Relating influenza virus membrane fusion kinetics to stoichiometry of neutralizing antibodies at the single-particle level.

The ability of antibodies binding the influenza hemagglutinin (HA) protein to neutralize viral infectivity is of key importance in the design of next-generation vaccines and for prophylactic and therapeutic use. The two antibodies CR6261 and CR8020 have recently been shown to efficiently neutralize influenza A infection by binding to and inhibiting the influenza A HA protein that is responsible for membrane fusion in the early steps of viral infection. Here, we use single-particle fluorescence microscopy to correlate the number of antibodies or antibody fragments (Fab) bound to an individual virion with the capacity of the same virus particle to undergo membrane fusion. To this end, individual, infectious virus particles bound by fluorescently labeled antibodies/Fab are visualized as they fuse to a planar, supported lipid bilayer. The fluorescence intensity arising from the virus-bound antibodies/Fab is used to determine the number of molecules attached to viral HA while a fluorescent marker in the viral membrane is used to simultaneously obtain kinetic information on the fusion process. We experimentally determine that the stoichiometry required for fusion inhibition by both antibody and Fab leaves large numbers of unbound HA epitopes on the viral surface. Kinetic measurements of the fusion process reveal that those few particles capable of fusion at high antibody/Fab coverage display significantly slower hemifusion kinetics. Overall, our results support a membrane fusion mechanism requiring the stochastic, coordinated action of multiple HA trimers and a model of fusion inhibition by stem-binding antibodies through disruption of this coordinated action.

A common solution to group 2 influenza virus neutralization.

The discovery and characterization of broadly neutralizing antibodies (bnAbs) against influenza viruses have raised hopes for the development of monoclonal antibody (mAb)-based immunotherapy and the design of universal influenza vaccines. Only one human bnAb (CR8020) specifically recognizing group 2 influenza A viruses has been previously characterized that binds to a highly conserved epitope at the base of the hemagglutinin (HA) stem and has neutralizing activity against H3, H7, and H10 viruses. Here, we report a second group 2 bnAb, CR8043, which was derived from a different germ-line gene encoding a highly divergent amino acid sequence. CR8043 has in vitro neutralizing activity against H3 and H10 viruses and protects mice against challenge with a lethal dose of H3N2 and H7N7 viruses. The crystal structure and EM reconstructions of the CR8043-H3 HA complex revealed that CR8043 binds to a site similar to the CR8020 epitope but uses an alternative angle of approach and a distinct set of interactions. The identification of another antibody against the group 2 stem epitope suggests that this conserved site of vulnerability has great potential for design of therapeutics and vaccines.

Ad26 vector-based COVID-19 vaccine encoding a prefusion-stabilized SARS-CoV-2 Spike immunogen induces potent humoral and cellular immune responses.

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).

A small-molecule fusion inhibitor of influenza virus is orally active in mice.

Recent characterization of broadly neutralizing antibodies (bnAbs) against influenza virus identified the conserved hemagglutinin (HA) stem as a target for development of universal vaccines and therapeutics. Although several stem bnAbs are being evaluated in clinical trials, antibodies are generally unsuited for oral delivery. Guided by structural knowledge of the interactions and mechanism of anti-stem bnAb CR6261, we selected and optimized small molecules that mimic the bnAb functionality. Our lead compound neutralizes influenza A group 1 viruses by inhibiting HA-mediated fusion in vitro, protects mice against lethal and sublethal influenza challenge after oral administration, and effectively neutralizes virus infection in reconstituted three-dimensional cell culture of fully differentiated human bronchial epithelial cells. Cocrystal structures with H1 and H5 HAs reveal that the lead compound recapitulates the bnAb hotspot interactions.

Universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin.

Broadly neutralizing antibodies against highly variable pathogens have stimulated the design of vaccines and therapeutics. We report the use of diverse camelid single-domain antibodies to influenza virus hemagglutinin to generate multidomain antibodies with impressive breadth and potency. Multidomain 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 electron microscopy structures of these antibodies with hemagglutinins from influenza A and B viruses reveal binding to highly conserved epitopes. Collectively, our findings demonstrate that multidomain antibodies targeting multiple epitopes exhibit enhanced virus cross-reactivity and potency. In combination with adeno-associated virus-mediated gene delivery, they may provide an effective strategy to prevent infection with influenza virus and other highly variable pathogens.

Potent peptidic fusion inhibitors of influenza virus.

