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1.
Current influenza virus vaccines protect mostly against homologous virus strains; thus, regular immunization with updated vaccine formulations is necessary to guard against the virus' hallmark remodeling of regions that mediate neutralization. Development of a broadly protective influenza vaccine would mark a significant advance in human infectious diseases research. Antibodies with broad neutralizing activity (nAbs) against multiple influenza virus strains or subtypes have been reported to bind the stalk of the viral hemagglutinin, suggesting that a vaccine based on this region could elicit a broadly protective immune response. Here we describe a hemagglutinin subunit 2 protein (HA2)-based synthetic peptide vaccine that provides protection in mice against influenza viruses of the structurally divergent subtypes H3N2, H1N1, and H5N1. The immunogen is based on the binding site of the recently described nAb 12D1, which neutralizes H3 subtype viruses, demonstrates protective activity in vivo, and, in contrast to a majority of described nAbs, appears to bind to residues within a single α-helical portion of the HA2 protein. Our data further demonstrate that the specific design of our immunogen is integral in the induction of broadly active anti-hemagglutinin antibodies. These results provide proof of concept for an HA2-based influenza vaccine that could diminish the threat of pandemic influenza disease and generally reduce the significance of influenza viruses as human pathogens.  相似文献   

2.
Despite widespread yearly vaccination, influenza leads to significant morbidity and mortality across the globe. To make a more broadly protective influenza vaccine, it may be necessary to elicit antibodies that can activate effector functions in immune cells, such as antibody-dependent cellular cytotoxicity (ADCC). There is growing evidence supporting the necessity for ADCC in protection against influenza and herpes simplex virus (HSV), among other infectious diseases. An HSV-2 strain lacking the essential glycoprotein D (gD), was used to create ΔgD-2, which is a highly protective vaccine against lethal HSV-1 and HSV-2 infection in mice. It also elicits high levels of IgG2c antibodies that bind FcγRIV, a receptor that activates ADCC. To make an ADCC-eliciting influenza vaccine, we cloned the hemagglutinin (HA) gene from an H1N1 influenza A strain into the ΔgD-2 HSV vector. Vaccination with ΔgD-2::HAPR8 was protective against homologous influenza challenge and elicited an antibody response against HA that inhibits hemagglutination (HAI+), is predominantly IgG2c, strongly activates FcγRIV, and protects against influenza challenge following passive immunization of naïve mice. Prior exposure of mice to HSV-1, HSV-2, or a replication-defective HSV-2 vaccine (dl5-29) does not reduce protection against influenza by ΔgD-2::HAPR8. This vaccine also continues to elicit protection against both HSV-1 and HSV-2, including high levels of IgG2c antibodies against HSV-2. Mice lacking the interferon-α/β receptor and mice lacking the interferon-γ receptor were also protected against influenza challenge by ΔgD-2::HAPR8. Our results suggest that ΔgD-2 can be used as a vaccine vector against other pathogens, while also eliciting protective anti-HSV immunity.

Influenza remains a global health threat. Seasonal strains of influenza A and B cause an estimated 5 million cases of severe infections and 500,000 deaths per year (1). Influenza pandemics have caused even greater morbidity and mortality. During the H1N1 pandemic of 1918 to 1919, 500 million people, approximately one-third of the world’s population at that time, were estimated to have been infected with this strain, leading to 50 million deaths (2). The H1N1 pandemic of 2009 is estimated to have caused up to 575,000 deaths (2). Currently, three types of influenza vaccines are offered annually in the United States: a recombinant virus expressing influenza proteins, chemically inactivated virus, and live attenuated virus (3). Regardless of the vaccine type, multiple strains are included to increase the chances of developing sufficient protection against major circulating influenza strains. However, these vaccines primarily elicit a neutralizing antibody response that is sensitive to changes in the influenza virus due to antigenic drift and shift (4). Antigenic drift results from an accumulation of random mutations in influenza antigens, like hemagglutinin (HA), altering sites recognized by the immune system (4). Influenza A strains can also undergo antigenic shift, whereby two different influenza strains infect the same cell to form a reassortant virus with new antigenic properties (4). Due to limited immunity in the population, these new strains are highly virulent, causing widespread epidemics and disease (4). With antigenic drift and shift, vaccine-mediated protection against circulating strains has been insufficient (5). Influenza vaccines that elicit more robust and long-term protection are therefore needed. Notably, if an influenza vaccine with ≥75% efficacy were to be broadly used in the United States, an estimated 19,500 deaths a year could be prevented and direct healthcare costs reduced by $3.5 billion (6).For many years, efforts to improve influenza vaccines have focused on eliciting an immune response for full, broad protection against both circulating and future strains of the virus. These studies have shown that, in general, neutralizing antibodies are sufficient for homologous protection (7). However, achieving heterologous protection may require more broadly neutralizing antibodies or nonneutralizing antibodies able to activate effector immune cells (5). Previous studies have found that passively transferred nonneutralizing monoclonal antibodies can be potently protective in a mouse influenza challenge model (810). Several novel strategies have attempted to generate a nonneutralizing response against influenza. For example, vaccines have been created to specifically target the conserved stem region of HA (1113).Nonneutralizing antibodies stimulate effector cell mechanisms, including antibody-mediated phagocytosis and antibody-dependent cellular cytotoxicity (ADCC), both of which require activation of the Fcγ receptors (FcγRs) (14). Specific isotypes of IgG antibodies are associated with FcγR modulation and subsequent ADCC activation, including the IgG1 and IgG3 subtypes in humans, as well as IgG2a and IgG2c subtypes in mice (1519). IgG2a and IgG2c isotypes are functionally equivalent and mouse strain-dependent, with IgG2c present in C57BL/6J mice (20). Recent studies have demonstrated that natural infection by influenza and vaccination elicit nonneutralizing antibodies with effector functions that contribute to protection (5, 9, 2127). In mouse and nonhuman primate challenge models, ADCC-mediating antibodies have demonstrated protection against both homologous and heterologous influenza challenge (9, 28).Recently, we developed a single-cycle herpes simplex virus (HSV) vaccine that completely protects against vaginal, skin, and ocular challenges by HSV-1 and HSV-2 (29, 30). Protection elicited by this vaccine, designated ΔgD-2 for its lack of the essential glycoprotein D (gD) gene, is transferable via passive infusion of immune sera to naïve wild-type mice but not to mice lacking the Fcγ common chain (30). The immune response elicited by ΔgD-2 primarily elicits nonneutralizing antibodies with high levels of FcγRIV-activating function.We asked whether ΔgD-2 could be used as a vaccine platform to induce broadly protective FcγRIV-activating antibodies against a heterologous antigen, such as influenza HA. In this study, we demonstrate that our recombinant vaccine, ΔgD-2::HAPR8, elicits protection against influenza with a high proportion of FcγRIV-activating antibodies. Additionally, anticipating the use of ΔgD-2 as a vaccine vector against other pathogens, we tested whether our construct would still be protective in mice lacking interferon (IFN) function. Many humans have inborn errors in their IFN signaling pathways, leading to more lethal outcomes in infection (31). Patients with such deficiencies are disproportionately represented among HSV encephalitis cases and are often diagnosed only after presenting with serious symptoms (3238). This at-risk population underscores the importance of eliciting protection against HSV in the absence of a functional IFN-α/β response. Additionally, many pathogens, such as dengue virus, require mouse models lacking IFN function, and for ease of testing, an efficacious vaccine should remain functional in these mice (3941). In this study, we demonstrate that ΔgD-2 is a versatile, immunogenic vaccine vector that provides a strong FcγRIV-activating immune response against heterologous pathogens, while maintaining its protective benefit against HSV, in both wild-type and IFN-deficient mice.  相似文献   

