A vaccine for hypertension based on virus-like particles: preclinical efficacy and phase I safety and immunogenicity

BACKGROUND: Despite the availability of efficacious drugs, the success of treating hypertension is limited by patients' inconsistent drug intake. Immunization against angiotensin II may offer a valuable alternative to conventional drugs for the treatment of hypertension, because vaccines induce relatively long-lasting effects and do not require daily dosing. Here we describe the preclinical development and the phase I clinical trial testing of a virus-like particle (VLP)-based antihypertensive vaccine. METHODS AND RESULTS: An angiotensin II-derived peptide was conjugated to the VLP Qbeta (AngQb). AngQb was highly immunogenic in mice and rats. To test for efficacy, spontaneously hypertensive rats (SHR) were immunized with 400 microg AngQb or VLP alone. Group mean systolic blood pressure (SBP) was reduced by up to 21 mmHg (159 +/- 2 versus 180 +/- 5 mmHg, P < 0.001), and total angiotensin II levels (antibody-bound and free) were increased ninefold (85 +/- 20 versus 9 +/- 1 pmol/l, P = 0.002) compared with VLP controls. SHR treated with the angiotensin-converting enzyme (ACE) inhibitor ramipril (1 mg/kg per day by mouth) reached an SBP of 155 +/- 2 mmHg. Twelve healthy volunteers of a placebo-controlled randomized phase I trial were injected once with 100 microg AngQb. Angiotensin II-specific antibodies were raised in all subjects (100% responder rate) and AngQb was well tolerated. CONCLUSIONS: AngQb reduces blood pressure in SHR to levels obtained with an ACE inhibitor, and is immunogenic and well tolerated in humans. Therefore, vaccination against angiotensin II has the potential to become a useful antihypertensive treatment providing long-lasting effects and improving patient compliance.     

人乳头瘤病毒16型表位嵌合病毒样颗粒的构建及免疫原性研究

目的 制备E7_49-57表位嵌合病毒样颗粒,并进行免疫原性分析。方法 利用分子模拟软件Discovery Studio预测HPV16 L1的E7_49-57最佳嵌合位点,将E7_49-57表位嵌入预测位点。构建pET28a-16L1-E7_49-57重组质粒,于大肠杆菌中诱导表达HPV16L1-E7_49-57蛋白并利用镍柱进行亲和层析纯化。于体外重组VLPs后,进行动态光散射粒径和透射电镜分析。用E7_49-57嵌合VLPs免疫小鼠,假病毒中和试验检测免疫血清中和抗体滴度。流式多因子法检测Th1和Th2型细胞因子水平。结果 HI loop区355/356为最佳表位嵌合位点。正确表达了HPV16 L1-E7_49-57蛋白并组装E7_49-57嵌合VLPs。E7_49-57嵌合VLPs免疫小鼠3次后,小鼠血清中和抗体滴度log10平均值达到4.23,略低于野生型VLPs(log10平均值为4.45)。但是,相对于野生型VLPs,E7_49-57嵌合VLPs诱导产生Th1型细胞因子(INF-γ, IL-2, TNF-α)的水平显著提高。结论 本研究制备的E7_49-57嵌合VLPs 能刺激机体同时产生较强的体液和细胞免疫应答,可能兼具预防和治疗双重功效,为宫颈癌治疗性疫苗的研制奠定基础。     

Comparison of whole gene and whole virus scrambled antigen approaches for DNA prime and fowlpox virus boost HIV type 1 vaccine regimens in macaques

T cell immunity plays a critical role in controlling HIV-1 viremia, and encoding a limited set of HIV-1 genes within DNA and poxvirus vectors can, when used sequentially, induce high levels of T cell immunity in primates. However, a limited breadth of T cell immunity exposes the host to potential infection with either genetically diverse HIV-1 strains or T cell escape variants of HIV-1. In an attempt to induce maximally broad immunity, we examined DNA and recombinant fowlpox virus (rFPV) vaccines encoding all HIV-1 genes derived from a global HIV-1 consensus sequence, but expressed as multiple overlapping scrambled 30-amino acid segments (scrambled antigen vaccines, or SAVINEs). Three groups of seven pigtail macaques were immunized with sets of DNA and rFPV expressing Gag/Pol antigens only, the whole genome SAVINE antigens, or no HIV-1 antigens and T cell immunity was monitored by ELISpot and intracellular cytokine staining. High levels of cross-subtype HIV-specific T cell immunity to Gag were consistently induced in the seven macaques primed with DNA and rFPV vaccines expressing Gag/Pol as intact proteins. It was, however, difficult to repeatedly boost immunity with further rFPV immunizations, presumably reflecting high levels of anti- FPV immunity. Unfortunately, this vaccine study did not consistently achieve a broadened level of T cell immunity to multiple HIV genes utilizing the novel whole-virus SAVINE approach, with only one of seven immunized animals generating broad T cell immunity to multiple HIV-1 proteins. Further refinements are planned with alternative vector strategies to evaluate the potential of the SAVINE technology.     

