Influenza virus presents a significant and persistent threat to public health worldwide and current vaccines provide immunity to viral isolates similar to the vaccine strain. High affinity antibodies against a conserved epitope could provide immunity to the diverse influenza subtypes and protection against future pandemic viruses. Co-crystal structures were determined at 2.2 and 2.7 Å resolutions for broadly neutralizing human antibody CR6261 Fab in complexes with the major surface antigen (hemagglutinin, HA) from viruses responsible for the 1918 H1N1 influenza pandemic and a recent lethal case of H5N1 avian influenza. In contrast to all other structurally characterized influenza antibodies, CR6261 recognizes a highly conserved helical region in the membrane-proximal stem of HA1/HA2. The antibody neutralizes the virus by blocking conformational rearrangements associated with membrane fusion. The CR6261 epitope identified here should accelerate the design and implementation of improved vaccines that can elicit CR6261-like antibodies, as well as antibodybased therapies for the treatment of influenza.Over the past century, three human influenza A pandemics (1918 H1N1 Spanish, 1957 H2N2 Asian, and 1968 have killed ∼50-100 million people worldwide. Each pandemic virus was derived, at least in part, from an avian influenza virus by direct interspecies transmission or exchange of genetic material between avian and human viruses (1-4). In each case, a novel hemagglutinin (HA) envelope glycoprotein was acquired that was antigenically distinct from the HAs of the human viruses in circulation at that time. HA is the primary target of neutralizing antibodies and rapidly and continuously accumulates mutations to escape recognition by the immune system. In pandemic years, HAs are shuffled from the vast reservoir of 16 HA subtypes in avian viruses into a circulating human virus to evade prevailing immunity in the human population. Thus, while many factors likely contribute to virulence and transmissibility, immune evasion is critical for the rapid spread of pandemic and epidemic viruses.Several small molecules are in use for treatment of influenza. Most notable are neuraminidase (NA) inhibitors, oseltamivir (Tamiflu) and zanamivir (Relenza), that prevent release of nascent virions, and amantadine (5) that interferes with the M2 channel proton conducting activity. However, excessive use leads to resistant viruses (6-8) that often show surprisingly little attenuation from the escape mutations, thereby contributing to rapid spread worldwide (6).

Identification of broadly neutralizing antibodies against influenza A viruses has raised hopes for the development of monoclonal antibody-based immunotherapy and ‘universal’ vaccines for influenza. However, a significant part of the annual flu burden is caused by two cocirculating, antigenically distinct lineages of influenza B viruses. Here we report human monoclonal antibodies, CR8033, CR8071 and CR9114, which protect mice against lethal challenge from both lineages. Antibodies CR8033 and CR8071 recognize distinct conserved epitopes in the head region of the influenza B hemagglutinin (HA), whereas CR9114 binds a conserved epitope in the HA stem and protects against lethal challenge with influenza A and B viruses. These antibodies may inform on development of monoclonal antibody-based treatments and a universal flu vaccine for all influenza A and B viruses.

Current flu vaccines provide only limited coverage against seasonal strains of influenza viruses. The identification of VH1-69 antibodies that broadly neutralize almost all influenza A group 1 viruses constituted a breakthrough in the influenza field. Here we report the isolation and characterization of a human monoclonal antibody CR8020 with broad neutralizing activity against most group 2 viruses, including H3N2 and H7N7, which cause severe human infection. The crystal structure of Fab CR8020 with the 1968 pandemic H3 hemagglutinin (HA) reveals a highly conserved epitope in the HA stalk distinct from the epitope recognized by the VH1-69 group 1 antibodies. Thus, a cocktail of two antibodies may be sufficient to neutralize most influenza A subtypes and, hence, enable development of a universal flu vaccine and broad spectrum antibody therapies.