3.
H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The monomer has some similarities to HA in the fusion pH conformation, and the monomer’s formation, which results from the presence of isopropanol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion.The initial steps in influenza virus infection involve sialic acid receptor binding and membrane fusion, both of which are functions of the hemagglutinin (HA) virus membrane glycoprotein. Anti-HA antibodies that block these functions neutralize virus infectivity. Such antibodies are induced by infection and by vaccination, and the immune pressure that they impose on subsequently infecting viruses is responsible for the antigenic drift for which influenza viruses are notorious. Zoonotic infections, which can lead to new pandemics, occur periodically, and H5N1, H7N9, and H10N8 avian viruses are recent examples of this sort. The threat that zoonotic infections present is based, in part, on the lack of immunity in the human population to the novel HAs that they contain. In attempts to substitute for this deficiency, human immune sera have been used successfully to treat severe infections (1), and monoclonal antibodies have been prepared from mice and from humans for potential use in immunotherapy.Analyses of antibodies produced by cloned immune cells derived from infected patients have revealed that antibodies are induced that are either subtype- or group-specific and others that cross-react with HAs of both groups (2). To date, cross-reactive antibodies have been shown to recognize both membrane-distal and membrane-proximal regions of HA (3). Subtype-specific antibodies, on the other hand, bind to the membrane-distal region, covering the receptor-binding site and, in some cases, inserting into it (4, 5).In the studies reported here, a human monoclonal antibody is described that recognizes the HAs of viruses of all three clades of the H5 subtype that have caused human infection and is shown to be effective in protecting mice from lethal challenge. EM and X-ray crystallography studies of HA-Fab complexes indicate that the antibody binds to a site containing residue 122, located on the membrane-distal surface of the HA trimer. We describe the antibody-binding site in detail to show that binding occurs at a distance from the receptor-binding site. Infectivity neutralization and receptor-binding experiments, together with these observations, lead to the conclusion that the antibody neutralizes viruses by blocking receptor binding in a way that is dependent on the Fc region of the bound antibody. We compare the site with similar sites reported by others (69) for antibodies that have not as yet given crystalline HA-Fab complexes.Under the conditions that we obtain crystals of the HA-Fab complex, the HA dissociates and reveals the structure of a monomeric HA. We consider the structure of the monomer in relation to the structure that HA has been shown to assume after exposure to the pH of membrane fusion.  相似文献   

4.
Development of an influenza vaccine that provides broadly cross-protective immunity has been a scientific challenge for more than half a century. This study presents an approach to overcome strain-specific protection by supplementing conventional vaccines with virus-like particles (VLPs) containing the conserved M2 protein (M2 VLPs) in the absence of adjuvants. We demonstrate that an inactivated influenza vaccine supplemented with M2 VLPs prevents disease symptoms without showing weight loss and confers complete cross protection against lethal challenge with heterologous influenza A viruses including the 2009 H1N1 pandemic virus as well as heterosubtypic H3N2 and H5N1 influenza viruses. Cross-protective immunity was long-lived, for more than 7 mo. Immune sera from mice immunized with M2 VLP supplemented vaccine transferred cross protection to naive mice. Dendritic and macrophage cells were found to be important for this cross protection mediated by immune sera. The results provide evidence that supplementation of seasonal influenza vaccines with M2 VLPs is a promising approach for overcoming the limitation of strain-specific protection by current vaccines and developing a universal influenza A vaccine.  相似文献   

5.
A vaccine which is effective against the HIV virus is considered to be the best solution to the ongoing global HIV/AIDS epidemic. In the past thirty years, numerous attempts to develop an effective vaccine have been made with little or no success, due, in large part, to the high mutability of the virus. More recent studies showed that a vaccine able to elicit broadly neutralizing antibodies (bnAbs), that is, antibodies that can neutralize a high fraction of global virus variants, has promise to protect against HIV. Such a vaccine has been proposed to involve at least three separate stages: First, activate the appropriate precursor B cells; second, shepherd affinity maturation along pathways toward bnAbs; and, third, polish the Ab response to bind with high affinity to diverse HIV envelopes (Env). This final stage may require immunization with a mixture of Envs. In this paper, we set up a framework based on theory and modeling to design optimal panels of antigens to use in such a mixture. The designed antigens are characterized experimentally and are shown to be stable and to be recognized by known HIV antibodies.

