Immunogenic display of diverse peptides,including a broadly cross-type neutralizing human papillomavirus L2 epitope,on virus-like particles of the RNA bacteriophage PP7

The immunogenicity of an antigen can be dramatically increased by displaying it in a dense, multivalent context, such as on the surface of a virus or virus-like particle (VLP). Here we describe a highly versatile VLP platform for peptide display based on VLPs of the RNA bacteriophage PP7. We show that this platform can be used for the engineered display of specific peptide sequences as well as for the construction of random peptide libraries. Peptides representing the FLAG epitope, the V3 loop of HIV gp120, and a broadly cross-type neutralizing epitope from L2, the minor capsid protein of Human Papillomavirus type 16 (HPV16), were inserted into an exposed surface loop of a form of PP7 coat protein in which the two identical polypeptides of coat were fused together to form a single-chain dimer. The recombinant proteins assembled into VLPs, displayed these peptides on their surfaces, and induced high-titer antibody responses. The single-chain dimer was also highly tolerant of random 6-, 8-, and 10-amino acid insertions. PP7 VLPs displaying the HPV16 L2 epitope generated robust anti-HPV16 L2 serum antibodies after intramuscular injection that protected mice from genital infection with HPV16 pseudovirus as well as a heterologous HPV pseudovirus type, HPV45. Thus, PP7 VLPs are well-suited for the display of a wide diversity of peptides in a highly immunogenic format.     

Adenovirus particles that display the Plasmodium falciparum circumsporozoite protein NANP repeat induce sporozoite-neutralizing antibodies in mice

Adenovirus particles can be engineered to display exogenous peptides on their surfaces by modification of viral capsid proteins, and particles that display pathogen-derived peptides can induce protective immunity. We constructed viable recombinant adenoviruses that display B-cell epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) in the major adenovirus capsid protein, hexon. Recombinants induced high-titer antibodies against CSP when injected intraperitoneally into mice. Serum obtained from immunized mice recognized both recombinant PfCSP protein and P. falciparum sporozoites, and neutralized P. falciparum sporozoites in vitro. Replicating adenovirus vaccines have provided economical protection against adenovirus disease for over three decades. The recombinants described here may provide a path to an affordable malaria vaccine in the developing world.     

含甲型H1N1流感病毒HA基因序列病毒样颗粒的制备

[目的]构建耐RNase酶的内含甲型H1N1流感病毒HA基因序列的病毒样颗粒。[方法]构建中间载体pET32a-MS2,将甲型H1N1流感病毒的HA基因片段连接中间载体上,构建原核表达载体pET32a-MS2-HA,转化宿主菌,诱导表达制备病毒样颗粒,对病毒样颗粒进行荧光定量RT-PCR检测和稳定性实验。[结果]表达载体经PCR、酶切鉴定分析后证实构建成功,荧光定量RT-PCR实验表明该病毒颗粒含有HA基因片段并且颗粒稳定性良好。[结论]成功构建了含甲型H1N1流感病毒HA基因序列的病毒样颗粒且稳定性良好,有望作为甲型H1N1流感病毒RNA检测的标准品和质控品。     

A papillomavirus-like particle (VLP) vaccine displaying HPV16 L2 epitopes induces cross-neutralizing antibodies to HPV11

Peptides of the papillomavirus L2 minor capsid protein can induce antibodies (Ab) that neutralize a broad range of human papillomavirus (HPV) genotypes. Unfortunately, L2 is antigenically subdominant to L1 in the virus capsid. To induce a strong anti-L2 Ab response with cross-neutralizing activity to other mucosal types, chimeric virus-like particles (VLP) were generated in which HPV16 L2 neutralization epitopes (comprising L2 residues 69-81 or 108-120) are inserted within an immunodominant surface loop (between residues 133 and 134) of the L1 major capsid protein of bovine papillomavirus type 1 (BPV1). These chimeras self-assembled into pentameric capsomers, or complete VLP similar to wild type (wt) L1 protein. Immunization of rabbits with assembled particle preparations induced L2-specific serum Ab with titers 10-fold higher than those induced by cognate synthetic L2 peptides coupled to KLH. Antisera to both chimeric proteins partially neutralized HPV16 pseudovirions, confirming that both HPV16 L2 peptides define neutralization epitopes. When analyzed for the ability to cross-neutralize infection by authentic HPV11 virions, using detection of early viral RNA by RT-PCR-assays as the readout, immune serum to chimeric protein comprising L2 residues 69-81, but not 108-120, was partially neutralizing. In addition, mouse-antiserum induced by vaccinations with synthetic L2 peptide 108-120, but not 69-81, was partially neutralizing in this assay. Induction of cross-neutralization Ab by L2 epitopes displayed on chimeric VLP represents a possible strategy for the generation of broad-spectrum vaccines to protect against relevant mucosal HPV and associated neoplasia.     

