Antibody-based protection against HIV infection by vectored immunoprophylaxis

Despite tremendous efforts, development of an effective vaccine against human immunodeficiency virus (HIV) has proved an elusive goal. Recently, however, numerous antibodies have been identified that are capable of neutralizing most circulating HIV strains. These antibodies all exhibit an unusually high level of somatic mutation, presumably owing to extensive affinity maturation over the course of continuous exposure to an evolving antigen. Although substantial effort has focused on the design of immunogens capable of eliciting antibodies de novo that would target similar epitopes, it remains uncertain whether a conventional vaccine will be able to elicit analogues of the existing broadly neutralizing antibodies. As an alternative to immunization, vector-mediated gene transfer could be used to engineer secretion of the existing broadly neutralizing antibodies into the circulation. Here we describe a practical implementation of this approach, which we call vectored immunoprophylaxis (VIP), which in mice induces lifelong expression of these monoclonal antibodies at high concentrations from a single intramuscular injection. This is achieved using a specialized adeno-associated virus vector optimized for the production of full-length antibody from muscle tissue. We show that humanized mice receiving VIP appear to be fully protected from HIV infection, even when challenged intravenously with very high doses of replication-competent virus. Our results suggest that successful translation of this approach to humans may produce effective prophylaxis against HIV.     

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.     

Neutralizing antibodies derived from the B cells of 1918 influenza pandemic survivors

Investigation of the human antibody response to influenza virus infection has been largely limited to serology, with relatively little analysis at the molecular level. The 1918 H1N1 influenza virus pandemic was the most severe of the modern era. Recent work has recovered the gene sequences of this unusual strain, so that the 1918 pandemic virus could be reconstituted to display its unique virulence phenotypes. However, little is known about adaptive immunity to this virus. We took advantage of the 1918 virus sequencing and the resultant production of recombinant 1918 haemagglutinin (HA) protein antigen to characterize at the clonal level neutralizing antibodies induced by natural exposure of survivors to the 1918 pandemic virus. Here we show that of the 32 individuals tested that were born in or before 1915, each showed seroreactivity with the 1918 virus, nearly 90 years after the pandemic. Seven of the eight donor samples tested had circulating B cells that secreted antibodies that bound the 1918 HA. We isolated B cells from subjects and generated five monoclonal antibodies that showed potent neutralizing activity against 1918 virus from three separate donors. These antibodies also cross-reacted with the genetically similar HA of a 1930 swine H1N1 influenza strain, but did not cross-react with HAs of more contemporary human influenza viruses. The antibody genes had an unusually high degree of somatic mutation. The antibodies bound to the 1918 HA protein with high affinity, had exceptional virus-neutralizing potency and protected mice from lethal infection. Isolation of viruses that escaped inhibition suggested that the antibodies recognize classical antigenic sites on the HA surface. Thus, these studies demonstrate that survivors of the 1918 influenza pandemic possess highly functional, virus-neutralizing antibodies to this uniquely virulent virus, and that humans can sustain circulating B memory cells to viruses for many decades after exposure-well into the tenth decade of life.     

含抗HIV广谱中和抗体2F5、4E10靶基因载体的构建及鉴定

目的:构建含HIV gp120,gp41序列中广谱中和抗体2F5,4E10作用靶基因的载体并进行鉴定,为后期重组载体表达产物诱导产生中和抗体及抗HIV亚单位疫苗的研究奠定基础.方法:根据NCBI中HIV gp120,gp41基因序列中可与2F5、4E10结合的区域设计引物并进行PCR反应,将PCR得到的目的片段插入到载体pET28a中,对重组载体进行PCR鉴定、酶切鉴定及DNA测序.结果:PCR鉴定、酶切鉴定及DNA测序结果证实重组载体构建成功.结论:成功构建了含HIV gp120,gp41序列中广谱中和抗体2F5,4E10作用靶基因的载体.     

Fc receptor but not complement binding is important in antibody protection against HIV

Most successful vaccines elicit neutralizing antibodies and this property is a high priority when developing an HIV vaccine. Indeed, passively administered neutralizing antibodies have been shown to protect against HIV challenge in some of the best available animal models. For example, antibodies given intravenously can protect macaques against intravenous or mucosal SHIV (an HIV/SIV chimaera) challenge and topically applied antibodies can protect macaques against vaginal SHIV challenge. However, the mechanism(s) by which neutralizing antibodies afford protection against HIV is not understood and, in particular, the role of antibody Fc-mediated effector functions is unclear. Here we report that there is a dramatic decrease in the ability of a broadly neutralizing antibody to protect macaques against SHIV challenge when Fc receptor and complement-binding activities are engineered out of the antibody. No loss of antibody protective activity is associated with the elimination of complement binding alone. Our in vivo results are consistent with in vitro assays indicating that interaction of Fc-receptor-bearing effector cells with antibody-complexed infected cells is important in reducing virus yield from infected cells. Overall, the data suggest the potential importance of activity against both infected cells and free virus for effective protection against HIV.     