Vaccines are the most important medical countermeasure for protecting entire populations against viruses, of which smallpox and measles vaccines are successful examples. In fact, a safe and effective HIV vaccine is considered to be the best way to end the global AIDS epidemic (1). However, how to produce a universal vaccine for highly antigenically variable viruses like HIV is a daunting and yet unsolved problem. The high variability of this virus allows it to elude the immune system, making the produced antibodies ineffective; that is, they are generally specific for a given strain of the virus but not for other strains resulting from mutation. In some cases, HIV-infected patients can elicit antibodies that can recognize and neutralize a broad range of different viral strains (2, 3). These broadly neutralizing antibodies (bnAbs) usually take a long time to appear naturally in infected patients and then only in a subset of such individuals.The reason that bnAbs can arise is that even highly variable pathogens have regions with a well-defined, relatively conserved structure, which is required for their function. In HIV, entry depends on the trimeric spike exposed on the external lipid membrane of the virion, a heterotrimer formed by the gp120 and gp41 glycoproteins produced by posttranslational cleavage of a gp160 precursor. This protein binds to the CD4 coreceptor on CD4 T lymphocytes during HIV infection, and it has some relatively conserved regions that can be used as a target for bnAbs. Indeed, many bnAbs target the CD4 binding site (CD4bs) (48). If naive B cells that can bind to one of these relatively conserved regions can be expanded upon exposure to different variants of the virus, antibodies could evolve to better recognize the conserved portions, while avoiding the variable ones. The resulting antibodies can acquire breadth in this way, thereby becoming bnAbs. A successful vaccine would contain immunogens that can guide the immune system to produce bnAbs, rather than strain-specific antibodies.In the past, numerous approaches for the development of an effective HIV vaccine have been tried. They include the use of cleverly chosen natural HIV proteins, the design of a consensus (9) or “center-of-tree” (10) antigens, and the creation of a mosaic protein from different HIV strains (11). All these methods used a single optimized antigen in the vaccine and were shown to be ineffective at eliciting bnAbs (12, 13). One possible reason for this is that, when exposed to a single antigen, the immune system will produce antibodies specific for that particular antigen, and neutralization escape variants can easily develop. A possible solution is to use more than one antigen in a vaccination protocol. This raises a number of questions: How many antigens are necessary? How different should they be from each other? And in what temporal order should they be administered? Answering such questions is far from trivial, in particular due to the limited mechanistic understanding of affinity maturation (AM) in vivo. Another problem is that bnAbs have an unusually high number of somatic mutations, not only in the complementarity-determining regions (CDRs) but also in the immunoglobulin framework regions (7, 14). Recent computational data on the flexibility of the antibody and the need for framework mutations in the simulated AM showed how important it is for a vaccination protocol to have a specific antigen that can prime a good antibody precursor B cell receptor (BCR) (15). Moreover, it has been shown that putative precursors of known classes of bnAbs are generally not able to neutralize HIV or recognize envelope (Env), often due to clashes of the antibody with the glycosylation shield that protects the HIV Env protein (8, 1618). For example, VRC01-class bnAbs are known to introduce a deletion or a mutation to a flexible glycine in the CDRL1 loop to avoid the glycan at N276 (19, 20).The above discussion led to the proposal of a vaccination strategy consisting of three steps. First, a special purpose antigen is used to activate the correct naïve or precursor B cell (17, 21). Since this precursor will generally not bind to native HIV, as a second step, one or more antigens are used as intermediates to induce somatic mutations and to allow recognition of the native virus. In the third step, one or more antigens are used in a mixture or in sequence to increase the breadth of the antibody population (19, 22, 23). Implementations of the first and second steps have already been shown to be promising in experiments (16, 21, 2427). However, much less is known about the third step. Some insights into this question can be obtained by in silico simulations of AM. Using coarse-grained models, it has been shown that, while administering a single mixture containing multiple antigens may induce too much frustration to lead to bnAbs formation, a sequential approach, in which antigens are administered one after another, seems to be more effective (23). It was also observed that the number of antigens required in a mixture is correlated with their sequence dissimilarity, and optimal breadth is obtained at an optimal number of antigens and dissimilarity (28). Given the coarse-grained nature of these studies, the actual antigen sequences to use in experiments cannot be obtained from them.In this work, we focus on the third step of the proposed vaccination protocol. In particular, we derive a set of empirical rules and protocols to select an optimal panel of antigens to maximize the breadth of the produced antibodies upon AM. To be able to do so, it is essential to understand, at an atomistic level of detail, the role of each antigen amino acid in the antibody/antigen interaction. This aspect will be presented in the next section based on an analysis of the available crystallographic structures of bnAbs bound to the gp160 Env glycoprotein. However, the structures do not provide information concerning HIV stability and function. For example, generating antigen sequences by introducing purely random mutations will likely lead to sequences that are lethal for the virus and/or are not representative of HIV in vivo. To overcome this problem, it is useful to consider the structural data together with a model of the gp160 fitness landscape (29), which is a measure of the ability of HIV to tolerate mutations in its gp160 sequence to escape immune pressure. Structural and fitness information together provide a classification of the antibody/antigen interface and indicate the residues to mutate and the amino acids that are more probable at those positions.While this analysis helps to reduce the number of antigen sequences to consider by highlighting the “hot spots” of antibody/antigen binding, it leaves open the question of how to select a combination of antigen sequences for use in a vaccine. Given rules of optimal sequence dissimilarity and optimal fitness according to the HIV landscape, a Pareto frontier approach will be described. It is able to select, from all possible panels of antigen sequences, the few that are predicted to best elicit antibodies with a broad activity spectrum. Experimental evidence of the viability of the designed antigens and of their immunogenic properties is presented in the final section.  相似文献   

