Three Candidate Epitope—Vaccines in Combination Inducing High Levels of Multiantibodies Against HIV—1

HIV-1 mutation results in immune evasion, which presents a serious challenge for conventional strategies for developing effective vaccines. So far, much experimental evidence indicates that HIV-1 particles in the blood of patients can be cleaned principally by neutralizing antibodies. Based on these facts, we prepared triple combination of epitope-vaccines with the objective of inducing antibodies with predefined multi-epitope-specificity against HW-1. According to the sequences of three neutralizing epitopes (RILAVERYLKD, ELDKWA and GPGRAFY, designated El, E2, and E3, respectively) on HIV-1 envelope proteins, three epitope-peptides ((E1)2: C-(RILAVERYLKDG)2; (E2)4: C-(ELDKWAG)4; and (E3)2:C-(GPGRAFY)2) were synthesized and then conjugated with carrier protein keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA), and used for immunizing rabbits. After the vaccine course, the triple combination of epitope-vaccines induced high levels of predefined multi-epitope-specific antibodies. An immunoblotting-analysis demonstrated that the antibodies could recognize the native epitopes on both gp41 protein and V3 loop peptide. Furthermore, we compared the immune responses of three doses of epitope-peptides in the candidate epitope-vaccine. Strong antibody responses to three epitopes were observed in a dose dependent manner, with increasing dose raising the immune response. This result indicated that immunotolerance did not occur using an epitope vaccine dose of 80 ~tg. Thus, our results demonstrate that epitope-vaccines in combination can synchronously induce high levels of antibodies with predefined multi-epitope-specificity against HIV-1, and may be used to develop effective vaccines against HIV as a new strategy.     

Characterization of Antibody Responses Against the 2F5 Epitope ELDKWA sing HIV-1 Env-Mediated Membrane Fusion and Neutralization Assays

The epitope ELDKWA, which is located in the membrane-proximal external region (MPER) of HIV-1 gp41, is an important neutralizing epitope. The human monoclonal antibody (mAb) 2F5 against this epitope shows broad neutralizing activity toward many HIV strains. However, several reports have shown that the epitope-specific mAbs induced by peptides containing MPER did not exhibit the same neutralizing activities as human mAb 2F5. In this study, four ELDKWA epitope specific mAbs (9E7, 7E10, 6B5, and 2B4) induced by immunization with the ELDKWA epitope in varied molecular contexts, all showed inhibitory activities with different potencies in HIV-1 Env-mediated membrane fusion assays and pseudovirus neutralization assays. This result indicates that though these antibodies recognize the epitope ELDKWA, their characterizations differ from that of neutralizing antibodies, implying that the neutralizing mAbs can be induced but also need to be screened, and the protective ability of a related vaccine antigen depends on the concentration of the neutralizing mAbs in the induced polyclonal antibodies.     

Predefined GPGRAFY-Epitope-Specific Monoclonal Antibodies with Different Activities for Recognizing Native HIV-1 gp120

A seven-amino acid epitope GPGRAFY at the tip of the V3 loop in HIV-1 gp120 is the principal neutralizing epitope, and a subset of anti-V3 antibodies specific for this epitope shows a broad range of neutralizing activity. GPGRAFY-epitope-specific neutralizing antibodies were produced using predefined GPGRAFY-epitope-specific peptides instead of a natural or recombinant gp120 bearing this epitope. All six monoclonal antibodies (mAbs) could recognize the GPGRAFY-epitope on peptides and two of the antibodies, 9D8 and 2D7, could recognize recombinant gp120 in enzymelinked immunosorkentassy (ELISA) assays. In the flow cytometry analysis, the mAbs 9D8 and 2D7 could bind to HIV-Env CHO-WT cells and the specific bindings could be inhibited by the GPGRAFY-epitope peptide, which suggests that these two mAbs could recognize the native envelope protein gp120 expressed on the cell membrane. However, in syncytium assays, none of the mAbs was capable of inhibiting HIV-Env-mediated cell membrane fusion. The different activities for recognizing native HIV-1 gp120 might be associated with different antibody affinities against the epitopes. The development of conformational mimics of the neutralization epitope in the gp120 V3 loop could elicit neutralizing mAbs with high affinity.     

