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.

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...

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...

Development of effective preventative interventions against SARS-CoV-2, the etiologic agent of COVID-19 is urgently needed. The viral surface spike (S) protein of SARS-CoV-2 is a key target for prophylactic measures as it is critical for the viral replication cycle and the primary target of neutralizing antibodies. We evaluated design elements previously shown for other coronavirus S protein-based vaccines to be successful, e.g., prefusion-stabilizing substitutions and heterologous signal peptides, for selection of a S-based SARS-CoV-2 vaccine candidate. In vitro characterization demonstrated that the introduction of stabilizing substitutions (i.e., furin cleavage site mutations and two consecutive prolines in the hinge region of S2) increased the ratio of neutralizing versus non-neutralizing antibody binding, suggestive for a prefusion conformation of the S protein. Furthermore, the wild-type signal peptide was best suited for the correct cleavage needed for a natively folded protein. These observations translated into superior immunogenicity in mice where the Ad26 vector encoding for a membrane-bound stabilized S protein with a wild-type signal peptide elicited potent neutralizing humoral immunity and cellular immunity that was polarized towards Th1 IFN-γ. This optimized Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in a phase I clinical trial (ClinicalTrials.gov Identifier: NCT04436276).

The utility of recombinant adenovirus serotype 5 (rAd5) vector-based vaccines for HIV-1 and other pathogens will likely be limited by the high prevalence of pre-existing Ad5-specific neutralizing Abs (NAbs) in human populations. However, the immunodominant targets of Ad5-specific NAbs in humans remain poorly characterized. In this study, we assess the titers and primary determinants of Ad5-specific NAbs in individuals from both the United States and the developing world. Importantly, median Ad5-specific NAb titers were >10-fold higher in sub-Saharan Africa compared with the United States. Moreover, hexon-specific NAb titers were 4- to 10-fold higher than fiber-specific NAb titers in these cohorts by virus neutralization assays using capsid chimeric viruses. We next performed adoptive transfer studies in mice to evaluate the functional capacity of hexon- and fiber-specific NAbs to suppress the immunogenicity of a prototype rAd5-Env vaccine. Hexon-specific NAbs were remarkably efficient at suppressing Env-specific immune responses elicited by the rAd5 vaccine. In contrast, fiber-specific NAbs exerted only minimal suppressive effects on rAd5 vaccine immunogenicity. These data demonstrate that functionally significant Ad5-specific NAbs are directed primarily against the Ad5 hexon protein in both humans and mice. These studies suggest a potential strategy for engineering novel Ad5 vectors to evade dominant Ad5-specific NAbs.

Broadly neutralizing antibodies against highly variable pathogens have stimulated the design of novel vaccines and therapeutics. Here, we report on diverse camelid single-domain antibodies to influenza hemagglutinin from which we generated multi-domain antibodies with unprecedented breadth and potency. Multi-domain antibody MD3606 protects mice against influenza A and B infection when administered intravenously or expressed locally from a recombinant adeno-associated virus vector. Crystal and single-particle EM structures of these antibodies with hemagglutinins from influenza A and B viruses reveal binding to highly conserved epitopes. Collectively, our findings demonstrate that multi-domain antibodies targeting multiple epitopes exhibit enhanced virus cross-reactivity and potency. In combination with adeno-associated virus-mediated gene delivery, they may provide a groundbreaking new strategy to prevent infection with influenza virus and other highly variable pathogens.

The presence of various levels of anti-adenovirus serotype 5 (Ad5)-neutralizing antibodies in humans is thought to contribute to the inconsistent clinical results obtained so far in diverse gene transfer and vaccination studies and might preclude universal dosing with recombinant Ad5. Prescreening of individuals eligible for Ad5 or alternative serotype treatment and subsequently tailoring the vector dose might aid in ensuring the consistency of clinical parameters. For this purpose, a qualified Ad neutralization assay is required. Here we have tested the different protocols used to date to determine anti-Ad neutralizing activity. Based on simplicity, speed, high throughput, sensitivity, and robustness, we propose a qualified assay in which Ad neutralization is monitored by luciferase reporter gene expression.