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.

A safe and effective vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be required to end the coronavirus disease 2019 (COVID-19) pandemic 1 – 8 . For global deployment and pandemic control, a vaccine that requires only a single immunization would be optimal. Here we show the immunogenicity and protective efficacy of a single dose of adenovirus serotype 26 (Ad26) vector-based vaccines expressing the SARS-CoV-2 spike (S) protein in nonhuman primates. 52 rhesus macaques were immunized with Ad26 vectors encoding S variants or sham control and were challenged with SARS-CoV-2 by the intranasal and intratracheal routes 9 , 10 . The optimal Ad26 vaccine induced robust neutralizing antibody responses and provided complete or near-complete protection in bronchoalveolar lavage and nasal swabs following SARS-CoV-2 challenge. Vaccine-elicited neutralizing antibody titers correlated with protective efficacy, suggesting an immune correlate of protection. These data demonstrate robust single-shot vaccine protection against SARS-CoV-2 in nonhuman primates. The optimal Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in clinical trials.

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).

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.

A novel plasmid-based adenovirus vector system that enables manufacturing of replication-incompetent (⌬E1) adenovirus type 11 (Ad11)-based vectors is described. Ad11 vectors are produced on PER.C6/55K cells yielding high-titer vector batches after purification. Ad11 seroprevalence proves to be significantly lower than that of Ad5, and neutralizing antibody titers against Ad11 are low. Ad11 seroprevalence among human immunodeficiency virus-positive (HIV ؉ ) individuals is as low as that among HIV ؊ individuals, independent of the level of immune suppression. The low level of coinciding seroprevalence between Ad11 and Ad35 in addition to a lack of correlation between high neutralizing antibody titers towards either adenovirus strongly suggest that the limited humoral cross-reactive immunity between these two highly related B viruses appears not to preclude the use of both vectors in the same individual. Ad11 transduces primary cells including smooth muscle cells, synoviocytes, and dendritic cells and cardiovascular tissues with higher efficiency than Ad5. Ad11 and Ad35 appear to have a similar tropism as judged by green fluorescent protein expression levels determined by using a panel of cancer cell lines. In addition, Ad5 preimmunization did not significantly affect Ad11-mediated transduction in C57BL/6 mice. We therefore conclude that the Ad11-based vector represents a novel and useful candidate gene transfer vehicle for vaccination and gene therapy.Adenovirus vectors are being developed for gene therapy purposes with the aim to treat inherited or acquired disease (6, 10, 15, 21) as well as for therapeutic and prophylactic vaccination strategies (5, 32). The use of adenovirus for vaccination has recently been fueled by highly promising results demonstrating protection against viruses and viral diseases in rodents and nonhuman primates (28,34,35) as well as induction of Tand B-cell responses in humans in early phase I vaccine trials with healthy volunteers (7). However, high seroprevalence and high neutralizing antibody (NAb) titers against the commonly used vectors, i.e., adenovirus type 5 (Ad5) and Ad2 (39), hamper the application of C group-based vectors, since circulating NAbs efficiently capture administered recombinant vectors obscuring therapeutic effect (19). It has been shown, at least in rodents (1, 42), nonhuman primates (3), and humans in early phase I clinical trials (7), that high levels of NAbs decrease gene transfer efficiency or blunt vaccine potency. Since levels of NAbs vary among individuals, overdosing with recombinant vector in an attempt to overcome the neutralizing activity may result in either excellent clinical results or severe vector-mediated toxicity. Thus, the presence of anti-Ad5 preexisting immunity does not allow accurate dose control and thereby may prevent the universal use of Ad5-based vectors as gene transfer vehicles in humans.Although many strategies are being pursued to avoid vector neutralization (3,5,19,23,41,43), a most viable strategy is the use of rare human adenovirus...

Adenoviral vectors based on adenovirus type 35 (rAd35) have the advantage of low natural vector immunity and induce strong, insert-specific T-and B-cell responses, making them prime-candidate vaccine carriers. However, severe vector-genome instability of E1-deleted rAd35 vectors was observed, hampering universal use. The instability of E1-deleted rAd35 vector proved to be caused by low pIX expression induced by removal of the pIX promoter, which was located in the E1B region of B-group viruses. Reinsertion of a minimal pIX promoter resulted in stable vectors able to harbour large DNA inserts (>5 kb). In addition, it is shown that replacement of the E4-Orf6 region of Ad35 by the E4-Orf6 region of Ad5 resulted in successful propagation of an E1-deleted rAd35 vector on existing E1-complementing cell lines, such as PER.C6 cells. The ability to produce these carriers on PER.C6 contributes significantly to the scale of manufacturing of rAd35-based vaccines. Next, a stable rAd35 vaccine was generated carrying Mycobacterium tuberculosis antigens Ag85A, Ag85B and TB10.4. The antigens were fused directly, resulting in expression of a single polyprotein. This vaccine induced dose-dependent CD4+ and CD8 + T-cell responses against multiple antigens in mice. It is concluded that the described improvements to the rAd35 vector contribute significantly to the further development of rAd35 carriers for mass-vaccination programmes for diseases such as tuberculosis, AIDS and malaria.

Interactions with receptors for the Fc region of IgG (FcγRs) have been shown to contribute to the in vivo protection against influenza A viruses provided by broadly neutralizing antibodies (bnAbs) that bind to the viral hemagglutinin (HA) stem. In particular, Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) has been shown to contribute to protection by stem-binding bnAbs. Fc-mediated effector functions appear not to contribute to protection provided by strain-specific HA head-binding antibodies. We used a panel of anti-stem and anti-head influenza A and B monoclonal antibodies with identical human IgG1 Fc domains and investigated their ability to mediate ADCC-associated FcγRIIIa activation. Antibodies which do not interfere with sialic acid binding of HA can mediate FcγRIIIa activation. However, the FcγRIIIa activation was inhibited when a mutant HA, unable to bind sialic acids, was used. Antibodies which block sialic acid receptor interactions of HA interfered with FcγRIIIa activation. The inhibition of FcγRIIIa activation by HA head-binding and sialic acid receptor-blocking antibodies was confirmed in plasma samples of H5N1 vaccinated human subjects. Together, these results suggest that in addition to Fc–FcγR binding, interactions between HA and sialic acids on immune cells are required for optimal Fc-mediated effector functions by anti-HA antibodies.