Influenza therapeutics with new targets and mechanisms of action are urgently needed to combat potential pandemics, emerging viruses, and constantly mutating strains in circulation. We report here on the design and structural characterization of potent peptidic inhibitors of influenza hemagglutinin. The peptide design was based on complementarity-determining region loops of human broadly neutralizing antibodies against the hemagglutinin (FI6v3 and CR9114). The optimized peptides exhibit nanomolar affinity and neutralization against influenza A group 1 viruses, including the 2009 H1N1 pandemic and avian H5N1 strains. The peptide inhibitors bind to the highly conserved stem epitope and block the low pH-induced conformational rearrangements associated with membrane fusion. These peptidic compounds and their advantageous biological properties should accelerate the development of new small molecule- and peptide-based therapeutics against influenza virus.

HA Antibody-Mediated FcγRIIIa Activity Is Both Dependent on FcR Engagement and Interactions between HA and Sialic Acids.

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.

The tumor-selective viral protein apoptin effectively kills human biliary tract cancer cells.

Biliary tract cancer, or cholangiocarcinoma, has a poor prognosis. Resection is the only curative treatment, but only a minority of patients are eligible. Chemotherapy and gamma-irradiation are merely palliative, as they are unable to remove the malignancy completely. The chicken anemia virus-derived protein apoptin induces apoptosis in a wide range of human tumor cells and is not hindered by mutations inactivating p53 or by overexpression of Bcl-2, changes known to frustrate chemotherapy and radiation therapy. We examined whether apoptin kills human biliary tract cancer cells. Expression of apoptin by means of plasmids caused extensive cell death in three independent cholangiocarcinoma cell lines, CC-LP, CC-SW, and Mz-ChA-1, regardless of their oncogenic mutations, which included inactivated p16 and p53 and the disruption of the transforming growth factor beta signaling pathway. In vitro delivery of apoptin by an adenoviral vector completely eradicated cholangiocarcinoma cells. Moreover, coexpression of the broad-spectrum caspase inhibitor p35 with apoptin only delayed the induced cell death. Changes in nuclear morphology still occurred early after transfection, and nuclei eventually disintegrated, suggesting that apoptin-induced cell death in these cells is not blocked by mutations in either the initiation or execution phase of apoptosis. The efficient induction of cell death by apoptin in cholangiocarcinoma cell lines makes apoptin an attractive candidate for molecular therapy of biliary tract cancer.

Adenovirus types 5 and 35 seroprevalence in AIDS risk groups supports type 35 as a vaccine vector.

The seroprevalence of adenovirus types 5 (Ad5) and 35 (Ad35) was investigated in patients at risk of AIDS. The seroprevalence of Ad5 was higher than Ad35 in HIV-infected patients from The Netherlands (60% versus 7%) and sub-Saharan Africa (90% versus 20%). The seroprevalence was similar among HIV-infected and uninfected individuals, and remained constant during progression to AIDS. Ad35 is less prone to neutralization than Ad5, encouraging the further development of Ad35 for vaccination against HIV.

A single short stretch of homology between adenoviral vector and packaging cell line can give rise to cytopathic effect-inducing, helper-dependent E1-positive particles.

An undesirable byproduct from recombinant adenoviral vectors is the emergence of replication competent adenovirus (RCA) that result from rare homologous recombination events between the viral E1-containing (permissive) mammalian host cell genome and the virus itself, restoring the E1 gene to the viral genome. To reduce or eliminate the problem of RCA, we evaluated production of a first generation Ad5 vector (Ad5FGF4) in the cell line PER.C6. This E1-transformed human cell line contains only Ad5 nucleotides 459-3510, which precludes double crossover-type homologous recombination because the Ad5FGF-4 only contains 5' Ad5 sequence up to nucleotide 453. The Ad5FGF4 vector does, however, retain 177 nucleotides of the 3' end of the E1B-55K gene that are also present in PER.C6. With only this single region of homology between vector and cell line, we were surprised to detect virus-specific cytopathic effects (CPE) in our cell-based assay for RCA. This CPE-inducing agent was amplified in nonpermissive A549 cells but also supported amplification of the parental Ad5FGF-4. Because we were unable to isolate the CPE-inducing agent in pure form we first identified it as atypical RCA. Polymerase chain reaction (PCR) and Southern blot experiments identified viral DNA segments in which recombination had occurred between the 177 nucleotides of E1B present in both Ad5FGF-4 and PER.C6. The atypical RCA genomes contain a copy of the original (PGK promoter-E1 gene carrying) plasmid used in the construction of the PER.C6 cell line and they retained the parental FGF-4 transgene. However, significant deletions occurred within the recombined genomes in compensation for the large insertion from PER.C6 sequences and resulted in the loss of essential viral genes. This deletion renders these recombinant viruses replication defective, requiring helper functions from remaining parental Ad5FGF-4 for amplification. These atypical RCA entities may be more properly designated as helper-dependent E1-positive particles (HDEPs). This finding shows the importance of avoiding the use of "nonmatched" vectors where any overlap exists between the recombinant vector and E1 sequences in the packaging cell line. The cloning of the FGF-4 transgene into an adenoviral vector specifically "matched" for PER.C6 (lacking the 177 nucleotide region of homology) has allowed extensive virus propagation (Ad5.1FGF-4) with no CPE- or HDEP-like events yet detected.