6.
Influenza pandemics require that a virus containing a hemagglutinin (HA) surface antigen previously unseen by a majority of the population becomes airborne-transmissible between humans. Although the HA protein is central to the emergence of a pandemic influenza virus, its required molecular properties for sustained transmission between humans are poorly defined. During virus entry, the HA protein binds receptors and is triggered by low pH in the endosome to cause membrane fusion; during egress, HA contributes to virus assembly and morphology. In 2009, a swine influenza virus (pH1N1) jumped to humans and spread globally. Here we link the pandemic potential of pH1N1 to its HA acid stability, or the pH at which this one-time-use nanomachine is either triggered to cause fusion or becomes inactivated in the absence of a target membrane. In surveillance isolates, our data show HA activation pH values decreased during the evolution of H1N1 from precursors in swine (pH 5.5–6.0), to early 2009 human cases (pH 5.5), and then to later human isolates (pH 5.2–5.4). A loss-of-function pH1N1 virus with a destabilizing HA1-Y17H mutation (pH 6.0) was less pathogenic in mice and ferrets, less transmissible by contact, and no longer airborne-transmissible. A ferret-adapted revertant (HA1-H17Y/HA2-R106K) regained airborne transmissibility by stabilizing HA to an activation pH of 5.3, similar to that of human-adapted isolates from late 2009–2014. Overall, these studies reveal that a stable HA (activation pH ≤ 5.5) is necessary for pH1N1 influenza virus pathogenicity and airborne transmissibility in ferrets and is associated with pandemic potential in humans.Wild aquatic birds are thought to be the natural reservoir of influenza A viruses (1). Influenza pandemics occur every few decades, and swine are widely believed to be a key factor in the genesis of pandemics by facilitating reassortment of the eight viral gene segments and replacing avian-like (α-2,3-linked) hemagglutinin (HA) sialic acid receptor-binding specificity with human-like (α-2,6-linked) (2). If the molecular adaptations that allow efficient human-to-human transmissibility are understood, then circulating viruses undergoing these changes (i.e., those with the greatest pandemic potential) could be identified.In 2009, pandemic (p) H1N1 emerged from swine and swiftly infected more than 60 million people, causing 12,000 US deaths in the first year (3). The pandemic strain originated by reassortment in swine, combining five genes (PB1, PB2, PA, NP, and NS) from North American triple-reassortant swine (TRS) viruses, two genes (NA and M) from Eurasian avian-like swine viruses, and an HA gene closely related to that of the classical swine lineage (4). pH1N1 viruses continue to circulate as seasonal H1N1 viruses. They retain several known pandemic traits, including α-2,6-linked sialic acid receptor-binding specificity of the HA, functional balance of HA and NA activity, and a polymerase adapted to the mammalian upper airway (5). Although these traits appear to be necessary for airborne transmissibility of influenza viruses, they do not appear to be sufficient. For example, H5N1 viruses engineered to have these traits were not air-transmissible among ferrets until a mutation increased HA thermostability and lowered the HA activation pH (68). The importance of HA stabilization in supporting the adaptation of influenza viruses to humans or enabling a human pandemic is not completely understood.After receptor binding and endocytosis, low pH triggers irreversible structural changes in the HA protein that fuse the viral envelope and host endosomal membrane (9). Measured HA activation pH values across all subtypes and species range from ∼5.0 to 6.0, trending higher in avian viruses (pH 5.6–6.0) and lower in human viruses (pH 5.0–5.5) (10).The goal of this study was to define the role of HA acid stability in pH1N1 pandemic capability. Our data show that HA activation pH decreased as H1N1 adapted from swine to humans. Complementary experiments in ferrets recapitulated this evolution, as we observed a loss-of-function pH1N1 virus acquired airborne transmissibility via stabilizing mutations. Overall, these studies link a fundamental molecular property, the barrier for activation of a membrane fusion protein (for influenza virus HA, its acid stability), to the interspecies adaptation of a ubiquitous respiratory virus.  相似文献   

7.
Four peptides have been synthesized, corresponding to different regions of the H3 influenza hemagglutinin, that are related to antigenic sites "A" and "B" of the molecule. The peptides consisted of the following sequences: 139-146, which forms the "loop" in the native hemagglutinin molecule, with either glycine or aspartic acid at position 144; 147-164, which contains part of antigenic determinant "B"; and 138-164, which comprises both the loop and the area 147-164. The peptides were conjugated to tetanus toxoid and used for immunization of rabbits and mice. All four conjugates elicited an immune response against the homologous peptides, but only the peptides 138-164 and 147-164 gave rise to antibodies that recognized and bound to the intact virus. Protection of mice against challenge infection with A/Eng/42/72 virus was achieved only by immunization with the conjugate (138-164)-TT, which led to partial protective effect. These data emphasize the role of molecular structure in determining the antigenic properties of synthetic peptides and indicate that the length of the peptide could be crucial for enforcing the right folding required to mimic the native structure.  相似文献   

8.