Immunization with PfEMP1-DBL1alpha generates antibodies that disrupt rosettes and protect against the sequestration of Plasmodium falciparum-infected erythrocytes

A family of parasite antigens known as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is believed to play an important role in the binding of infected erythrocytes to host receptors in the micro-vasculature. Available data advocates the existence of a subset of very adhesive (rosetting, auto-agglutinating) and antigenic PfEMP1s implicated as virulence factors. Serum antibodies that disrupt rosettes are rarely found in children with severe malaria but are frequent in those with mild disease suggesting that they may be protective. Here we have developed a Semliki forest virus (SFV) vaccine construct with a recombinant gene (mini-var gene) encoding a mini-PfEMP1 (DBL1alpha-TM-ATS) obtained from a particularly antigenic and rosetting parasite (FCR3S1.2). The mini-PfEMP1 is presented to the host mimicking the location of the native molecule at the infected erythrocyte surface. Antibodies generated by a regimen of priming with SFV RNA particles and boosting with a recombinant protein recognize the infected erythrocyte surface (immuno-fluorescence/rosette-disruption) and prevent the sequestration of P. falciparum-infected erythrocytes in an in vivo model of severe malaria. The data prove the involvement of DBL1alpha in the adhesion of infected- and uninfected erythrocytes and the role of rosette-disruptive antibodies in preventing these cellular interactions. The work supports the use of DBL1alpha in a vaccine again severe malaria.     

A prime-boost immunisation regimen using DNA followed by recombinant modified vaccinia virus Ankara induces strong cellular immune responses against the Plasmodium falciparum TRAP antigen in chimpanzees

Two chimpanzees were vaccinated intramuscularly against malaria using plasmid DNA expressing the pre-erythrocytic antigens thrombospondin related adhesion protein (PfTRAP) and liver stage specific antigen-1 (PfLSA-1) of Plasmodium falciparum together with GM-CSF protein. A recombinant modified vaccinia virus Ankara (MVA) expressing PfTRAP was injected intramuscularly 6 weeks later to boost the immune response. This sequence of antigen delivery induced a specific and long-lasting T cell and antibody response to PfTRAP as detected by ELISPOT assay and ELISA. Antibody responses were detected after four DNA injections, and were boosted by injection of recombinant MVA expressing PfTRAP. Interferon-gamma secreting antigen-specific T cells were detected in both animals, but only after boosting with recombinant MVA. By screening a panel of PfTRAP-derived peptides, an epitope was identified that was recognized by cytotoxic T lymphocytes in one of the chimpanzees studied. T cells specific for this epitope were present in PBMCs and liver-infiltrating lymphocytes at a frequency of between 1 in 200 and 1 in 500. The high immunogenicity of this prime-boost regimen in chimpanzees supports further assessment of this delivery strategy for the induction of protection against P. falciparum malaria in humans.     