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.     

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.     

Critical role of an eight-amino acid sequence of VP1 in neutralization of poliovirus type 3

The three serotypes of poliovirus are members of the picornaviradae, a group of viruses which cause a variety of diseases of considerable importance in man and animals. We have previously used antigenic mutants resistant to neutralizing monoclonal antibodies to identify a single antigenic site for the neutralization of poliovirus type 3 (ref. 1). Evidence based on oligonucleotide mapping suggested that this site corresponded largely to one physicochemical region of the capsid protein viral polypeptide 1 (VP1). We now present conclusive evidence that most of the mutations conferring resistance to neutralization are confined to an eight-amino acid region of VP1, specified by a sequence of viral RNA 277-300 bases from the start of the region coding for VP1. These data strongly suggest that this small region constitutes a major antigenic site involved in virus neutralization and they provide the most detailed information currently available on the antigenic site of a human virus.     

Repeated epitope in the recombinant epitope-peptide could enhance ELDKWA-epitope-specific antibody response

Based on the hypothesis suggested by us that epitope-vaccine may be a new strategy against HIV mutation, we have studied several neutralizing epitopes on HIV envelope proteins. However we do not know whether a repeated epitope in a recombinant epitope-peptide can enhance epitope-specific antibody response or not. ELDKWA-epitope (aa669-674) on the C-domain of HIV-1 gp41 is a neutralizing epitope defined by the monoclonal antibody (mAb) 2F5 with broad neutralizing activity. In this study, we designed and prepared a series of the recombinant epitope-peptides bearing 1, 4 and 8 copies of ELDKWA-epitope respectively. In the comparison of the antisera induced by the three recombinant antigens, an obviously increased titre of ELDKWA-epitope-specific antibody was observed in the case of four and eight repeated epitopes. In flow cytometry analysis, the epitope-specific antibodies in both antisera showed stronger activity to bind the transfected CHO-WT cells that stably express HIV-1 envelope glycoprotein on the cell surfaces. These experimental results indicated that repeated epitope in the recombinant epitope-peptide could enhance ELDKWA-epitope-specific antibody response, which could contribute to designing an effective recombinant epitope-vaccine.     

Rapid cloning of high-affinity human monoclonal antibodies against influenza virus

Pre-existing neutralizing antibody provides the first line of defence against pathogens in general. For influenza virus, annual vaccinations are given to maintain protective levels of antibody against the currently circulating strains. Here we report that after booster vaccination there was a rapid and robust influenza-specific IgG+ antibody-secreting plasma cell (ASC) response that peaked at approximately day 7 and accounted for up to 6% of peripheral blood B cells. These ASCs could be distinguished from influenza-specific IgG+ memory B cells that peaked 14-21 days after vaccination and averaged 1% of all B cells. Importantly, as much as 80% of ASCs purified at the peak of the response were influenza specific. This ASC response was characterized by a highly restricted B-cell receptor (BCR) repertoire that in some donors was dominated by only a few B-cell clones. This pauci-clonal response, however, showed extensive intraclonal diversification from accumulated somatic mutations. We used the immunoglobulin variable regions isolated from sorted single ASCs to produce over 50 human monoclonal antibodies (mAbs) that bound to the three influenza vaccine strains with high affinity. This strategy demonstrates that we can generate multiple high-affinity mAbs from humans within a month after vaccination. The panel of influenza-virus-specific human mAbs allowed us to address the issue of original antigenic sin (OAS): the phenomenon where the induced antibody shows higher affinity to a previously encountered influenza virus strain compared with the virus strain present in the vaccine. However, we found that most of the influenza-virus-specific mAbs showed the highest affinity for the current vaccine strain. Thus, OAS does not seem to be a common occurrence in normal, healthy adults receiving influenza vaccination.     

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.     

Characterization of the Antigenicity and Immunogenicity of the Escherichia Coli Produced RNase Domain of the Classical Swine Fever Virus Glycoprotein E

E^rns is a highly glycosylated envelope protein of classical swine fever virus (CSFV) with RNase activity. E^rns can induce neutralizing antibodies and provide immune protection against CSFV infection. In this study, the RNase domain of the E^rns was produced in Escherichia coli. Its reactivity with CSFV-positive sera and its ability to induce antibodies and to provide protective immunity were then investigated. The serological tests showed that the prokaryotically expressed RNase domain of the E^rns retained its antigenicity and induced high titers of humoral responses. However, only partial protection and a limited amount of neutralizing antibodies were demonstrated by an in vitro neutralization test and an immunization/challenge test. The results suggest that other essential factors rather than simply enhancing the immunogenicity of E^rns should be taken into consideration when E^rns is enrolled as one of the components of a candidate vaccine.     