Background  

Maternal antibody is the major form of protection against disease in early life; however, its presence interferes with active immunization of offspring. In order to overcome the immunosuppression caused by maternal antibody, several immune strategies were explored in this paper using mouse model and influenza vaccines.  相似文献   

9.
Major ME 《Viruses》2009,1(2):144-165
Studies in patients and chimpanzees that spontaneously clear Hepatitis C Virus (HCV) have demonstrated that natural immunity to the virus is induced during primary infections and that this immunity can be cross protective. These discoveries led to optimism regarding prophylactic HCV vaccines and a number of studies in the chimpanzee model have been performed, all of which resulted in modified infections after challenge but did not always prevent persistence of the virus. Therapeutic vaccine strategies have also been pursued in an effort to reduce the costs and side effects associated with anti-viral drug treatment. This review summarizes the studies performed thus far in both patients and chimpanzees for prophylactic and therapeutic vaccination, assesses the progress made and future perspectives.  相似文献   

10.
We previously reported that some of the rare broadly reactive, HIV-1 neutralizing antibodies are polyreactive, leading to the hypothesis that induction of these types of neutralizing antibody may be limited by immunologic tolerance. However, the notion that such antibodies are sufficiently autoreactive to trigger B cell tolerance is controversial. To test directly whether rare neutralizing HIV-1 antibodies can activate immunologic tolerance mechanisms, we generated a knock-in mouse in which the Ig heavy chain (HC) variable region rearrangement (VHDJH) from the polyreactive and broadly neutralizing human monoclonal antibody 2F5 was targeted into the mouse Igh locus. In vitro, this insertion resulted in chimeric human/mouse 2F5 antibodies that were functionally similar to the human 2F5 antibody, including comparable reactivity to human and murine self-antigens. In vivo, the 2F5 VHDJH insertion supported development of large- and small pre-B cells that expressed the chimeric human/mouse Igμ chain but not the production of immature B cells expressing membrane IgM. The developmental arrest exhibited in 2F5 VHDJH knock-in mice is characteristic of other knock-in strains that express the Ig HC variable region of autoreactive antibodies and is consistent with the loss of immature B cells bearing 2F5 chimeric antibodies to central tolerance mechanisms. Moreover, homozygous 2F5 VHDJH knock-in mice support reduced numbers of residual splenic B cells with low surface IgM density, severely diminished serum IgM levels, but normal to elevated quantities of serum IgGs that did not react with autoantigens. These features are consistent with elimination of 2F5 HC autoreactivity by additional negative selection mechanism(s) in the periphery.  相似文献   

11.
Human cytomegalovirus (CMV) utilizes different glycoproteins to enter into fibroblast and epithelial cells. A trimer of glycoproteins H, L, and O (gH/gL/gO) is required for entry into all cells, whereas a pentamer of gH/gL/UL128/UL130/UL131A is selectively required for infection of epithelial, endothelial, and some myeloid-lineage cells, but not of fibroblasts. Both complexes are of considerable interest for vaccine and immunotherapeutic development but present a conundrum: gH/gL-specific antibodies have moderate potency yet neutralize CMV entry into all cell types, whereas pentamer-specific antibodies are more potent but do not block fibroblast infection. Which cell types and neutralizing activities are important for protective efficacy in vivo remain unclear. Here, we present evidence that certain CMV strains have evolved polymorphisms in gO to evade trimer-specific neutralizing antibodies. Using luciferase-tagged variants of strain TB40/E in which the native gO is replaced by gOs from other strains, we tested the effects of gO polymorphisms on neutralization by monoclonal antibodies (mAbs) targeting four independent epitopes in gH/gL that are common to both trimer and pentamer. Neutralization of fibroblast entry by three mAbs displayed a range of potencies that depended on the gO type, a fourth mAb failed to neutralize fibroblast entry regardless of the gO type, while neutralization of epithelial cell entry by all four mAbs was potent and independent of the gO type. Thus, specific polymorphisms in gO protect the virus from mAb neutralization in the context of fibroblast but not epithelial cell entry. No influence of gO type was observed for protection against CMV hyperimmune globulin or CMV-seropositive human sera, suggesting that antibodies targeting protected gH/gL epitopes represent a minority of the polyclonal neutralizing repertoire induced by natural infection.  相似文献   

12.
The conserved oligomannose epitope, Man9GlcNAc2, recognized by the broadly neutralizing human mAb 2G12 is an attractive prophylactic vaccine candidate for the prevention of HIV-1 infection. We recently reported total chemical synthesis of a series of glycopeptides incorporating one to three copies of Man9GlcNAc2 coupled to a cyclic peptide scaffold. Surface plasmon resonance studies showed that divalent and trivalent, but not monovalent, compounds were capable of binding 2G12. To test the efficacy of the divalent glycopeptide as an immunogen capable of inducing a 2G12-like neutralizing antibody response, we covalently coupled the molecule to a powerful immune-stimulating protein carrier and evaluated immunogenicity of the conjugate in two animal species. We used a differential immunoassay to demonstrate induction of high levels of carbohydrate-specific antibodies; however, these antibodies showed poor recognition of recombinant gp160 and failed to neutralize a panel of viral isolates in entry-based neutralization assays. To ascertain whether antibodies produced during natural infection could recognize the mimetics, we screened a panel of HIV-1-positive and -negative sera for binding to gp120 and the synthetic antigens. We present evidence from both direct and competitive binding assays that no significant recognition of the glycopeptides was observed, although certain sera did contain antibodies that could compete with 2G12 for binding to recombinant gp120.  相似文献   

13.
目的 以宠物犬为研究对象,监测在自然感染条件下其体内乙型脑炎病毒(JEV)中和抗体随流行季节的变化规律, 进而评估JEV感染人的风险性。方法 按月采集不同年龄的宠物犬血液,分离血清,用微量细胞中和试验检测血清JEV中和抗体。结果 共得到宠物犬血清253份,中和抗体阳性率达到28.1%。不同季节间犬JEV中和抗体阳性率差异显著,JEV流行季节(6-9月)之前(3-5月)和之后(10-12月)的3个月中和抗体阳性率分别为16.3%和43.4%,差异有统计学意义(χ2=14.432, P<0.01)。小于1岁与大于1岁的宠物犬中和抗体阳性率分别为24.1%和39.0%,二者差异显著(χ2=5.633, P<0.05)。而犬的性别间中和抗体阳性率显著无统计学意义(χ2=1.860, P>0.05)。结论 在JE流行季节宠物犬受到JEV自然感染的机率较高,可作为哨兵动物来评估JEV感染人的风险。  相似文献   