BackgroundThe hemagglutinin (HA) glycoprotein is the principal target of protective humoral immune responses to influenza virus infections but such antibody responses only provide efficient protection against a narrow spectrum of HA antigenic variants within a given virus subtype. Avian influenza viruses such as H5N1 are currently panzootic and pose a pandemic threat. These viruses are antigenically diverse and protective strategies need to cross protect against diverse viral clades. Furthermore, there are 16 different HA subtypes and no certainty the next pandemic will be caused by an H5 subtype, thus it is important to develop prophylactic and therapeutic interventions that provide heterosubtypic protection.Methods and FindingsHere we describe a panel of 13 monoclonal antibodies (mAbs) recovered from combinatorial display libraries that were constructed from human IgM+ memory B cells of recent (seasonal) influenza vaccinees. The mAbs have broad heterosubtypic neutralizing activity against antigenically diverse H1, H2, H5, H6, H8 and H9 influenza subtypes. Restriction to variable heavy chain gene IGHV1-69 in the high affinity mAb panel was associated with binding to a conserved hydrophobic pocket in the stem domain of HA. The most potent antibody (CR6261) was protective in mice when given before and after lethal H5N1 or H1N1 challenge.ConclusionsThe human monoclonal CR6261 described in this study could be developed for use as a broad spectrum agent for prophylaxis or treatment of human or avian influenza infections without prior strain characterization. Moreover, the CR6261 epitope could be applied in targeted vaccine strategies or in the design of novel antivirals. Finally our approach of screening the IgM+ memory repertoire could be applied to identify conserved and functionally relevant targets on other rapidly evolving pathogens.

BackgroundExperimental animal data show that protection against severe acute respiratory syndrome coronavirus (SARS-CoV) infection with human monoclonal antibodies (mAbs) is feasible. For an effective immune prophylaxis in humans, broad coverage of different strains of SARS-CoV and control of potential neutralization escape variants will be required. Combinations of virus-neutralizing, noncompeting mAbs may have these properties.Methods and FindingsHuman mAb CR3014 has been shown to completely prevent lung pathology and abolish pharyngeal shedding of SARS-CoV in infected ferrets. We generated in vitro SARS-CoV variants escaping neutralization by CR3014, which all had a single P462L mutation in the glycoprotein spike (S) of the escape virus. In vitro experiments confirmed that binding of CR3014 to a recombinant S fragment (amino acid residues 318–510) harboring this mutation was abolished. We therefore screened an antibody-phage library derived from blood of a convalescent SARS patient for antibodies complementary to CR3014. A novel mAb, CR3022, was identified that neutralized CR3014 escape viruses, did not compete with CR3014 for binding to recombinant S1 fragments, and bound to S1 fragments derived from the civet cat SARS-CoV-like strain SZ3. No escape variants could be generated with CR3022. The mixture of both mAbs showed neutralization of SARS-CoV in a synergistic fashion by recognizing different epitopes on the receptor-binding domain. Dose reduction indices of 4.5 and 20.5 were observed for CR3014 and CR3022, respectively, at 100% neutralization. Because enhancement of SARS-CoV infection by subneutralizing antibody concentrations is of concern, we show here that anti-SARS-CoV antibodies do not convert the abortive infection of primary human macrophages by SARS-CoV into a productive one.ConclusionsThe combination of two noncompeting human mAbs CR3014 and CR3022 potentially controls immune escape and extends the breadth of protection. At the same time, synergy between CR3014 and CR3022 may allow for a lower total antibody dose to be administered for passive immune prophylaxis of SARS-CoV infection.

Recombinant adenovirus serotype 5 (rAd5) vector-based vaccines are currently being developed for both human immunodeficiency virus type 1 and other pathogens. The potential limitations associated with rAd5 vectors, however, have led to the construction of novel rAd vectors derived from rare Ad serotypes. Several rare serotype rAd vectors have already been described, but a detailed comparison of multiple rAd vectors from subgroups B and D has not previously been reported. Such a comparison is critical for selecting optimal rAd vectors for advancement into clinical trials. Here we describe the construction of three novel rAd vector systems from Ad26, Ad48, and Ad50. We report comparative seroprevalence and immunogenicity studies involving rAd11, rAd35, and rAd50 vectors from subgroup B; rAd26, rAd48, and rAd49 vectors from subgroup D; and rAd5 vectors from subgroup C. All six rAd vectors from subgroups B and D exhibited low seroprevalence in a cohort of 200 individuals from sub-Saharan Africa, and they elicited Gag-specific cellular immune responses in mice both with and without preexisting anti-Ad5 immunity. The rAd vectors from subgroup D were also evaluated using rhesus monkeys and were shown to be immunogenic after a single injection. The rAd26 vectors proved the most immunogenic among the rare serotype rAd vectors studied, although all rare serotype rAd vectors were still less potent than rAd5 vectors in the absence of anti-Ad5 immunity. These studies substantially expand the portfolio of rare serotype rAd vectors that may prove useful as vaccine vectors for the developing world.Replication-incompetent, recombinant adenovirus serotype 5 (rAd5) vectors have been demonstrated to elicit potent antigen-specific cellular immune responses in both preclinical and clinical studies (2,7,25,26,28). In particular, rAd5 vectorbased vaccines for human immunodeficiency virus type 1 (HIV-1) and other pathogens are currently being advanced into large-scale clinical studies. However, the immunogenicity and clinical utility of rAd5 vectors may be limited by the high prevalence of preexisting anti-Ad5 immunity in human populations, particularly in the developing world (13,19,25,30,31,33,35). Preexisting anti-Ad5 immunity has already been shown to suppress the immunogenicity of rAd5 vector-based vaccines in mice (3,14,15,22,30,36), rhesus monkeys (6, 22), and humans (7, 25). Moreover, immunization with rAd5 vectors generates potent antivector immunity that substantially inhibits the utility of homologous vector readministration (3,6,24).The generation of novel rAd vectors that circumvent antiAd5 immunity is therefore an important research priority. Strategies that are currently being explored include constructing hexon-chimeric rAd5 vectors (22), generating rAd vectors from nonhuman Ad serotypes (8,11,21,34), and developing rAd vectors from rare human Ad serotypes (12,14,25,35). Such novel rAd vectors may prove useful as vaccine vectors in populations in the developing world with high levels of preexisting anti-Ad5 immunity. Nov...

