 1) diabetes, autoreactive lymphocytes destroy pancreatic β-cells responsible for insulin synthesis. To assess the feasibility of gene therapy for type 1 diabetes, recombinant vaccinia virus (rVV) vectors were constructed expressing pancreatic islet autoantigens proinsulin (INS) and a 55-kDa immunogenic peptide from glutamic acid decarboxylase (GAD), and the immunomodulatory cytokine interleukin (IL)-10. To augment the beneficial effects of recombinant virus therapy, the INS and GAD genes were fused to the C terminus of the cholera toxin B subunit (CTB). Five-week-old non-obese diabetic (NOD) mice were injected once with rVV. Humoral antibody immune responses and hyperglycemia in the infected mice were analyzed. Only 20% of the mice inoculated with rVV expressing the CTB::INS fusion protein developed hyperglycemia, in comparison to 70% of the mice in the uninoculated animal group. Islets from pancreatic tissues isolated from euglycemic mice from this animal group showed no sign of inflammatory lymphocyte invasion. Inoculation with rVV producing CTB::GAD or IL-10 was somewhat less effective in reducing diabetes. Humoral antibody isotypes of hyperglycemic and euglycemic mice from all treated groups possessed similar IgG1/IgG2c antibody titer ratios from 19 to 32 wk after virus inoculation. In comparison with uninoculated mice, 11-wk-old NOD mice injected with virus expressing CTB::INS were delayed in diabetes onset by more than 4 wk. The experimental results demonstrate the feasibility of using rVV expressing CTB::INS fusion protein to generate significant protection and therapy against type 1 diabetes onset and progression.     

Expansion and diversification of virus-specific T cells following immunization of human immunodeficiency virus type 1 (HIV-1)-infected individuals with a recombinant modified vaccinia virus Ankara/HIV-1 Gag vaccine

Affordable therapeutic strategies that induce sustained control of human immunodeficiency virus type 1 (HIV-1) replication and are tailored to the developing world are urgently needed. Since CD8(+) and CD4(+) T cells are crucial to HIV-1 control, stimulation of potent cellular responses by therapeutic vaccination might be exploited to reduce antiretroviral drug exposure. However, therapeutic vaccines tested to date have shown modest immunogenicity. In this study, we performed a comprehensive analysis of the changes in virus-specific CD8(+) and CD4(+) T-cell responses occurring after vaccination of 16 HIV-1-infected individuals with a recombinant modified vaccinia virus Ankara-vectored vaccine expressing the consensus HIV-1 clade A Gag p24/p17 sequences and multiple CD8(+) T-cell epitopes during highly active antiretroviral therapy. We observed significant amplification and broadening of CD8(+) and CD4(+) gamma interferon responses to vaccine-derived epitopes in the vaccinees, without rebound viremia, but not in two unvaccinated controls followed simultaneously. Vaccine-driven CD8(+) T-cell expansions were also detected by tetramer reactivity, predominantly in the CD45RA(-) CCR7(+) or CD45RA(-) CCR7(-) compartments, and persisted for at least 1 year. Expansion was associated with a marked but transient up-regulation of CD38 and perforin within days of vaccination. Gag-specific CD8(+) and CD4(+) T-cell proliferation also increased postvaccination. These data suggest that immunization with MVA.HIVA is a feasible strategy to enhance potentially protective T-cell responses in individuals with chronic HIV-1 infection.     

Differential immunogenicity of various heterologous prime-boost vaccine regimens using DNA and viral vectors in healthy volunteers

Heterologous prime-boost vaccination has been shown to be an efficient way of inducing T cell responses in animals and in humans. We have used three vaccine vectors, naked DNA, modified vaccinia virus Ankara (MVA), and attenuated fowlpox strain, FP9, for prime-boost vaccination approaches against Plasmodium falciparum malaria in humans. In this study, we characterize, using two types of ELISPOT assays and FACS analysis, cell-mediated immune responses induced by different prime-boost combinations where all vectors encode a multiepitope string fused to the pre-erythrocytic Ag thrombospondin-related adhesion protein. We show that these different vectors need to be used in a specific order for an optimal ex vivo IFN-gamma response. From the different combinations, DNA priming followed by MVA boosting and FP9 priming followed by MVA boosting were most immunogenic and in both cases the IFN-gamma response was of broad specificity and cross-reactive against two P. falciparum strains (3D7 and T9/96). Immunization with all three vectors showed no improvement over optimal two vector regimes. Strong ex vivo IFN-gamma responses peaked 1 wk after the booster dose, but cultured ELISPOT assays revealed longer-lasting T cell memory responses for at least 6 mo. In the DNA-primed vaccinees the IFN-gamma response was mainly due to CD4(+) T cells, whereas in the FP9-primed vaccinees it was mainly due to CD4-dependent CD8(+) T cells. This difference may be of importance for the protective efficacy of these vaccination approaches against various diseases.     

Influenza A virus nucleoprotein derived from Escherichia coli or recombinant vaccinia (Tiantan) virus elicits robust cross-protection in mice