Induction of multi-epitopespecific antibodies against HIV-1 by multi-epitopevaccines

Some neutralizing antibodies against HIV-1 envelope proteins were highly effective to inhibit the infection of different strainsin vitro, and existed in the infected individuals with very low levels. We suggested multi-epitope-vaccine as a new strategy to increase levels of neutralizing antibodies and the abilities against HIV mutationin vivo. Two candidate multi-epitope-vaccines induced antibodies with predefined multi-epitope-specificity in rhesus macaque. These antibodies recognized corresponding neutralizing epitopes on epitope-peptides, gp41 peptides, V3 loop peptide, rsgp41 and rgp120. Besides, three candidate epitope-vaccines in combination (another kind of multi-epitopevaccines) showed similar potency to induce predefined multiple immune responses in rabbits. These results suggest that multi-epitope-vaccines may be a new strategy to induce multi-antiviral activities against HIV-1 infection and mutations.     

Antibody neutralization and escape by HIV-1

Neutralizing antibodies (Nab) are a principal component of an effective human immune response to many pathogens, yet their role in HIV-1 infection is unclear. To gain a better understanding of this role, we examined plasma from patients with acute HIV infection. Here we report the detection of autologous Nab as early as 52 days after detection of HIV-specific antibodies. The viral inhibitory activity of Nab resulted in complete replacement of neutralization-sensitive virus by successive populations of resistant virus. Escape virus contained mutations in the env gene that were unexpectedly sparse, did not map generally to known neutralization epitopes, and involved primarily changes in N-linked glycosylation. This pattern of escape, and the exceptional density of HIV-1 envelope glycosylation generally, led us to postulate an evolving 'glycan shield' mechanism of neutralization escape whereby selected changes in glycan packing prevent Nab binding but not receptor binding. Direct support for this model was obtained by mutational substitution showing that Nab-selected alterations in glycosylation conferred escape from both autologous antibody and epitope-specific monoclonal antibodies. The evolving glycan shield thus represents a new mechanism contributing to HIV-1 persistence in the face of an evolving antibody repertoire.     

An engineered poliovirus chimaera elicits broadly reactive HIV-1 neutralizing antibodies

The Sabin type 1 vaccine strain of poliovirus is probably the safest and most successful live-attenuated vaccine virus used in humans. Its widespread use since the early 1960s has contributed significantly to the virtual eradication of poliomyelitis in developed countries. We have reported previously the construction of an intertypic antigen chimaera of poliovirus, based on the Sabin 1 strain, and proposed that this virus could be modified to express on its surface antigenic determinants from other pathogens. We describe here the construction and characterization of a poliovirus antigen chimaera containing an epitope from the transmembrane glycoprotein (gp41) of human immunodeficiency virus type 1 (HIV-1). In antibody absorption experiments, the virus chimaera inhibited neutralization of HIV-1 by antipeptide monoclonal antibodies specific for the gp41 epitope and significantly reduced the group specific neutralizing activity of HIV-1-positive human sera. Rabbit antisera raised by subcutaneous injection of the polio/HIV chimaera in adjuvant was shown to be specific for HIV-1 gp41 in peptide-binding assays and by western blotting. Moreover, the antisera neutralized a wide range of American and African HIV-1 isolates and also inhibited virus-induced cell fusion. Monoclonal antibodies against the HIV-1 derived regions of the chimaera also neutralized HIV-1. These results establish the potential of using poliovirus for the presentation of foreign antigens and suggest that Sabin 1 poliovirus/HIV chimaeras could offer an approach to the development of an HIV vaccine.     

利用搭桥PCR法实现轮状病毒VP7表位片段的重组

选择了RV VP7的3个高度保守可诱发高水平体液免疫反应的中和性抗原表位,人工合成了这3个表位片段。采用了一种不需要限制性内切酶和连接酶的重组DNA方法-搭桥PCR法,方便快速地构建了轮状病毒VF7蛋白的重组表位基因,将其T／A克隆入PBS-T载体,经过测序与报道序列同源性达100％。结果表明：搭桥法PCR简单易行,快速准确,尤其是在构建2个或多个小基因片段的融合时,比酶切、连接方法更具优越性。     

HIV-1 gag-specific cytotoxic T lymphocytes defined with recombinant vaccinia virus and synthetic peptides

Current candidate vaccines fail to protect primates against challenge with human immunodeficiency virus (HIV) in the presence of antibody responses; this underlines the importance of studying cell-mediated immunity to HIV and identifying specific epitopes that stimulate cytotoxic T lymphocytes (CTL). Using a recombinant vaccinia virus to express the gag protein of HIV-1 we found HLA class-I-restricted gag-specific CTL in thirteen out of fifteen healthy HIV seropositive patients. We then used short synthetic peptides in the lysis assay to screen for gag CTL epitopes. In one patient we have identified a peptide in p24 that is recognized by CTL in association with HLA-B27. This peptide, and further peptide sequences defined by these methods, could be incorporated in vaccines designed to induce cell-mediated immunity against HIV.     