The identification of human broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem revitalized hopes of developing a universal influenza vaccine. Using a rational design and library approach, we engineered stable HA stem antigens ("mini-HAs") based on an H1 subtype sequence. Our most advanced candidate exhibits structural and bnAb binding properties comparable to those of full-length HA, completely protects mice in lethal heterologous and heterosubtypic challenge models, and reduces fever after sublethal challenge in cynomolgus monkeys. Antibodies elicited by this mini-HA in mice and nonhuman primates bound a wide range of HAs, competed with human bnAbs for HA stem binding, neutralized H5N1 viruses, and mediated antibody-dependent effector activity. These results represent a proof of concept for the design of HA stem mimics that elicit bnAbs against influenza A group 1 viruses.

Replication-deficient human adenovirus type 5 (Ad5) can be produced to high titers in complementing cell lines, such as PER.C6, and is widely used as a vaccine and gene therapy vector. However, preexisting immunity against Ad5 hampers consistency of gene transfer, immunological responses, and vector-mediated toxicities. We report the identification of human Ad35 as a virus with low global prevalence and the generation of an Ad35 vector plasmid system for easy insertion of heterologous genes. In addition, we have identified the minimal sequence of the Ad35-E1B region (molecular weight, 55,000 [55K]), pivotal for complementation of fully E1-lacking Ad35 vector on PER.C6 cells. After stable insertion of the 55K sequence into PER.C6 cells a cell line was obtained (PER.C6/55K) that efficiently transcomplements both Ad5 and Ad35 vectors. We further demonstrate that transduction with Ad35 is not hampered by preexisting Ad5 immunity and that Ad35 efficiently infects dendritic cells, smooth muscle cells, and synoviocytes, in contrast to Ad5.It has been shown in diverse in vivo models that recombinant adenovirus type 5 (Ad5) has potential as a vehicle to transfer genes for treatment or prevention of disease (49, 52). Although encouraging, the extrapolation from animal models to humans faces at least one extra hurdle, i.e., the presence of anti-Ad5 neutralizing activity (NA) in sera from human individuals. The humoral response to Ad5 is strong and has been found to impede, depending on the administration route, the infection efficiency in animal models as well as in humans (7,9,18,29,30,35,37,42,45). Concomitant with the decrease in transduction, high NA against the vector also abolishes Ad5-mediated toxicity (8). Importantly, when very high vector doses were used in preimmunized nonhuman primates, new toxic effects were found that were not observed in naive animals (54). These findings show that preexisting immunity severely hampers accurate dose control, since human individuals differ in their NA against Ad5-based vectors. Strategies to bypass NA to Ad5 viruses include switching of adenovirus type (28,32,36) and use of animal adenoviruses (13,25,34). Animal adenoviruses have the advantage that NA is predicted to be absent in humans. Disadvantages of this strategy include the lack of knowledge regarding the biology of these viruses including tropism on human cells, potential difficulties in manufacturing, and the possibility of in vivo recombination with human types leading to unknown disease. Human adenoviruses on the other hand are better characterized and their subclinical disease association in humans is known (10,17,55). However, recent knowledge on the prevalence of NA towards human adenoviruses worldwide is not available and therefore it is difficult to predict which type would be the best alternative for Ad5. To identify human adenovirus types with low seroprevalence, an extensive screen was performed using most human adenovirus types and serum samples derived from healthy blood donors from 6 different geographica...