Immunity to conserved viral antigens is an attractive approach to develop a universal vaccine against epidemic and pandemic influenza. A nucleoprotein (NP)-based vaccine has been explored and preliminary studies have shown promise. However, no study has explored the immunity and cross-protective efficacy of recombinant NP derived from Escherichia coli compared with recombinant vaccinia virus (Tiantan). Recombinant NP protein (rNP) from influenza virus A/Jingke/30/95(H3N2) was obtained from E. coli and recombinant vaccinia virus (Tiantan) RVJ1175NP. Purified rNP without adjuvant and RVJ1175NP were used to immunize BALB/c mice intramuscularly. Humoral immune responses were detected by ELISA, while cell-mediated immune responses were measured by ex vivo IFN-γ ELISPOT and in vivo cytotoxicity assays. The cross-protective efficacy was assessed by a challenge with a heterosubtype of influenza virus A/PR/8/34(H1N1). Our results demonstrate that a high dose (90 μg) of rNP induced NP-specific antibodies and T cell responses that were comparable with those of RVJ1175NP in mice. Importantly, the survival ratio (36, 73, and 78%) of the vaccinated mice after the influenza virus A/PR/8/34(H1N1) challenge was rNP vaccine dose-dependent (10, 30, and 90 μg, respectively), and no significant differences were observed between the rNP- and RVJ1175NP-immunized (91%) mice. Influenza A virus NP derived from E. coli or recombinant vaccinia (Tiantan) virus elicited cross-protection against influenza virus in mice, and the immune response and protective efficacy of rNP were comparable to RVJ1175NP. These data provide a basis for the use of prokaryotically expressed NP as a candidate universal influenza vaccine.     

Determination of functional effects of mutations in the steroid 21-hydroxylase gene (CYP21) using recombinant vaccinia virus

Steroid 21-hydroxylase (P450c21) is absent or defective in more than 90% of patients with congenital adrenal hyperplasia. This disorder of cortisol biosynthesis occurs in a wide spectrum of clinical severity; specific mutations in the 21-hydroxylase gene (CYP21) have been found in association with particular clinical phenotypes. To determine the functional effects of mutations causing amino acid substitutions, normal P450c21 and three mutagenized P450c21 enzymes were expressed at high levels in cultured COS-1 cells using recombinant vaccinia virus. A single amino acid substitution (Val281----Leu) present in patients with mild "nonclassical" 21-hydroxylase deficiency resulted in an enzyme with 20-50% of normal activity. A mutation (Ile172----Asn) identified in patients with the "simple virilizing" form (poor cortisol synthesis but adequate aldosterone synthesis) resulted in an enzyme with less than 2% of normal activity. Finally, a cluster mutation (Ile-Val-Glu-Met234-238----Asn-Glu-Glu-Lys) found in a patient with severe "salt wasting" 21-hydroxylase deficiency (inadequate aldosterone synthesis) results in an enzyme with no detectable activity. These data indicate that the severity of 21-hydroxylase deficiency correlates with the degree of enzymatic compromise.     

Additional evidence for the augmented induction of tumor-specific resistance in vaccinia virus-primed mice by immunization with vaccinia virus-modulated syngeneic tumor cells

The augmenting effect of vaccinia virus infection of tumor cells on induction of tumor-specific resistance was examined in mice. C3H/HeN mice were primed intraperitoneally (ip) with live vaccinia virus after whole-body irradiation with 250 rad of X-rays. Three weeks later the mice were immunized ip 3 times at weekly intervals with syngeneic murine hepatoma MH134 or spontaneous myeloma X5563 which had been infected in vitro with vaccinia virus and subsequently irradiated with 7000 rad of X-rays. One week after the third immunization, the mice were challenged with 1 X 10(5) viable cells of MH134 or X5563 ip or 1 X 10(6) tumor cells intradermally (id). On ip challenge with viable MH134 cells all mice that had not been pretreated died within 3 weeks due to ascites tumor out-growth, whereas all mice that had been vaccinia virus-primed and immunized with vaccinia virus-infected MH134 cells survived. On ip challenge with X5563 cells, the percentage survival of vaccinia virus-primed and vaccinia virus-modified tumor-immunized mice was 80%. On id challenge with MH134 and X5563 tumor cells, in un-treated mice tumors grew to more than 5 mm in diameter within 3 weeks, whereas 90% and 60%, respectively, of the mice that had been vaccinia virus-primed and immunized with vaccinia virus-infected tumor cells showed no tumor out-growth. Pretreatment by only immunization with vaccinia virus-infected cells or vaccinia virus-priming and immunization with virus non-infected tumor cells were not effective for preventing induction of tumor-resistance to either ip or id challenge with MH134 or X5563 tumor cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

重组腺病毒载体乙肝疫苗的免疫学研究

目的比较腺病毒(Adenovirus,Ad)载体乙型肝炎病毒(Hapotitis B virus,HBV)疫苗鼻腔黏膜、皮下注射接种小鼠免疫效果.方法以腺病毒为载体、以HBV preS2/S为目的基因构建重组疫苗,然后将重组疫苗分别鼻黏膜和皮下接种BALB/c小鼠,与阴性对照组(载体对照组、生理盐水对照组)比较接种重组疫苗35、72 d的免疫效果.结果鼻腔接种Ad HBV疫苗组动物在72 d时血清抗体终末效价为1/2 470,同时可提高NK细胞杀伤能力和干扰素-γ、IL-2含量升高.皮下注射Ad HBV在免疫35 d和72 d、1∶200血清稀释度均未测到阳性抗体(A值,P/N值＜2.1).结论重组疫苗黏膜接种诱导小鼠产生特异抗体和细胞免疫反应高于皮下接种途径.     

Development of a novel recombinant influenza vaccine in insect cells

Influenza is a highly contagious viral respiratory illness which is best prevented through vaccination. Currently, all US licensed influenza vaccines are produced in embryonated chicken eggs. The Baculovirus Expression Vector System (BEVS) technology offers several advantages over existing technology, including an exact match between the circulating virus and the antigen in the vaccine, speed, safety, versatility, and reliable scale-up. The expresSF+ insect cells are grown in the absence of serum and have been extensively qualified for safety according to ICH and US FDA guidance and for suitability for the production of recombinant proteins using BEVS. FluBlok^®, a recombinant hemagglutinin influenza vaccine, is composed of purified hemagglutinin protein produced using the BEVS technology. FluBlok has been shown to be safe, effective, and efficacious in human clinical studies.