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Broadly Reactive Human Monoclonal Antibodies Elicited following Pandemic H1N1 Influenza Virus Exposure Protect Mice against Highly Pathogenic H5N1 Challenge.

ABSTRACT: Broadly cross-reactive antibodies (Abs) that recognize conserved epitopes within the influenza virus hemagglutinin (HA) stalk domain are of particular interest for their potential use as therapeutic and prophylactic agents against multiple influenza virus subtypes, including zoonotic virus strains. Here, we characterized four human HA stalk-reactive monoclonal antibodies (MAbs) for their binding breadth and affinity, in vitro neutralization capacity, and in vivo protective potential against an highly pathogenic avian influenza virus. The monoclonal antibodies were isolated from individuals shortly following infection with (70-1F02 and 1009-3B05) or vaccination against (05-2G02 and 09-3A01) A(H1N1)pdm09. Three of the MAbs bound HAs from multiple strains of group 1 viruses, and one MAb, 05-2G02, bound to both group 1 and group 2 influenza A virus HAs. All four antibodies prophylactically protected mice against a lethal challenge with the highly pathogenic A/Vietnam/1203/04 (H5N1) strain. Two MAbs, 70-1F02 and 09-3A01, were further tested for their therapeutic efficacy against the same strain and showed good efficacy in this setting as well. One MAb, 70-1F02, cocrystallized with H5 HA and showed heavy-chain-only interactions similar to those seen with the previously described CR6261 anti-stalk antibody. Finally, we show that antibodies that compete with these MAbs are prevalent in serum from an individual recently infected with the A(H1N1)pdm09 virus. The antibodies described here can be developed into broad-spectrum antiviral therapeutics that could be used to combat infections by zoonotic or emerging pandemic influenza viruses.IMPORTANCE The rise in zoonotic infections of humans by emerging influenza viruses is a worldwide public health concern. The majority of recent zoonotic human influenza cases were caused by H7N9 and H5Nx viruses and were associated with high morbidity and mortality. In addition, seasonal influenza viruses are estimated to cause up to 650,000 deaths annually worldwide. Currently available antiviral treatment options include only neuraminidase inhibitors, but some influenza viruses are naturally resistant to these drugs, and others quickly develop resistance-conferring mutations. Alternative therapeutics are urgently needed. Broadly protective antibodies that target the conserved "stalk" domain of the hemagglutinin represent potential potent antiviral prophylactic and therapeutic agents that can assist pandemic preparedness. Here, we describe four human monoclonal antibodies that target conserved regions of influenza HA and characterize their binding spectrum as well as their protective capacity in prophylactic and therapeutic settings against a lethal challenge with a zoonotic influenza virus.

SUBMITTER: Nachbagauer R

PROVIDER: S-EPMC6069173 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Broadly Reactive Human Monoclonal Antibodies Elicited following Pandemic H1N1 Influenza Virus Exposure Protect Mice against Highly Pathogenic H5N1 Challenge.

Nachbagauer Raffael R Shore David D Yang Hua H Johnson Scott K SK Gabbard Jon D JD Tompkins S Mark SM Wrammert Jens J Wilson Patrick C PC Stevens James J Ahmed Rafi R Krammer Florian F Ellebedy Ali H AH

Journal of virology 20180731 16

Broadly cross-reactive antibodies (Abs) that recognize conserved epitopes within the influenza virus hemagglutinin (HA) stalk domain are of particular interest for their potential use as therapeutic and prophylactic agents against multiple influenza virus subtypes, including zoonotic virus strains. Here, we characterized four human HA stalk-reactive monoclonal antibodies (MAbs) for their binding breadth and affinity, <i>in vitro</i> neutralization capacity, and <i>in vivo</i> protective potentia ...[more]

PMID: 29899095

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Project description:UnlabelledNeuraminidase (NA), an influenza virus envelope glycoprotein, removes sialic acid from receptors for virus release from infected cells. For this study, we used a baculovirus-insect cell expression system to construct and purify recombinant NA (rNA) proteins of H5N1 (A/Vietnam/1203/2004) and pandemic H1N1 (pH1N1) (A/Texas/05/2009) influenza viruses. BALB/c mice immunized with these proteins had high titers of NA-specific IgG and NA-inhibiting (NI) antibodies against H5N1, pH1N1, H3N2, and H7N9 viruses. H5N1 rNA immunization resulted in higher quantities of NA-specific antibody-secreting B cells against H5N1 and heterologous pH1N1 viruses in the spleen. H5N1 rNA and pH1N1 rNA immunizations both provided complete protection against homologous virus challenges, with H5N1 rNA immunization providing better protection against pH1N1 virus challenges. Cross-reactive NI antibodies were further dissected via pH1N1 rNA protein immunizations with I149V (NA with a change of Ile to Val at position 149), N344Y, and I365T/S366N NA mutations. The I365T/S366N mutation of pH1N1 rNA enhanced cross-reactive NI antibodies against H5N1, H3N2, and H7N9 viruses. It is our hope that these findings provide useful information for the development of an NA-based universal influenza vaccine.ImportanceNeuraminidase (NA) is an influenza virus enzymatic protein that cleaves sialic acid linkages on infected cell surfaces, thus facilitating viral release and contributing to viral transmission and mucus infection. In currently available inactivated or live, attenuated influenza vaccines based on the antigenic content of hemagglutinin proteins, vaccine efficacy can be contributed partly through NA-elicited immune responses. We investigated the NA immunity of different recombinant NA (rNA) proteins associated with pH1N1 and H5N1 viruses. Our results indicate that H5N1 rNA immunization induced more potent cross-protective immunity than pH1N1 rNA immunization, and three mutated residues, I149V, I365T, and S366N, near the NA enzyme active site(s) are linked to enhanced cross-reactive NA-inhibiting antibodies against heterologous and heterosubtypic influenza A viruses. These findings provide useful information for the development of an NA-based universal influenza vaccine.