Antibodies induced by multi?epitope vaccine showed inhibitory activity against heterologous influenza A virus (H3N2)

In this study, recognition of 4 recombinant viral proteins (GST?NHA1) by the antibodies induced by multi?epitope vaccine was testified. Inhibitory activities of these antibodies were also investigated in vitro against four heterologous influenza A viruses (H3N2). Three epitope?specific antibodies purified by affinity chromatography could reduce the plaque formation. Interestingly, the three neutralizing antibodies in combination showed obvious enhancement of inhibitory activity, suggesting that the development of recombinant multi?epitope vaccine might be an effective way against viral mutation.     

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.     

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.     

Location and primary structure of a major antigenic site for poliovirus neutralization

We have determined a major antigenic site for virus neutralization on the capsid protein VP1 of poliovirus type 3. Antigenic mutant viruses selected for resistance to individual monoclonal antibodies had point mutations concentrated in a region 277-294 bases downstream from the start of the region of viral RNA coding for VP1. These findings provide the basis for an improved understanding of the molecular basis of virus neutralization.     

T-cell responses to human AIDS virus in macaques immunized with recombinant vaccinia viruses

There is much interest in developing vaccines against acquired immune deficiency syndrome (AIDS), which is caused by a retrovirus termed human immunodeficiency virus (HIV). Isolates of this virus include human T-lymphotropic virus type III (HTLV-III), lymphadenopathy-associated virus (LAV), and AIDS-associated retrovirus (ARV). Several approaches towards the development of an AIDS vaccine result in the production of antibodies in subprimates. These methods involve the use of: antigens isolated from the AIDS virus; viral antigens expressed by transfected cells or by recombinant vaccinia viruses; and particular synthetic peptides of viral antigens. Because T-cell-mediated immunity (in addition to antibodies) is involved in resistance to diseases and death caused by various enveloped viruses, we sought to determine whether potential AIDS vaccines can induce T-cell responses against the AIDS virus. Here we report that immunization of non-human primates, Macaca fascicularis (macaques), with recombinant vaccinia viruses that express LAV envelope glycoproteins gp41 and gp110 results not only in the production of antibodies against the LAV envelope antigens but also in the generation of T-cells that proliferate and produce the lymphokine interleukin-2 (IL-2), in response to stimulation with purified LAV. We believe this is the first report demonstrating T-cell-mediated immunity to the virus that causes AIDS.     

Polyreactivity increases the apparent affinity of anti-HIV antibodies by heteroligation

During immune responses, antibodies are selected for their ability to bind to foreign antigens with high affinity, in part by their ability to undergo homotypic bivalent binding. However, this type of binding is not always possible. For example, the small number of gp140 glycoprotein spikes displayed on the surface of the human immunodeficiency virus (HIV) disfavours homotypic bivalent antibody binding. Here we show that during the human antibody response to HIV, somatic mutations that increase antibody affinity also increase breadth and neutralizing potency. Surprisingly, the responding naive and memory B cells produce polyreactive antibodies, which are capable of bivalent heteroligation between one high-affinity anti-HIV-gp140 combining site and a second low-affinity site on another molecular structure on HIV. Although cross-reactivity to self-antigens or polyreactivity is strongly selected against during B-cell development, it is a common serologic feature of certain infections in humans, including HIV, Epstein-Barr virus and hepatitis C virus. Seventy-five per cent of the 134 monoclonal anti-HIV-gp140 antibodies cloned from six patients with high titres of neutralizing antibodies are polyreactive. Despite the low affinity of the polyreactive combining site, heteroligation demonstrably increases the apparent affinity of polyreactive antibodies to HIV.     

Soluble CD4 molecules neutralize human immunodeficiency virus type 1

Human immunodeficiency virus (HIV) infection can bring about total collapse of the immune system by infecting helper T lymphocytes which express CD4, the molecule which mediates interaction between the cell surface and viral envelope glycoprotein gp120 (refs 3-10). HIV apparently escapes the effects of neutralizing antibodies in vivo by generating new variants which must still interact with CD4 to maintain a cycle of infection. One route to block HIV infection, therefore, could use solubilized CD4 protein to inhibit attachment of the virus to its target cell. We have used recombinant DNA techniques to generate soluble forms of CD4, and show here that these are potent inhibitors of HIV infection in vitro.     

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.