14.
The efficacy of vaccination against Haemonchus contortus infection with two recombinant proteins, rHco-gal-m and rHco-gal-f, was studied in 9-10-month-old goats. Vaccination with 100 microg protein reduced faecal egg output and worm burdens by 37.25% and 41.1%, respectively. Corresponding reductions with 200 microg protein were 48.03% and 46.19%. Vaccinated groups had significantly higher IgG levels than the negative and positive controls. Significant negative correlations were detected between IgG level, mucosal homogenate IgA concentration, haemoglobin and abomasal worm burden at necropsy. By contrast a positive correlation was found between the percentage of B cells, monocytes and abomasal worm burden. These findings suggested that vaccination with a combination of recombinant rHco-gal-m/f proteins had a role in protecting goats against H. contortus infection.  相似文献   

15.
Influenza prophylaxis would benefit from a simple method to administer influenza vaccine into skin without the need for hypodermic needles. In this study, solid metal microneedle arrays (MNs) were investigated as a system for cutaneous vaccine delivery using influenza virus antigen. The MNs with 5 monument-shaped microneedles per array were produced and coated with inactivated influenza virus A/PR/8/34 (IIV). As much as 10 μg of viral proteins could be coated onto an array of 5 microneedles, and the coated IIV was delivered into skin at high efficiency within minutes. The coated MNs were used to immunize mice in comparison with conventional intramuscular injection at the same dose. Analysis of immune responses showed that a single immunization with IIV-coated MNs induced strong antibody responses against influenza virus, with significant levels of hemagglutination inhibition activities (>1:40), which were comparable to those induced by conventional intramuscular immunization. Moreover, mice immunized by a single dose of IIV coated on MNs were effectively protected against lethal challenge by a high dose of mouse-adapted influenza virus A/PR/8/34. These results show that MNs are highly effective as a simple method of vaccine delivery to elicit protective immune responses against virus infection.  相似文献   

16.
Ebola hemorrhagic fever is an acute and often deadly disease caused by Ebola virus (EBOV). The possible intentional use of this virus against human populations has led to design of vaccines that could be incorporated into a national stockpile for biological threat reduction. We have evaluated the immunogenicity and efficacy of an EBOV vaccine candidate in which the viral surface glycoprotein is biomanufactured as a fusion to a monoclonal antibody that recognizes an epitope in glycoprotein, resulting in the production of Ebola immune complexes (EICs). Although antigen-antibody immune complexes are known to be efficiently processed and presented to immune effector cells, we found that codelivery of the EIC with Toll-like receptor agonists elicited a more robust antibody response in mice than did EIC alone. Among the compounds tested, polyinosinic:polycytidylic acid (PIC, a Toll-like receptor 3 agonist) was highly effective as an adjuvant agent. After vaccinating mice with EIC plus PIC, 80% of the animals were protected against a lethal challenge with live EBOV (30,000 LD(50) of mouse adapted virus). Surviving animals showed a mixed Th1/Th2 response to the antigen, suggesting this may be important for protection. Survival after vaccination with EIC plus PIC was statistically equivalent to that achieved with an alternative viral vector vaccine candidate reported in the literature. Because nonreplicating subunit vaccines offer the possibility of formulation for cost-effective, long-term storage in biothreat reduction repositories, EIC is an attractive option for public health defense measures.  相似文献   