Interleukin-12 gene adjuvant increases the immunogenicity of virus-like particles of human papillomavirus type 16 regional variant strain

ObjectivesTo analyze the immunogenicity of virus-like particles (VLP) of human papillomavirus type 16 (HPV16) isolated in East China and the adjuvant potential of interleukin-12 (IL-12).MethodsThe variant HPV16 L1VLP expressed in sf9 insect cells were purified with cesium chloride gradient centrifugation. BALB/c mice were vaccinated with VLP (L1N), VLP with Freund's adjuvant (L1A) or VLP with IL-12 recombinant plasmid (L1P). HPV16 VLP specific IgG and IFN-γ level in the serum were detected by ELISA, and the percentage of CD4⁺ and CD8⁺ in spleen cells was detected with flow cytometry.ResultsThe titers of serum IgG antibodies in vaccinated groups were higher than in negative control and the serum antibodies mainly recognized conformation-dependent HPV16 VLP epitopes. Splenic CD4⁺ and CD8⁺ T cell subsets increased after vaccination in every experimental group, and CD8⁺ increased obviously in L1P group. The ratio of CD4⁺/CD8⁺ decreased in L1P group and increased in the other two groups, compared to control group. Vaccination induced specific secretion of IFN-γ in the serum of vaccinated group (p < 0.05), especially in the L1P group.ConclusionsVLP of HPV16 variant strain isolated in East China could induce humoral immunity and cellular immunity in mice, and IL-12 recombinant plasmid can enhance cellular immunity.     

A vaccine for HIV type 1: The antibody perspective

Antibodies that bind well to the envelope spikes of immunodeficiency viruses such as HIV type 1 (HIV-1) and simian immunodeficiency virus (SIV) can offer protection or benefit if present at appropriate concentrations before viral exposure. The challenge in antibody-based HIV-1 vaccine design is to elicit such antibodies to the viruses involved in transmission in humans (primary viruses). At least two major obstacles exist. The first is that very little of the envelope spike surface of primary viruses appears accessible for antibody binding (low antigenicity), probably because of oligomerization of the constituent proteins and a high degree of glycosylation of one of the proteins. The second is that the mature oligomer constituting the spikes appears to stimulate only weak antibody responses (low immunogenicity). Viral variation is another possible obstacle that appears to present fewer problems than anticipated. Vaccine design should focus on presentation of an intact mature oligomer, increasing the immunogenicity of the oligomer and learning from the antibodies available that potently neutralize primary viruses.     

Long-term effects of antiretroviral combination therapy on HIV type 1 DNA levels.

HIV-1 RNA and DNA levels were measured in 58 patients, of whom 37 and 11, respectively, received triple or double combination therapy. At baseline, strong correlations were found between the number of HIV-1 DNA copies per 106 CD4+ cells, the plasma HIV-1 RNA levels, and the isolation rate of HIV-1 from blood cells. In comparison with HIV-1 RNA, a much slower decline in HIV-1 DNA levels was seen, which probably represents a long lifetime of provirus-bearing CD4+ cells. In 10 untreated patients, the proviral load was stable. In patients receiving triple therapy, a significant decline in HIV-1 DNA was seen independent of whether the proviral load was expressed as copies per 106 CD4+ cells or as copies per milliliter of blood. In contrast, a decline was seen only when the proviral load was expressed as copies per 10(6) CD4+ cells in patients given two drugs. The difference between the two patient categories is likely to be due to a more frequent infection of CD4+ cells during therapy in the patients with double therapy. Interpretation of changes in HIV-1 DNA levels is thus dependent on how the proviral burden is expressed. Further studies should evaluate whether changes in HIV-1 DNA load can be used as markers of viral replication during efficient antiretroviral combination therapy.     