A group specific anamnestic immune reaction against HIV-1 induced by a candidate vaccine against AIDS

The first experimental immunization of humans against the AIDS retrovirus, HIV-1, was started in a series of HIV seronegative, healthy volunteers in November 1986. For the primary vaccination recombinant vaccinia virus (V25) expressing the complete gp160 env protein of the HTLV-IIIB strain of HIV-1 was introduced by scarification. This elicited a weak primary response which we subsequently attempted to enhance by additional immunizations (boosting), using four different immunization protocols. We report here that intravenous injection of paraformaldehyde-fixed autologous cells infected in vitro with V25 (individual D.Z.) gave the best results. This individual received second and third boosts of intramuscular gp160 derived from an HTLV-IIIB clone using the hybrid vaccinia virus/bacteriophage T7 expression system. An anamnestic humoral and cellular immune reaction was achieved for over one year after the original vaccination, with high levels of antibodies to the viral envelope, and neutralizing antibodies against divergent HIV-1 strains such as HTLV-IIIB and HTLV-IIIRF (also called HTLV-III HAT) after the first boost. In addition, group-specific cell-mediated immunity and cell-mediated cytotoxicity against infected T4 cells were obtained after the primary vaccine and enhanced by the boosts. Finally, skin tests showed both immediate and delayed hypersensitivity to gp160 in vivo. Although this protocol is not practical for a large scale vaccine trial, our results show for the first time that an immune state against HIV can be obtained in man.     

Immune responses of a designed HIV-1 DNA vaccine on rhesus monkeys

According to the UNAIDS/WHO report (http:// www.unaids.org/Epi2005/doc/report.html), there are about 40.3 million HIV/AIDS survivors, and the new HIV infection number amounts to about 4.9 million, while the death number is some 3.1 million in the world in…     

Structural characteristics and biological functions of the HIV-1 gp120 V3 region

Recent studies demonstrate that the V3 loop of HIV-1 gp120 plays an important role in the attachment of HIV-1 to the target cells. Several amino acids in this domain are involved in the interaction of gp120 with the co-receptors. The V3 loop elicits one of the earliest antiviral antibody responses in HIV-1 infection and has been identified as the principal neutralizing determinant (PND). A subset of antibodies to V3 loop show a broad range of neutralizing activity. Unfortunately, this loop undergoes broad mutation and is one of the hypervariable regions. Mutations of some amino acids in this PND could affect syncytium formation, virus infectivity and neutralization. Knowing the structural characteristics and biological functions of the V3 region could help us to understand mechanism of HIV infection and to develop new strategy against HIV-1. In this review, the structural characteristics, variation and biological functions of the V3 loop as well as immunological responses to the V3 loop are discussed.     

Structural basis of West Nile virus neutralization by a therapeutic antibody

West Nile virus is a mosquito-borne flavivirus closely related to the human epidemic-causing dengue, yellow fever and Japanese encephalitis viruses. In establishing infection these icosahedral viruses undergo endosomal membrane fusion catalysed by envelope glycoprotein rearrangement of the putative receptor-binding domain III (DIII) and exposure of the hydrophobic fusion loop. Humoral immunity has an essential protective function early in the course of West Nile virus infection. Here, we investigate the mechanism of neutralization by the E16 monoclonal antibody that specifically binds DIII. Structurally, the E16 antibody Fab fragment engages 16 residues positioned on four loops of DIII, a consensus neutralizing epitope sequence conserved in West Nile virus and distinct in other flaviviruses. The E16 epitope protrudes from the surface of mature virions in three distinct environments, and docking studies predict Fab binding will leave five-fold clustered epitopes exposed. We also show that E16 inhibits infection primarily at a step after viral attachment, potentially by blocking envelope glycoprotein conformational changes. Collectively, our results suggest that a vaccine strategy targeting the dominant DIII epitope may elicit safe and effective immune responses against flaviviral diseases.     