Potential for broad-spectrum protection against feline calicivirus using an attenuated myxoma virus expressing a chimeric FCV capsid protein

It has previously been demonstrated that recombinant myxoma viruses expressing FCV capsid protein are capable of eliciting protective responses against virulent FCV challenge, following vaccination, in cats. An attempt was made to produce a bivalent myxoma recombinant expressing the capsid protein genes of both FCV strains F9 and LS015. The FCV capsid protein genes were inserted into the myxoma growth factor gene (MGF) locus, and the serine protease inhibitor (SERP 2) gene locus. Subsequent recombination between myxoma-FCV viruses resulted in a recombinant expressing a chimeric form of the capsid protein. Nonetheless, cats immunised with this myxoma-FCV recombinant demonstrate high levels of serum neutralising antibodies against both F9 and LS015 strains. Such a chimeric vaccine may provide effective protection against a wide range of FCV strains.     

A prime-boost immunisation regimen using DNA followed by recombinant modified vaccinia virus Ankara induces strong cellular immune responses against the Plasmodium falciparum TRAP antigen in chimpanzees

Two chimpanzees were vaccinated intramuscularly against malaria using plasmid DNA expressing the pre-erythrocytic antigens thrombospondin related adhesion protein (PfTRAP) and liver stage specific antigen-1 (PfLSA-1) of Plasmodium falciparum together with GM-CSF protein. A recombinant modified vaccinia virus Ankara (MVA) expressing PfTRAP was injected intramuscularly 6 weeks later to boost the immune response. This sequence of antigen delivery induced a specific and long-lasting T cell and antibody response to PfTRAP as detected by ELISPOT assay and ELISA. Antibody responses were detected after four DNA injections, and were boosted by injection of recombinant MVA expressing PfTRAP. Interferon-gamma secreting antigen-specific T cells were detected in both animals, but only after boosting with recombinant MVA. By screening a panel of PfTRAP-derived peptides, an epitope was identified that was recognized by cytotoxic T lymphocytes in one of the chimpanzees studied. T cells specific for this epitope were present in PBMCs and liver-infiltrating lymphocytes at a frequency of between 1 in 200 and 1 in 500. The high immunogenicity of this prime-boost regimen in chimpanzees supports further assessment of this delivery strategy for the induction of protection against P. falciparum malaria in humans.     

Towards an RTS,S-based, multi-stage, multi-antigen vaccine against falciparum malaria: progress at the Walter Reed Army Institute of Research

The goal of the Malaria Vaccine Program at the Walter Reed Army Institute of Research (WRAIR) is to develop a licensed multi-antigen, multi-stage vaccine against Plasmodium falciparum able to prevent all symptomatic manifestations of malaria by preventing parasitemia. A secondary goal is to limit disease in vaccinees that do develop malaria. Malaria prevention will be achieved by inducing humoral and cellular immunity against the pre-erythrocytic circumsporozoite protein (CSP) and the liver stage antigen-1 (LSA-1). The strategy to limit disease will target immune responses against one or more blood stage antigens, merozoite surface protein-1 (MSP-1) and apical merozoite antigen-1 (AMA-1). The induction of T- and B-cell memory to achieve a sustained vaccine response may additionally require immunization with an adenovirus vector such as adenovirus serotype 35. RTS,S, a CSP-derived antigen developed by GlaxoSmithKline Biologicals in collaboration with the Walter Reed Army Institute of Research over the past 17 years, is the cornerstone of our program. RTS,S formulated in AS02A (a GSK proprietary formulation) is the only vaccine candidate shown in field trials to prevent malaria and, in one instance, to limit disease severity. Our vaccine development plan requires proof of an individual antigen's efficacy in a Phase 2 laboratory challenge or field trial prior to its integration into an RTS,S-based, multi-antigen vaccine. Progress has been accelerated through extensive partnerships with industrial, academic, governmental, and non-governmental organizations. Recent safety, immunogenicity, and efficacy trials in the US and Africa are presented, as well as plans for the development of a multi-antigen vaccine.     