单纯疱疹病毒2型多表位复合DNA疫苗的构建与免疫原性

目的 构建单纯疱疹病毒2型(HSV-2)糖蛋白gD、gB、gC和早期表达蛋白ICP27的多表位复合DNA疫苗,探讨其诱导机体免疫应答的能力.方法 将HSV-2野生株经PCR鉴定后,利用PCR技术从其基因组扩增出HSV-2 ICP27 377-459、gD146-179、gD223-306、gB529-606、gC247-282和gD1-77等6个基因片段.采用多基冈片段一步酶切连接法获得构建的6个片段的复合DNA疫苗(HV)融合基冈片段,经pGEMT载体克隆后,定向插入真核表达质粒pcDNA3.1载体中,构建重组真核表达质粒HV-pcDNA3.1,并对其进行酶切分析及测序鉴定.10只C57/BL6小鼠均分为脂质体包裹pcDNA3.1空载体质粒对照组和脂质体包裹HV-pcDNA3.1质粒免疫组.ELISA检测小鼠血清HSV-2特异性IgG、IL-2和IFN-γ,乳酸脱氢酶法检测CTL功能.数据行t检验.结果 酶切重组质粒HV-pcDNA3.1,电泳可见两条带,分别为融合基因HV(1.3 kb)和线性质粒pcDNA3.1(5.4 kb).测序结果表明,克隆基因插入方向正确,与基因库HSV-2相关序列同源性达99.9%.与pcDNA3.1空载体质粒组相比,HV-pcDNA3.1免疫组小鼠血清中HSV-2特异性IgG(0.29±0.14比0.11±0.04,P＜0.05)、IL-2(1 246.8±157.0比685.2±104.2,P＜0.05)和IFN-γ(1 340.3±108.5比547.7±189.3,P＜0.05)表达明显增加,CTL活性增强(31.82±10.12比8.30±2.91,P＜0.05).结论 成功构建了HSV-2糖蛋白gD、gB、gC和早期表达蛋白ICP27的多表位复合DNA疫苗,能有效引起小鼠机体特异性体液免疫和细胞免疫应答.     

Phase 1 safety and immunogenicity evaluation of a multiclade HIV-1 DNA candidate vaccine

BACKGROUND: Gene-based vaccine delivery is an important strategy in the development of a preventive vaccine for acquired immunodeficiency syndrome (AIDS). Vaccine Research Center (VRC) 004 is the first phase 1 dose-escalation study of a multiclade HIV-1 DNA vaccine. METHODS: VRC-HIVDNA009-00-VP is a 4-plasmid mixture encoding subtype B Gag-Pol-Nef fusion protein and modified envelope (Env) constructs from subtypes A, B, and C. Fifty healthy, uninfected adults were randomized to receive either placebo (n=10) or study vaccine at 2 mg (n=5), 4 mg (n=20), or 8 mg (n=15) by needle-free intramuscular injection. Humoral responses (measured by enzyme-linked immunosorbant assay, Western blotting, and neutralization assay) and T cell responses (measured by enzyme-linked immunospot assay and intracellular cytokine staining after stimulation with antigen-specific peptide pools) were measured. RESULTS: The vaccine was well tolerated and induced cellular and humoral responses. The maximal CD4(+) and CD8(+) T cell responses occurred after 3 injections and were in response to Env peptide pools. The pattern of cytokine expression by vaccine-induced HIV-specific T cells evolved over time, with a diminished frequency of interferon- gamma -producing T cells and an increased frequency of interleukin-2-producing T cells at 1 year. CONCLUSIONS: DNA vaccination induced antibody to and T cell responses against 3 major HIV-1 subtypes and will be further evaluated as a potential component of a preventive AIDS vaccine regimen.     

Excellent safety and tolerability of the human immunodeficiency virus type 1 pGA2/JS2 plasmid DNA priming vector vaccine in HIV type 1 uninfected adults

A vaccine consisting of DNA priming followed by recombinant modified vaccinia Ankara (rMVA) boosting has achieved long-term control of a pathogenic challenge with a chimera of simian and human immunodeficiency viruses (SHIV-89.6P) in rhesus macaques. Based on these results, clade B HIV-1 DNA and rMVA immunogens have been developed for trials in humans. We conducted a first-time in humans phase I safety trial using the pGA2/JS2 (JS2) HIV-1 DNA priming vector expressing Gag, Pol, Env, Tat, Rev, and Vpu. Thirty HIV-uninfected adults were vaccinated with 0.3 or 3 mg of JS2 DNA, or a saline placebo, by intramuscular injection at months 0 and 2. Both doses of DNA were safe and well-tolerated with no differences between the control, 0.3 mg, or 3 mg groups (n = 6, 12, and 12, respectively) through 12 months of postvaccination follow- up. A chromium-release assay using fresh peripheral blood mononuclear cells (PBMCs) and a validated IFN-gamma ELISpot assay with frozen PBMCs failed to detect CD4(+) or CD8(+) HIV-1-specific T cell responses. HIV-specific neutralizing antibodies were also not detected. The vaccine is being further developed as a priming vector for a combined DNA plus rMVA prime/boost HIV vaccination regimen.     