Project description:Influenza viruses have caused numerous pandemics throughout human history. The 1957 influenza pandemic was initiated by an H2N2 influenza virus. This H2N2 influenza virus was the result of a reassortment event between a circulating H2N2 avian virus and the seasonal H1N1 viruses in humans. Previously, our group has demonstrated the effectiveness of hemagglutinin (HA) antigens derived using computationally optimized broadly reactive antigen (COBRA) methodology against H1N1, H3N2, and H5N1 viruses. Using the COBRA methodology, H2 HA COBRA antigens were designed using sequences from H2N2 viruses isolated from humans in the 1950s and 1960s, as well as H2Nx viruses isolated from avian and mammalian species between the 1950s and 2016. In this study, the effectiveness of H2 COBRA HA antigens (Z1, Z3, Z5, and Z7) was evaluated in DBA/2J mice and compared to that of wild-type H2 HA antigens. The COBRA HA vaccines elicited neutralizing antibodies to the majority of viruses in our H2 HA panel and across all three clades as measured by hemagglutination inhibition (HAI) and neutralization assays. Comparatively, several wild-type HA vaccines elicited antibodies against a majority of the viruses in the H2 HA panel. DBA/2J mice vaccinated with COBRA vaccines showed increase survival for all three viral challenges compared to the wild-type H2 vaccines. In particular, the Z1 COBRA is a promising candidate for future work toward a pandemic H2 influenza vaccine.IMPORTANCE H2N2 influenza has caused at least one pandemic in the past. Given that individuals born after 1968 have not been exposed to H2N2 influenza viruses, a future pandemic caused by H2 influenza is likely. An effective H2 influenza vaccine would need to elicit broadly cross-reactive antibodies to multiple H2 influenza viruses. Choosing a wild-type virus to create a vaccine may elicit a narrow immune response and not protect against multiple H2 influenza viruses. COBRA H2 HA vaccines were developed and evaluated in mice along with wild-type H2 HA vaccines. Multiple COBRA H2 HA vaccines protected mice from all three viral challenges and produced broadly cross-reactive neutralizing antibodies to H2 influenza viruses.

Project description:Influenza virus neuraminidase (NA) is an interesting target of small-molecule antiviral drugs. We isolated a set of H5N1 NA-specific single-domain antibodies (N1-VHHm) and evaluated their in vitro and in vivo antiviral potential. Two of them inhibited the NA activity and in vitro replication of clade 1 and 2 H5N1 viruses. We then generated bivalent derivatives of N1-VHHm by two methods. First, we made N1-VHHb by genetically joining two N1-VHHm moieties with a flexible linker. Second, bivalent N1-VHH-Fc proteins were obtained by genetic fusion of the N1-VHHm moiety with the crystallizable region of mouse IgG2a (Fc). The in vitro antiviral potency against H5N1 of both bivalent N1-VHHb formats was 30- to 240-fold higher than that of their monovalent counterparts, with 50% inhibitory concentrations in the low nanomolar range. Moreover, single-dose prophylactic treatment with bivalent N1-VHHb or N1-VHH-Fc protected BALB/c mice against a lethal challenge with H5N1 virus, including an oseltamivir-resistant H5N1 variant. Surprisingly, an N1-VHH-Fc fusion without in vitro NA-inhibitory or antiviral activity also protected mice against an H5N1 challenge. Virus escape selection experiments indicated that one amino acid residue close to the catalytic site is required for N1-VHHm binding. We conclude that single-domain antibodies directed against influenza virus NA protect against H5N1 virus infection, and when engineered with a conventional Fc domain, they can do so in the absence of detectable NA-inhibitory activity.Highly pathogenic H5N1 viruses are a zoonotic threat. Outbreaks of avian influenza caused by these viruses occur in many parts of the world and are associated with tremendous economic loss, and these viruses can cause very severe disease in humans. In such cases, small-molecule inhibitors of the viral NA are among the few treatment options for patients. However, treatment with such drugs often results in the emergence of resistant viruses. Here we show that single-domain antibody fragments that are specific for NA can bind and inhibit H5N1 viruses in vitro and can protect laboratory mice against a challenge with an H5N1 virus, including an oseltamivir-resistant virus. In addition, plant-produced VHH fused to a conventional Fc domain can protect in vivo even in the absence of NA-inhibitory activity. Thus, NA of influenza virus can be effectively targeted by single-domain antibody fragments, which are amenable to further engineering.