Development, expression, and murine testing of a multistage Plasmodium falciparum malaria vaccine candidate

A synthetic gene encoding twelve B cell epitopes, six T-cell proliferative epitopes, and three cytotoxic T lymphocyte (CTL) epitopes from nine stage-specific antigens, representing the sporozoite, liver stage, asexual blood-stage, and sexual-stage antigens of Plasmodium falciparum, was constructed by assembling overlapping oligonucleotides followed by PCR extension and annealing. A three-step PCR protocol using twelve long oligonucleotides was employed to generate a 1053 base-pair synthetic gene, the identity of which was confirmed by sequencing. This synthetic gene, named CDC/NII MAL VAC-1, was cloned, and the recombinant protein was expressed in the Baculovirus Expression Vector System (BEVS). The selection of malarial epitopes for inclusion in this vaccine construct was based on immunoepidemiological studies in malaria endemic area, in vitro, and in vivo protection studies in model systems. The 41 kDa BEVS-expressed recombinant protein reacted with mouse antibodies specific for individual B cell epitopes in the vaccine construct and with sera from clinically immune Kenyan adults. An immunization study in three strains of mice that differ at the H-2 locus demonstrated that the BEVS-expressed recombinant protein is immunogenic; the candidate vaccine antigen induced high titer antibodies, and lymphocyte proliferative and IFN-gamma responses. These results demonstrate that individual B and T cell epitopes can be assembled to create synthetic genes that encode proteins capable of eliciting specific antibody and T cell responses.     

Differences in epitope recognition, isotype and titer of antisera to Plasmodium falciparum merozoite surface protein 4 raised by different modes of DNA or protein immunization

Plasmodium falciparum merozoite surface protein 4 (MSP4) is being developed as a component of a subunit vaccine against asexual stages of malaria. Three DNA constructs were produced that induced expression of MSP4 either in the cytoplasm of transfected cells or secreted from cells under the control of the human tissue plasminogen activator (TPA) signal or the native P. falciparum MSP4 signal. Only the construct containing the TPA signal induced detectable antibodies in mice, although gene expression was demonstrated in all constructs and MSP4 was shown to be secreted using either signal by in vitro transient transfection of COS cells. Two recombinant MSP4 proteins that encoded the same sequence as the plasmid DNA were produced in E. coli (EcMSP4-His) and S. cerevisiae (yMSP4-His) and used to raise antibodies in mice. Comparison of the antibodies elicited by these various antigen formulations showed differences in titer, isotype and epitope recognition. The titer of antibodies induced by DNA vaccination was lower than that induced by yMSP4-His, which in turn was lower than that induced by EcMSP4-His. The isotype profiles of the antibodies were also different, the plasmid DNA induced predominantly IgG(2a) responses whereas the two proteins induced predominantly IgG(1) responses. The antibodies induced by DNA and yMSP4-His recognized predominantly the C-terminal epidermal growth factor (EGF)-like domain of the protein, whereas EcMSP4-His induced antibodies recognizing all domains of the protein equally. The antibodies induced by DNA vaccination were directed almost extensively to conformational epitopes so that reactivity with native MSP4 was abolished after disulfide bonds in the protein were disrupted. Antibodies induced by recombinant proteins recognized linear epitopes as well and reactivity to native MSP4 was preserved after reduction and alkylation of parasite proteins.     

一起急性胃肠炎疫情中重组诺如病毒基因特征分析

目的 明确成都市2018年5月某建筑工地发生的一起急性胃肠炎疫情中的诺如病毒的基因型别,并对其遗传进化和分子流行病学特征进行分析研究。方法 对实时荧光定量PCR检测结果为阳性的标本采用常规RT - PCR扩增其RNA依赖的RNA聚合酶区和衣壳蛋白区并测序,与国内外参考株进行序列比对和构建进化树等基因特征分析。结果 序列比对和进化树分析结果显示:此次疫情中检出的诺如病毒核苷酸同源性为100%;其RNA依赖的RNA聚合酶区与北京株处于同一分支,同源性95.4%;而衣壳蛋白区与莫斯科株同源性最高,为96.4%。结合SimPlot对其进行分析,重组可能位点在213 bp处,定位在ORF1/2交叠区。结论 引起本次急性胃肠炎疫情的重组诺如病毒基因型为GI.P4 - GI.5,这是成都市首次检出诺如病毒GI群重组株。     

人乳头瘤病毒16型晚期蛋白在昆虫细胞中的表达

目的 构建HPV16型晚期蛋白重组杆状病毒,并使其在昆虫细胞中获得高效表达.方法 首先用线性化的杆状病毒DNA与重组杆状病毒转移质粒共转染sf9昆虫细胞进行同源重组,获得2株重组杆状病毒.结果 经鉴定该重组病毒中有目的DNA存在且可表达晚期蛋白;电镜观察发现重组杆状病毒感染的sf9细胞核内有乳头瘤病毒VLPs.结论 HPV16型晚期蛋白在昆虫细胞中的成功表达为HPV16型预防性基因工程亚单位疫苗的研制和诊断试剂的研究开发奠定了基础.     