Broad neutralization coverage of HIV by multiple highly potent antibodies

Broadly neutralizing antibodies against highly variable viral pathogens are much sought after to treat or protect against global circulating viruses. Here we probed the neutralizing antibody repertoires of four human immunodeficiency virus (HIV)-infected donors with remarkably broad and potent neutralizing responses and rescued 17 new monoclonal antibodies that neutralize broadly across clades. Many of the new monoclonal antibodies are almost tenfold more potent than the recently described PG9, PG16 and VRC01 broadly neutralizing monoclonal antibodies and 100-fold more potent than the original prototype HIV broadly neutralizing monoclonal antibodies. The monoclonal antibodies largely recapitulate the neutralization breadth found in the corresponding donor serum and many recognize novel epitopes on envelope (Env) glycoprotein gp120, illuminating new targets for vaccine design. Analysis of neutralization by the full complement of anti-HIV broadly neutralizing monoclonal antibodies now available reveals that certain combinations of antibodies should offer markedly more favourable coverage of the enormous diversity of global circulating viruses than others and these combinations might be sought in active or passive immunization regimes. Overall, the isolation of multiple HIV broadly neutralizing monoclonal antibodies from several donors that, in aggregate, provide broad coverage at low concentrations is a highly positive indicator for the eventual design of an effective antibody-based HIV vaccine.     

Molecular architecture of native HIV-1 gp120 trimers

The envelope glycoproteins (Env) of human and simian immunodeficiency viruses (HIV and SIV, respectively) mediate virus binding to the cell surface receptor CD4 on target cells to initiate infection. Env is a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120), and forms trimers on the surface of the viral membrane. Using cryo-electron tomography combined with three-dimensional image classification and averaging, we report the three-dimensional structures of trimeric Env displayed on native HIV-1 in the unliganded state, in complex with the broadly neutralizing antibody b12 and in a ternary complex with CD4 and the 17b antibody. By fitting the known crystal structures of the monomeric gp120 core in the b12- and CD4/17b-bound conformations into the density maps derived by electron tomography, we derive molecular models for the native HIV-1 gp120 trimer in unliganded and CD4-bound states. We demonstrate that CD4 binding results in a major reorganization of the Env trimer, causing an outward rotation and displacement of each gp120 monomer. This appears to be coupled with a rearrangement of the gp41 region along the central axis of the trimer, leading to closer contact between the viral and target cell membranes. Our findings elucidate the structure and conformational changes of trimeric HIV-1 gp120 relevant to antibody neutralization and attachment to target cells.     

Variable and conserved neutralization antigens of human immunodeficiency virus

Human immunodeficiency virus type 1 (HIV-1, HTLV-III/LAV), the retrovirus responsible for acquired immune deficiency syndrome (AIDS), shows a high degree of genetic polymorphism, particularly in the env gene. We have examined sera from rabbits and guinea pigs immunized with gp130, a recombinant env glycoprotein, and sera from HIV-1-infected subjects, to test their capacity to neutralize a panel of genetically divergent HIV-1 isolates. The sera raised against recombinant antigen specifically neutralized the virus strain from which the env gene was cloned (HTLV-IIIB), but not an independent isolate (HTLV-IIIRF). One rabbit serum tested on seven isolates cross-neutralized two at lower titres. In contrast, human sera from Britain and Uganda, chosen for ability to neutralize HTLV-IIIRF, cross-neutralized six other HIV-1 isolates. When serum and isolate were derived from the same subject, the serum was in some cases effective at slightly lower concentrations (higher titres). Human complement did not affect neutralization titres. These findings indicate that genetically diverse HIV-1 isolates carry both variable and widely conserved antigenic epitopes for neutralizing antibodies. The identification of shared epitopes may help the development of protective vaccines.     