Project description:Carbapenem-resistant (CR) sequence type 258 (ST258) Klebsiella pneumoniae has become an urgent health care threat, causing an increasing number of high-mortality infections. Its resistance to numerous antibiotics and threat to immunocompromised patients necessitate finding new therapies to combat these infections. Previous successes in the laboratory, as well as the conservation of capsular polysaccharide (CPS) among the members of the ST258 clone, suggest that monoclonal antibody (MAb) therapy targeting the outer polysaccharide capsule of K. pneumoniae could serve as a valuable treatment alternative for afflicted patients. Here, we isolated several IgG antibodies from mice inoculated with a mixture of CR K. pneumoniae CPS conjugated to anthrax protective antigen. Two of these MAbs, 17H12 and 8F12, bind whole and oligosaccharide epitopes of the CPS of clade 2 ST258 CR K. pneumoniae, which is responsible for the most virulent CR K. pneumoniae infections in the United States. These antibodies were shown to agglutinate all clade 2 strains and were also shown to promote extracellular processes killing these bacteria, including biofilm inhibition, complement deposition, and deployment of neutrophil extracellular traps. Additionally, they promoted opsonophagocytosis and intracellular killing of CR K. pneumoniae by human-derived neutrophils and cultured murine macrophages. Finally, when mice were intratracheally infected with preopsonized clade 2 CR K. pneumoniae, these MAbs reduced bacterial dissemination to organs. Our data suggest that broadly reactive anticapsular antibodies and vaccines against clade 2 ST258 CR K. pneumoniae are possible. Such MAbs and vaccines would benefit those susceptible populations at risk of infection with this group of multidrug-resistant bacteria.IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae is an enteric bacterium that has been responsible for an increasing number of deadly outbreaks and hospital-acquired infections. The pathogen's resistance to numerous antibiotics, including new drugs, leaves few therapeutic options available for infected patients, who often are too sick to fight the infection themselves. Immunotherapy utilizing monoclonal antibodies has been successful in other medical fields, and antibodies targeting the outer polysaccharide capsule of these bacteria could be a valuable treatment alternative. This study presents two anticapsular antibodies, 17H12 and 8F12, that were found to be protective against the most virulent carbapenem-resistant K. pneumoniae clinical strains. These antibodies are shown to promote the killing of these strains through several extracellular and intracellular processes and prevent the spread of infection in mice from the lungs to distal organs. Thus, they could ultimately treat or protect patients infected or at risk of infection by this multidrug-resistant bacterium.

Project description:We investigated the protective efficacy of two intranasal chitosan (CSN and TM-CSN) adjuvanted H5N1 Influenza vaccines against highly pathogenic avian Influenza (HPAI) intratracheal and intranasal challenge in a ferret model. Six groups of 6 ferrets were intranasally vaccinated twice, 21 days apart, with either placebo, antigen alone, CSN adjuvanted antigen, or TM-CSN adjuvanted antigen. Homologous and intra-subtypic antibody cross-reacting responses were assessed. Ferrets were inoculated intratracheally (all treatments) or intranasally (CSN adjuvanted and placebo treatments only) with clade 1 HPAI A/Vietnam/1194/2004 (H5N1) virus 28 days after the second vaccination and subsequently monitored for morbidity and mortality outcomes. Clinical signs were assessed and nasal as well as throat swabs were taken daily for virology. Samples of lung tissue, nasal turbinates, brain, and olfactory bulb were analysed for the presence of virus and examined for histolopathological findings. In contrast to animals vaccinated with antigen alone, the CSN and TM-CSN adjuvanted vaccines induced high levels of antibodies, protected ferrets from death, reduced viral replication and abrogated disease after intratracheal challenge, and in the case of CSN after intranasal challenge. In particular, the TM-CSN adjuvanted vaccine was highly effective at eliciting protective immunity from intratracheal challenge; serologically, protective titres were demonstrable after one vaccination. The 2-dose schedule with TM-CSN vaccine also induced cross-reactive antibodies to clade 2.1 and 2.2 H5N1 viruses. Furthermore ferrets immunised with TM-CSN had no detectable virus in the respiratory tract or brain, whereas there were signs of virus in the throat and lungs, albeit at significantly reduced levels, in CSN vaccinated animals. This study demonstrated for the first time that CSN and in particular TM-CSN adjuvanted intranasal vaccines have the potential to protect against significant mortality and morbidity arising from infection with HPAI H5N1 virus.

Dataset Information

Broadly Reactive Human Monoclonal Antibodies Elicited following Pandemic H1N1 Influenza Virus Exposure Protect Mice against Highly Pathogenic H5N1 Challenge.

Publications

Broadly Reactive Human Monoclonal Antibodies Elicited following Pandemic H1N1 Influenza Virus Exposure Protect Mice against Highly Pathogenic H5N1 Challenge.

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