Recombinant Liver Stage Antigen-1 (LSA-1) formulated with AS01 or AS02 is safe,elicits high titer antibody and induces IFN-γ/IL-2 CD4+ T cells but does not protect against experimental Plasmodium falciparum infection

Plasmodium falciparum Liver Stage Antigen 1 (LSA-1) is a pre-erythrocytic stage antigen. Our LSA-1 vaccine candidate is a recombinant protein with full-length C- and N-terminal flanking domains and two of the 17 amino acid repeats from the central repeat region termed “LSA-NRC.” We describe the first Phase I/II study of this recombinant LSA-NRC protein formulated with either the AS01 or AS02 adjuvant system. We conducted an open-label Phase I/II study. Thirty-six healthy malaria-naïve adults received one of four formulations by intra-deltoid injection on a 0 and 1 month schedule; low dose (LD) LSA-NRC/AS01:10 μg LSA-NRC/0.5 ml AS01 (n = 5), high dose (HD) LSA-NRC/AS01: 50 μg LSA-NRC/0.5 ml AS01 (n = 13); LD LSA-NRC/AS02: 10 μg LSA-NRC/0.5 ml AS02 (n = 5) and HD LSA-NRC/AS02: 50 μg LSA-NRC/0.5 ml AS02 (n = 13). Two weeks post-second immunization, the high dose vaccinees and 6 non-immunized infectivity controls underwent experimental malaria sporozoite challenge.     

Soluble and glyco-lipid modified baculovirus Plasmodium falciparum C-terminal merozoite surface protein 1, two forms of a leading malaria vaccine candidate

Recombinant homologues of the Plasmodium merozoite surface protein 1 C-terminus are leading blood stage malaria vaccine candidates. MSP1 is anchored to the merozoite plasma membrane in vivo by a glycosyl-phosphatidyl-inositol (GPI) moiety, implicated in malaria pathology. Two types of recombinant Plasmodium falciparum MSP1p19 (PfMSP1p19) expressed in baculovirus/insect cells are described here: (1) a soluble, secreted form (PfMSP1p19S) and (2) detergent soluble cellular form(s) (PfMSP1p19+A), released from the infected cell surface by treatment with GPI specific phosphatidyl-inositol phospholipase C (PI-PLC). Soluble and cellular PfMSP1p19 were purified and characterized using SDS-PAGE, mass spectrometry (MS), N-terminal amino acid sequencing, gel filtration and glycan analyses. Quantitative inositol dosage suggested that surface GPI processed entities constituted only 14% of the purified cellular PfMSP1p19+A, with GPI unprocessed forms likely recovered in the endoplasmic reticulum. Nevertheless, this preparation has dramatic immuno-stimulatory activity to be described elsewhere. The interest of these results for both malaria specific and generic vaccine development are discussed.     

Immunogenicity and in vitro protective efficacy of a polyepitope Plasmodium falciparum candidate vaccine constructed by epitope shuffling