17.
The four dengue virus (DENV) serotypes, DENV-1, -2, -3, and -4, are endemic throughout tropical and subtropical regions of the world, with an estimated 390 million acute infections annually. Infection confers long-term protective immunity against the infecting serotype, but secondary infection with a different serotype carries a greater risk of potentially fatal severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. The single most effective measure to control this threat to global health is a tetravalent DENV vaccine. To date, attempts to develop a protective vaccine have progressed slowly, partly because the targets of type-specific human neutralizing antibodies (NAbs), which are critical for long-term protection, remain poorly defined, impeding our understanding of natural immunity and hindering effective vaccine development. Here, we show that the envelope glycoprotein domain I/II hinge of DENV-3 and DENV-4 is the primary target of the long-term type-specific NAb response in humans. Transplantation of a DENV-4 hinge into a recombinant DENV-3 virus showed that the hinge determines the serotype-specific neutralizing potency of primary human and nonhuman primate DENV immune sera and that the hinge region both induces NAbs and is targeted by protective NAbs in rhesus macaques. These results suggest that the success of live dengue vaccines may depend on their ability to stimulate NAbs that target the envelope glycoprotein domain I/II hinge region. More broadly, this study shows that complex conformational antibody epitopes can be transplanted between live viruses, opening up similar possibilities for improving the breadth and specificity of vaccines for influenza, HIV, hepatitis C virus, and other clinically important viral pathogens.The four dengue virus serotypes (DENV-1, -2, -3, and -4), transmitted by Aedes spp. mosquitoes, are endemic throughout tropical and subtropical regions of the world, with an estimated 390 million new infections annually (1). Primary infection with one serotype confers long-term immunity against that serotype, but repeat infection with a different serotype has an increased risk of potentially fatal severe dengue disease (2), including dengue hemorrhagic fever and dengue shock syndrome. This risk has been attributed, at least in part, to the ability of some cross-reactive antibodies to enhance infection of Fc-receptor bearing cells. The consensus is that, to be safe and effective, any dengue vaccine must simultaneously induce neutralizing antibodies (NAbs) to all four serotypes. However, DENV vaccine development has progressed slowly, highlighted by the disappointing results of the live-attenuated Sanofi Pasteur tetravalent DENV vaccine trial in Thailand (3). Progress is hindered, in part, because the epitopes targeted by the type-specific human NAbs critical for long-term protection (4, 5) remain poorly defined, limiting our understanding of natural DENV immunity and slowing effective vaccine development.The DENV envelope glycoprotein (E) (Fig. 1A) is the major surface-exposed DENV antigen and the principle target of NAbs. The E structure consists of three distinct domains: I, II, and III (EDI, EDII, and EDIII) (6, 7); EDIII is a continuous peptide extending from domain I and forming an Ig-like fold, whereas EDI and EDII are discontinuous and connect by four peptide linkers that form the EDI/EDII hinge. We and others have recently described potent human DENV NAbs that bind to epitopes around the EDI/EDII hinge (8, 9). To more fully explore the significance of this antigenic region, we used reverse genetics and synthetic biology to transplant the EDI/EDII antigenic region from DENV-4 into a DENV-3 background and showed by both gain- and loss-of-function assays that the EDI/EDII hinge region is the primary target of the long-lived DENV serotype-specific NAb response.Open in a separate windowFig. 1.(A) Cartoon representation of the DENV-3 E dimer with EDI (red), EDII (yellow), and EDIII (blue). The EDI/EDII hinge region is circled. Location of the critical residues associated with escape mutations and mutagenesis-mapped 5J7 residues are shown. Residues critical for 5J7 binding identified by shotgun mutagenesis loss of binding of E glycoprotein expressed in HEK-293T cells are shown in magenta. Mutations associated with both viral escape from 5J7 and loss of binding in HEK-293T cells (L53 and K128) are shown in orange, and the single residue identified by escape mutation alone (Q269K270_insK) is shown in cyan. All critical residues were individually identified but are shown on a single E dimer for simplicity. (B) Cartoon representation of the DENV E dimer with the locations of variable EDI/EDII hinge residues transplanted between rDENV-3 and rDENV-4 to make rDENV-3/4 shown in green. (C) Primary sequence alignment and secondary structure of rDENV-3 E, rDENV-3/4 E, and rDENV-4 E. Secondary structure is indicated above the primary sequence and color-coded to the corresponding domains on the tertiary structure (A and B). Arrows indicate β-sheets, cylinders indicate helices, and lines indicate spanning loops and strands. Binding, escape, and hinge residues shown in A and B are indicated by corresponding colors in the rDENV-3 sequence. Amino acid residues transplanted between rDENV-3/4 and rDENV-4 are indicated in green for both sequences.  相似文献   

18.
The four dengue virus serotypes (DENV1–4) are mosquito-borne flaviviruses that infect ∼390 million people annually; up to 100 million infections are symptomatic, and 500,000 cases progress to severe disease. Exposure to a heterologous DENV serotype, the specific infecting DENV strains, and the interval of time between infections, as well as age, ethnicity, genetic polymorphisms, and comorbidities of the host, are all risk factors for severe dengue. In contrast, neutralizing antibodies (NAbs) are thought to provide long-lived protection against symptomatic infection and severe dengue. The objective of dengue vaccines is to provide balanced protection against all DENV serotypes simultaneously. However, the association between homotypic and heterotypic NAb titers and protection against symptomatic infection remains poorly understood. Here, we demonstrate that the titer of preinfection cross-reactive NAbs correlates with reduced likelihood of symptomatic secondary infection in a longitudinal pediatric dengue cohort in Nicaragua. The protective effect of NAb titers on infection outcome remained significant when controlled for age, number of years between infections, and epidemic force, as well as with relaxed or more stringent criteria for defining inapparent DENV infections. Further, individuals with higher NAb titers immediately after primary infection had delayed symptomatic infections compared with those with lower titers. However, overall NAb titers increased modestly in magnitude and remained serotype cross-reactive in the years between infections, possibly due to reexposure. These findings establish that anti-DENV NAb titers correlate with reduced probability of symptomatic DENV infection and provide insights into longitudinal characteristics of antibody-mediated immunity to DENV in an endemic setting.Dengue virus (DENV) is a mosquito-borne flavivirus that infects up to 390 million individuals each year (1). Although most infections are inapparent, ∼25% of infections cause acute febrile illness, which progresses to severe disease in half a million individuals annually (2). DENV consists of four evolutionarily distinct, antigenically related DENV serotypes, DENV1–4, and neutralizing antibodies (NAbs) against the four serotypes are considered a critical component of the protective immune response (3, 4). Primary (1°) DENV infection induces a NAb response that is described as increasingly type-specific over time, providing long-term protection against the 1° infecting serotype, but only transient protection against other DENV serotypes (5, 6). Cross-serotype protection against symptomatic infection is observed for up to 2 years after 1° infection, after which point individuals are at increased risk of symptomatic infection and severe dengue upon subsequent heterologous infection (710). Over time, cross-serotype–reactive antibodies are thought to decay to subneutralizing levels, binding, but not neutralizing, DENV and contributing to enhanced replication during heterologous infection by facilitating virus entry into target cells expressing Fc receptors (11). However, after subsequent infection with a different serotype, the NAb response becomes broadly neutralizing and is thought to reduce incidence of severe disease (12).There has been limited success in establishing the relationship between the level of preinfection NAb titers to DENV and risk of disease upon subsequent DENV infection in endemic settings. In recent vaccine trials, symptomatic disease was observed in individuals with relatively high NAb titers, raising concerns that the current immunologic assays do not measure the NAbs critical for protection (13). In studies of infants, who receive IgG antibodies by transplacental transfer from DENV-immune mothers, infants with higher NAb titers at birth generally, although not always, experienced symptomatic disease later than those with lower titers (1416). Recent studies in children and adults have made important advances in demonstrating an association between the quantity of cross-reactive preinfection NAb titers and reduced risk of symptomatic secondary (2°) infection, defined as two or more infections, but have not been conclusive: the association did not hold for all DENV serotypes (15, 17); exposure could not be proven for DENV-negative individuals (18); or the magnitude of preinfection NAb titers was not directly studied (12, 19). Thus, there is an urgent need to definitively establish whether NAb titers correlate with protection in endemic settings. Here, we estimated the relationship between preinfection NAb titers and probability of symptomatic infection and characterized determinants of long-term protection in children with multiple DENV infections in a pediatric dengue cohort study in Nicaragua.  相似文献   