Soluble CD4 blocks the infectivity of diverse strains of HIV and SIV for T cells and monocytes but not for brain and muscle cells

The CD4 antigen has been subverted as a receptor by the human and simian immunodeficiency viruses (HIV-1, HIV-2 and SIV). Several groups have reported that recombinant, soluble forms of the CD4 molecule (sCD4) block the infection of T lymphocytes by HIV-1, as CD4 binds the HIV envelope glycoprotein, gp120, with high affinity. We now report that sCD4 blocks diverse strains of HIV-1, HIV-2 and SIV, but is less effective for HIV-2. The blocking effect is apparent even after adsorption of virions to CD4 cells. Soluble CD4 prevents HIV infection of T-lymphocytic and myelomonocytic cell lines, but neither sCD4 nor anti-CD4 antibodies inhibit infection of glioma and rhabdomyosarcoma cell lines.     

Structural definition of a conserved neutralization epitope on HIV-1 gp120

The remarkable diversity, glycosylation and conformational flexibility of the human immunodeficiency virus type 1 (HIV-1) envelope (Env), including substantial rearrangement of the gp120 glycoprotein upon binding the CD4 receptor, allow it to evade antibody-mediated neutralization. Despite this complexity, the HIV-1 Env must retain conserved determinants that mediate CD4 binding. To evaluate how these determinants might provide opportunities for antibody recognition, we created variants of gp120 stabilized in the CD4-bound state, assessed binding of CD4 and of receptor-binding-site antibodies, and determined the structure at 2.3 A resolution of the broadly neutralizing antibody b12 in complex with gp120. b12 binds to a conformationally invariant surface that overlaps a distinct subset of the CD4-binding site. This surface is involved in the metastable attachment of CD4, before the gp120 rearrangement required for stable engagement. A site of vulnerability, related to a functional requirement for efficient association with CD4, can therefore be targeted by antibody to neutralize HIV-1.     

对虾白斑病毒多抗原决定簇重组蛋白的抗原性分析

为研究重组多表位蛋白制备疫苗的方法的可行性,研究通过利用目前有研究证实与对虾白斑综合征病毒(WSSV)感染与致病力相关的重要决定因子囊膜蛋白VP28、VP19和被膜蛋白VP26,从这三种蛋白基因中筛选表位基因,将这些筛选到的抗原表位通过最优化的方式组合以及密码子优化,组成新的表位基因序列,命名为Y1798G,长度为509 bp。将该基因克隆到p ET-32a原核表达载体,生产抗原表位蛋白。将纯化的抗原蛋白注射到小白鼠体内,获得抗体;并通过蛋白免疫印迹(western blot)实验验证获得的抗体能否识别结合WSSV。结果表明所构建的抗原表位重组蛋白对WSSV具有特异抗原性,可见此种制备对虾白斑病毒疫苗的策略具有可行性。     

HIV infection is blocked in vitro by recombinant soluble CD4

The T-cell surface glycoprotein, CD4 (T4), acts as the cellular receptor for human immunodeficiency virus, type 1 (HIV-1), the first member of the family of viruses that cause acquired immunodeficiency syndrome. HIV recognition of CD4 is probably mediated through the virus envelope glycoprotein (gp120) as shown by co-immunoprecipitation of CD4 and gp120 (ref.5) and by experiments using recombinant gp120 as a binding probe. Here we demonstrate that recombinant soluble CD4(rsT4) purified from the conditioned medium of a stably transfected Chinese hamster ovary cell line is a potent inhibitor of both virus replication and virus-induced cell fusion (syncytium formation). These results suggest that rsT4 is sufficient to bind HIV, and that it represents a potential anti-viral therapy for HIV infection.     

Evolution and transmission of stable CTL escape mutations in HIV infection.

Increasing evidence indicates that potent anti-HIV-1 activity is mediated by cytotoxic T lymphocytes (CTLs); however, the effects of this immune pressure on viral transmission and evolution have not been determined. Here we investigate mother-child transmission in the setting of human leukocyte antigen (HLA)-B27 expression, selected for analysis because it is associated with prolonged immune containment in adult infection. In adults, mutations in a dominant and highly conserved B27-restricted Gag CTL epitope lead to loss of recognition and disease progression. In mothers expressing HLA-B27 who transmit HIV-1 perinatally, we document transmission of viruses encoding CTL escape variants in this dominant Gag epitope that no longer bind to B27. Their infected infants target an otherwise subdominant B27-restricted epitope and fail to contain HIV replication. These CTL escape variants remain stable without reversion in the absence of the evolutionary pressure that originally selected the mutation. These data suggest that CTL escape mutations in epitopes associated with suppression of viraemia will accumulate as the epidemic progresses, and therefore have important implications for vaccine design.