Maternal HIV type 1 infection suppresses MMP-1 expression in endothelial cells of uninfected newborns: nonviral vertical transmission of HIV type 1-related effects

HIV-1 infection is associated with vascular alterations. This is accompanied by an increased risk of cardiovascular diseases and Kaposi's sarcoma, an endothelial cell-derived tumor. We investigated the impact of maternal HIV-1 infection on phenotype and gene expression of endothelial cells in newborns. For this reason endothelial precursor cells and differentiated endothelial cells were isolated from cord blood as well as from umbilical veins and arteries of noninfected infants born to HIV-1-infected (H-group) and noninfected (Ngroup) mothers. No apparent differences in proliferation, capillary formation, and expression of endothelial cell markers were detected in these cells. Interestingly, the expression of matrix metalloproteinase was repressed significantly (X2 analysis, p < 0.002) and consistently at the RNA, the protein, and the secretory levels in the H-group as compared to the N-group. Neither treatment with zidovudine (AZT), mutations in the matrix metalloproteinase-1 (MMP-1) promoter, nor epigenetic changes in the promoter methylation pattern were responsible for the repression of MMP-1 expression in H-group endothelial cells. The reduced MMP-1 expression may contribute to the impaired cardiac function that has been observed in children of HIV-1-infected women. Most interestingly, our findings indicate that HIV-1-related effects can be transferred from mother to child in the absence of HIV-1 transmission.     

Phase I trial of a therapeutic HIV type 1 vaccine,Vacc-4x,in HIV type 1-infected individuals with or without antiretroviral therapy

Highly active antiretroviral therapy (HAART) can effectively suppress HIV-1 replication but, as soon as the drugs are withdrawn, there is a rapid rebound of replicating virus. Severe metabolic toxicities and therapy failures due to the appearance of resistant virus are becoming an increasing problem that precludes long-term continuous medication. Therapeutic immunizations represent a feasible and attractive means of supplementing or, alternatively, replacing current therapies, thereby reducing the potential for emergence of drug-resistant HIV-1 strains. We have performed an open, single-center, phase I safety study of a candidate therapeutic HIV-1 vaccine, Vacc-4x, given to 11 HIV-1-infected individuals with or without antiretroviral therapy. The immunogen consists of four synthetic peptides based on the major core protein p24. To ensure optimal exposure of the immunogen to the antigen-presenting cells (APCs), the vaccine was given intradermally together with granulocyte-macrophage colony-stimulating factor (GM-CSF). Responses to the immunization protocol were determined by delayed-type hypersensitivity (DTH) reaction, interferon gamma-secreting cells in the enzyme-linked immunospot (ELISpot) assay, and antibody production to the p24 protein and the peptides. The vaccine was safe and in general well tolerated. Plasma HIV RNA levels and CD4(+) cell counts did not change appreciably during the study. All patients showed a positive DTH response. For two of the patients, the immunization protocol induced responses to one or two of the tested peptides whereas a third patient showed reactivity to one of the peptides before immunization. A weak antibody response in the peptide-specific enzyme-linked immunosorbent assay could be seen in seven patients.     

Determination of HIV type 1 CRF01_AE gag p17 and env-V3 consensus sequences for HIV/AIDS vaccine design

A molecular epidemiological study of the gag p17 and env-V3 regions on HIV-infected drug users and blood donors was carried out in northern Thailand from 1998 through 2002 to determine the predominant subtype and consensus sequence (CS) for circulating HIV-1 strains. CRF01_AE was concluded to be a predominant strain and the nucleotide CSs in gag p17 and env-V3 showed only 1.26% and no difference from CS in the Los Alamos database, respectively. Our env-V3 CS was identical to the previously published CSs, suggesting that the CS was very conserved from 1990 through 2002 in Thailand. Gag p17 and env-V3 nucleotide sequences of seroconvertors in our subjects were quite similar to the CS and conserved for at least 9 and 6 years postinfection, respectively. These results suggest that the CS approach to the HIV-1 antigen design could overcome HIV diversity and help us develop an effective HIV/AIDS vaccine.