Mechanisms of hemagglutinin targeted influenza virus neutralization.

Human monoclonal antibodies have been identified which neutralize broad spectra of influenza A or B viruses. Here, we dissect the mechanisms by which such antibodies interfere with infectivity. We distinguish four mechanisms that link the conserved hemagglutinin (HA) epitopes of broadly neutralizing antibodies to critical processes in the viral life cycle. HA-stem binding antibodies can act intracellularly by blocking fusion between the viral and endosomal membranes and extracellularly by preventing the proteolytic activation of HA. HA-head binding antibodies prevent viral attachment and release. These insights into newly identified ways by which the human immune system can interfere with influenza virus infection may aid the development of novel universal vaccines and antivirals.

Highly conserved protective epitopes on influenza B viruses.

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 substantial 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, that 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 highly conserved neutralizing epitope on group 2 influenza A viruses.

Current flu vaccines provide only limited coverage against seasonal strains of influenza viruses. The identification of V(H)1-69 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 V(H)1-69 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.

High-level expression from two independent expression cassettes in replication-incompetent adenovirus type 35 vector.

Replication-incompetent adenovirus type 35 (rAd35) represents a potent vaccine carrier that elicits strong, antigen-specific T- and B-cell responses in diverse preclinical models. Moreover, Ad35 is rare in human populations, resulting in the absence of neutralizing antibodies against this carrier, in contrast to the commonly used rAd5. Therefore, rAd35 is being investigated as a vaccine carrier for a number of diseases for which an effective vaccine is needed, including malaria, AIDS and tuberculosis. However, it can be perceived that effective immunization will require insertion of multiple antigens into adenoviral vectors. We therefore wanted to create rAd35 vectors carrying double expression cassettes, to expand within one vector the number of insertion sites for foreign DNA encoding antigenic proteins. We show that it is possible to generate rAd35 vectors carrying two cytomegalovirus promoter-driven expression cassettes, provided that the polyadenylation signals in each expression cassette are not identical. We demonstrate excellent rAd35 vector stability and show that expression of a transgene is not influenced by the presence of a second expression cassette. Moreover, by using two model vaccine antigens, i.e. the human immunodeficiency virus-derived Env-gp120 protein and the Plasmodium falciparum-derived circumsporozoite protein, we demonstrate that potent T- and B-cell responses are induced to both antigens expressed from a single vector. Such rAd35 vectors thus expand the utility of rAd35 vaccine carriers for the development of vaccines against, for example, malaria, AIDS and tuberculosis.

Protective immune responses to a recombinant adenovirus type 35 tuberculosis vaccine in two mouse strains: CD4 and CD8 T-cell epitope mapping and role of gamma interferon.

There is an urgent need for an efficacious vaccine against tuberculosis (TB). Cellular immune responses are key to an effective protective response against TB. Recombinant adenovirus (rAd) vectors are especially suited to the induction of strong T-cell immunity and thus represent promising vaccine vehicles for the prevention of TB. We have previously reported on rAd vector serotype 35, the serotype of choice due to low preexisting immunity worldwide, which expresses a unique fusion protein of Mycobacterium tuberculosis antigens Ag85A, Ag85B, and TB10.4 (Ad35-TBS). Here, we demonstrate that Ad35-TBS confers protection against M. tuberculosis when administered to mice through either an intranasal or an intramuscular route. Histological evaluation of lung tissue corroborated the protection and, in addition, demonstrated differences between two mouse strains, with diffuse inflammation in BALB/c mice and distinct granuloma formation in C57BL/6 mice. Epitope mapping analysis in these mouse strains showed that the major T-cell epitopes are conserved in the artificial fusion protein, while three novel CD8 peptides were discovered. Using a defined set of T-cell epitopes, we reveal differences between the two mouse strains in the type of protective immune response, demonstrating that different antigen-specific gamma interferon (IFN-gamma)-producing T cells can provide protection against M. tuberculosis challenge. While in BALB/c (H-2(d)) mice, a dominant CD8 T-cell response was detected, in C57BL/6 (H-2(b)) mice, more balanced CD4/CD8 T-cell responses were observed, with a more pronounced CD4 response in the lungs. These results unify conflicting reports on the relative importance of CD4 versus CD8 T-cell responses in protection and emphasize the key role of IFN-gamma.

Comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups B and D.

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.

Generation of a novel replication-incompetent adenoviral vector derived from human adenovirus type 49: manufacture on PER.C6 cells, tropism and immunogenicity.

Recombinant adenoviral vectors based on type 5 (rAd5) show great promise as a vaccine carrier. However, neutralizing activity against Ad5 is prevalent and high-titred among human populations, and significantly dampens Ad5-based vaccine modalities. The generation of alternative adenoviral vectors with low seroprevalence thus receives much research attention. Here, it is shown that a member from human adenovirus subgroup D, i.e. Ad49, does not cross-react with Ad5 neutralizing activity, making it a candidate serotype for vector development. Therefore, a plasmid system that allows formation of replication-incompetent adenovirus serotype 49 vaccine vectors (rAd49) was constructed and it was demonstrated that rAd49 can be successfully propagated to high titres on existing Ad5.E1-complementing cell lines such as PER.C6. Using an rAd49 vector carrying the luciferase marker gene, detailed seroprevalence studies were performed, demonstrating that rAd49 has low seroprevalence and neutralizing antibody titres worldwide. Also, we have initiated rAd49 vector receptor usage suggesting that rAd49 utilizes hCD46 as a cellular receptor. Finally, the immunogenicity of the rAd49 vector was assessed and it was shown that an rAd49.SIVGag vaccine induces strong anti-SIVGag CD8+ T-lymphocytes in naïve mice, albeit less than an rAd5.SIVGag vaccine. However, in mice with high anti-Ad5 immunity the rAd5.SIVGag vaccine was severely blunted, whereas the anti-SIVGag response was not significantly suppressed using the rAd49.SIVGag vaccine. These data demonstrate the potential of a replication deficient human group D adenoviral vector for vaccination purposes.

Age dependence of adenovirus-specific neutralizing antibody titers in individuals from sub-Saharan Africa.

We assessed neutralizing antibody titers to adenovirus serotype 5 (Ad5) and six rare adenovirus serotypes, serotypes 11, 35, 50, 26, 48, and 49, in pediatric populations in sub-Saharan Africa. We observed a clear age dependence of Ad5-specific neutralizing antibody titers. These data will help to guide the development of Ad vector-based vaccines for human immunodeficiency virus type 1 and other pathogens.

Immunogenicity of heterologous prime-boost regimens involving recombinant adenovirus serotype 11 (Ad11) and Ad35 vaccine vectors in the presence of anti-ad5 immunity.

The high prevalence of preexisting immunity to adenovirus serotype 5 (Ad5) in human populations will likely limit the immunogenicity and clinical utility of recombinant Ad5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 and other pathogens. A potential solution to this problem is to utilize rAd vaccine vectors derived from rare Ad serotypes such as Ad35 and Ad11. We have previously reported that rAd35 vectors were immunogenic in the presence of anti-Ad5 immunity, but the immunogenicity of heterologous rAd prime-boost regimens and the extent that cross-reactive anti-vector immunity may limit this approach have not been fully explored. Here we assess the immunogenicity of heterologous vaccine regimens involving rAd5, rAd35, and novel rAd11 vectors expressing simian immunodeficiency virus Gag in mice both with and without anti-Ad5 immunity. Heterologous rAd prime-boost regimens proved significantly more immunogenic than homologous regimens, as expected. Importantly, all regimens that included rAd5 were markedly suppressed by anti-Ad5 immunity. In contrast, rAd35-rAd11 and rAd11-rAd35 regimens elicited high-frequency immune responses both in the presence and in the absence of anti-Ad5 immunity, although we also detected clear cross-reactive Ad35/Ad11-specific humoral and cellular immune responses. Nevertheless, these data suggest the potential utility of heterologous rAd prime-boost vaccine regimens using vectors derived from rare human Ad serotypes.

Neutralizing antibodies to adenovirus serotype 5 vaccine vectors are directed primarily against the adenovirus hexon protein.

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.