19.
A DNA copy of the influenza virus hemagglutinin gene, derived from influenza virus A/Jap/305/57 (H2N2) was inserted into the genome of vaccinia virus under the control of an early vaccinia virus promoter. Tissue culture cells infected with the purified recombinant virus synthesized influenza hemagglutinin, which was glycosylated and transported to the cell surface where it could be cleaved with trypsin into HA1 and HA2 subunits. Rabbits and hamsters inoculated intradermally with recombinant virus produced circulating antibodies that inhibited hemagglutination by influenza virus. Furthermore, vaccinated hamsters achieved levels of antibody similar to those obtained upon primary infection with influenza virus and were protected against respiratory infection with the A/Jap/305/57 influenza virus.  相似文献   

20.
The ability of viruses to mutate and evade the human immune system and neutralizing antibodies remains an obstacle to antiviral and vaccine development. Many neutralizing antibodies, including some approved for emergency use authorization (EUA), reduced or lost activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. Here, we introduce a geometric deep learning algorithm that efficiently enhances antibody affinity to achieve broader and more potent neutralizing activity against such variants. We demonstrate the utility of our approach on a human antibody P36-5D2, which is effective against SARS-CoV-2 Alpha, Beta, and Gamma but not Delta. We show that our geometric neural network model optimizes this antibody’s complementarity-determining region (CDR) sequences to improve its binding affinity against multiple SARS-CoV-2 variants. Through iterative optimization of the CDR regions and experimental measurements, we enable expanded antibody breadth and improved potency by ∼10- to 600-fold against SARS-CoV-2 variants, including Delta. We have also demonstrated that our approach can identify CDR changes that alleviate the impact of two Omicron mutations on the epitope. These results highlight the power of our deep learning approach in antibody optimization and its potential application to engineering other protein molecules. Our optimized antibodies can potentially be developed into antibody drug candidates for current and emerging SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide over the past 2 y, causing hundreds of millions of confirmed infections and millions of deaths (1). The receptor-binding domain (RBD) of the SARS-CoV-2 virus spike protein initiates binding to the host receptor, angiotensin converting enzyme 2 (ACE2) (26), and serves as an initial essential step in viral–cell membrane fusion, as well as a potential target for neutralizing antibodies (710). Neutralizing antibodies that target RBD have already shown therapeutic and clinical value (1117).However, reduced sensitivity of SARS-CoV-2 variants to antibody and serum neutralization has been widely observed (1821). For example, the B.1.617 lineage, also known as the Delta variant, contains two mutations (L452R and T478K) in the RBD that facilitate viral escape—the ability of viruses to evade the immune system and cause disease (22). The L452R mutation is located at the periphery of the receptor binding motif (RBM) and is found to reduce neutralizing activity by antibodies. The T478K mutation in the RBD, located within the epitope region in the RBM, is also associated with antibody escape. There has been striking evidence of antibodies that have been greatly affected, or even have lost their neutralizing activity altogether, by viral escape (2326).Experimental methods to improve antibody binding and neutralization have been developed. In vitro affinity maturation methods, such as random mutagenesis with display technologies, has been shown to improve antibody binding against target proteins, but such approaches are time consuming and labor intensive (2732). Targeted optimization toward one particular variant may also result in loss of neutralizing activity against other variants. Efficient optimization of antibodies that confer broad and potent neutralizing activity against diverse variants is therefore urgently needed.Here, we develop and apply a deep learning framework to efficiently optimize antibodies to achieve broader and more potent neutralizing activity against SARS-CoV-2 variants. Based on a large collection of antibody–antigen complex structures and binding affinity data, we trained a geometric neural network model, recently developed in computer vision, that effectively extracts interresidue interaction features and makes predictions of changes in binding affinity due to single or multiple amino acid substitutions to the antigen. To search for favorable complementarity-determining region (CDR) mutations that potentially improve antibody binding, we also simulate an in silico ensemble of predicted complex structures with CDR mutations to obtain a robust estimation of the free energy change, also known as ΔΔG. Compared to traditional approaches, the deep learning search space is theoretically much larger and is also easily applicable in targeting multiple variants simultaneously via multiobjective optimization.To demonstrate the utility of our approach, we sought to optimize a human neutralizing antibody P36-5D2, which was initially isolated from a convalescent patient, and demonstrated reasonably strong potency and breadth against Alpha, Beta, and Gamma (33) but not Delta, due to Delta’s L452R but not T478K mutation through computational structure analysis. We applied our deep learning model to predict CDR sequences that potentially improved binding affinity against the Delta variant while maintaining activity against Alpha, Beta, and Gamma. Through an iterative process of modeling and experimental validation, we were able to obtain six optimized antibodies with substantially improved potency of about 10- to 600-fold against multiple variants, including Delta. We also provide initial promising studies on Omicron. These results highlight the power of deep learning approaches for antibody optimization and their potential application to a wide range of other protein molecules. The optimized antibodies presented here also have the potential to be further developed as antibody drug candidates against SARS-CoV-2 variants.  相似文献   

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