A polyepitope chimeric antigen incorporating multiple protective and conservative epitopes from multiple antigens of Plasmodium falciparum has been considered to be more effective in inducing multiple layers of immunity against malaria than a single stage- or single antigen-based vaccine. By modifying the molecular breeding approach to epitope shuffling, we have constructed a polyepitope chimeric gene that encodes 11 B-cell and T-cell proliferative epitope peptides derived from eight key antigens mostly in the blood stage of Plasmodium falciparum. A 35-kDa antigen encoded by this gene, named Malaria RCAg-1, was purified from an E. coli expression system. Immunization of rabbits and mice with the purified protein in the presence of Freund's adjuvant strongly generated long-lasting antibody responses that recognized the corresponding individual epitope peptide in this vaccine as well as blood stage parasites. CD4(+) T-cell responses were also elicited as shown by the enhancement of T-cell proliferation, IFN-gamma and IL-4 level. In vitro assay of protection revealed that vaccine-elicited antibodies could efficiently inhibit the growth of blood-stage parasites. Additionally, the chimeric antigen was recognized by human serum specimens from malaria patients and individuals living in the endemic area. Our studies indicate the potential of M.RCAg-1 recombinant protein as malaria candidate vaccines as well as the rationale of the epitope shuffling technology applied in designing malaria vaccines.     

Genetically highly divergent RNA virus with astrovirus-like (5′-end) and hepevirus-like (3′-end) genome organization in carnivorous birds,European roller (Coracias garrulus)

Astroviruses (family Astroviridae) and hepeviruses (family Hepeviridae) are small, non-enveloped viruses with genetically diverse +ssRNA genome thought to be enteric pathogens infecting vertebrates including humans. Recently, many novel astro- and hepatitis E virus-like +ssRNA viruses have been described from lower vertebrate species. The non-structural proteins of astro- and hepeviruses are highly diverse, but the structural/capsid proteins represent a common phylogenetic position shed the light of their common origin by inter-viral recombination. In this study, a novel astrovirus/hepevirus-like virus with +ssRNA genome (Er/SZAL5/HUN/2011, MK450332) was serendipitously identified and characterized from 3 (8.5%) out of 35 European roller (Coracias garrulus) faecal samples by RT-PCR in Hungary. The complete genome of Er/SZAL5/HUN/2011 (MK450332) is 8402 nt-long and potentially composed three non-overlapping open reading frames (ORFs): ORF1a (4449 nt/1482aa), ORF1b (1206 nt/401aa) and ORF2 (1491 nt/496aa). The ORF1ab has an astrovirus-like genome organization containing the non-structural conserved elements (TM, CC, NLS, VPg) and enzyme residues (trypsine-like protease, RNA-dependent RNA-polymerase) with low amino acid sequence identity, 15% (ORF1a) and 44% (ORF1b), to astroviruses. Supposedly the ORF2 is a capsid protein but neither the astrovirus-like subgenomic RNA promoter (sgRNA) nor the astrovirus-like capsid characteristics have been identifiable. However, the predicted capsid protein (ORF2) showed 26% identity to the corresponding protein of hepevirus-like novel Rana hepevirus (MH330682). This novel +ssRNA virus strain Er/SZAL5/HUN/2011 with astrovirus-like genome organization in the non-structural genome regions (ORF1a and ORF1b) and Rana hepevirus-related capsid (ORF2) protein represent a potentially recombinant virus species and supports the common origin hypothesis, although, the taxonomic position of the studied virus is still under discussion.     

Chimeric hepatitis E virus-like particle as a carrier for oral-delivery

Oral delivery with virus-like particles (VLPs) is advantageous because of the inherited entry pathway from their parental viral capsids, which enables VLP to withstand the harsh and enzymatic environment associated with human digestive tract. However, the repeat use of this system is challenged by the self-immunity. In order to overcome this problem, we engineered the recombinant capsid protein of hepatitis E virus by inserting p18 peptide, derived from the V3 loop of HIV-1 gp120, into the antibody-binding site. The chimeric VLP resembled the tertiary and quaternary structures of the wild type VLP and specifically reacted with an HIV-1 antibody against V3 loop. Different from the wild type VLP, the chimeric VLP was vulnerable to trypsin cleavage although it appeared as intact particle, suggesting that the intermolecular forces of attraction between the recombinant capsid proteins are strong enough to maintain the VLP icosahedral arrangement. Importantly, this VLP containing the V3 loop did not react with anti-HEV antibodies, in correspondence to the mutation at its antibody-binding site. Therefore, the insertion of peptides at the surface antigenic site could allow VLPs to escape pre-existing anti-HEV humoral immunity.