Structure of a Classical Broadly Neutralizing Stem Antibody in Complex with a Pandemic H2 Influenza Virus Hemagglutinin

We report the structural characterization of the first antibody identified to cross-neutralize multiple subtypes of influenza A viruses. The crystal structure of mouse antibody C179 bound to the pandemic 1957 H2N2 hemagglutinin (HA) reveals that it targets an epitope on the HA stem similar to those targeted by the recently identified human broadly neutralizing antibodies. C179 also inhibits the low-pH conformational change of the HA but uses a different angle of approach and both heavy and light chains.     

HIV-1 Neutralizing Antibody Signatures and Application to Epitope-Targeted Vaccine Design

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Antibody Persistence in Adults Two Years after Vaccination with an H1N1 2009 Pandemic Influenza Virus-Like Particle Vaccine

The influenza virus is a human pathogen that causes epidemics every year, as well as potential pandemic outbreaks, as occurred in 2009. Vaccination has proven to be sufficient in the prevention and containment of viral spreading. In addition to the current egg-based vaccines, new and promising vaccine platforms, such as cell culture-derived vaccines that include virus-like particles (VLPs), have been developed. VLPs have been shown to be both safe and immunogenic against influenza infections. Although antibody persistence has been studied in traditional egg-based influenza vaccines, studies on antibody response durations induced by VLP influenza vaccines in humans are scarce. Here, we show that subjects vaccinated with an insect cell-derived VLP vaccine, in the midst of the 2009 H1N1 influenza pandemic outbreak in Mexico City, showed antibody persistence up to 24 months post-vaccination. Additionally, we found that subjects that reported being revaccinated with a subsequent inactivated influenza virus vaccine showed higher antibody titres to the pandemic influenza virus than those who were not revaccinated. These findings provide insights into the duration of the antibody responses elicited by an insect cell-derived pandemic influenza VLP vaccine and the possible effects of subsequent influenza vaccination on antibody persistence induced by this VLP vaccine in humans.     

Protection from the 2009 H1N1 Pandemic Influenza by an Antibody from Combinatorial Survivor-Based Libraries

Influenza viruses elude immune responses and antiviral chemotherapeutics through genetic drift and reassortment. As a result, the development of new strategies that attack a highly conserved viral function to prevent and/or treat influenza infection is being pursued. Such novel broadly acting antiviral therapies would be less susceptible to virus escape and provide a long lasting solution to the evolving virus challenge. Here we report the in vitro and in vivo activity of a human monoclonal antibody (A06) against two isolates of the 2009 H1N1 pandemic influenza virus. This antibody, which was obtained from a combinatorial library derived from a survivor of highly pathogenic H5N1 infection, neutralizes H5N1, seasonal H1N1 and 2009 “Swine” H1N1 pandemic influenza in vitro with similar potency and is capable of preventing and treating 2009 H1N1 influenza infection in murine models of disease. These results demonstrate broad activity of the A06 antibody and its utility as an anti-influenza treatment option, even against newly evolved influenza strains to which there is limited immunity in the general population.     

Pandemic Influenza H1N1 2009, Innate Immunity,and the Impact of Immunosenescence on Influenza Vaccine

Seasonal and pandemic strains of influenza have widespread implications for the global economy and global health. This has been highlighted recently as the epidemiologic characteristics for hospitalization and mortality for pandemic influenza H1N1 2009 are now emerging. While treatment with neuraminidase inhibitors are effective for seasonal and pandemic influenza, prevention of morbidity and mortality through effective vaccines requires a rigorous process of research and development. Vulnerable populations such as older adults (i.e., > age 65 years) suffer the greatest impact from seasonal influenza yet do not have a consistent seroprotective response to seasonal influenza vaccines due to a combination of factors. This short narrative review will highlight the emerging epidemiologic characteristics of pandemic H1N1 2009 and focus on immunosenescence, innate immune system responses to influenza virus infection and vaccination, and influenza vaccine responsiveness as it relates to seasonal and H1N1 pandemic influenza vaccines.     

A Novel Bivalent Vaccine Based on a PB2-Knockout Influenza Virus Protects Mice from Pandemic H1N1 and Highly Pathogenic H5N1 Virus Challenges

Vaccination is an effective means to protect against influenza virus. Although inactivated and live-attenuated vaccines are currently available, each vaccine has disadvantages (e.g., immunogenicity and safety issues). To overcome these problems, we previously developed a replication-incompetent PB2-knockout (PB2-KO) influenza virus that replicates only in PB2 protein-expressing cells. Here, we generated two PB2-KO viruses whose PB2-coding regions were replaced with the HA genes of either A/California/04/2009 (H1N1pdm09) or A/Vietnam/1203/2004 (H5N1). The resultant viruses comparably, or in some cases more efficiently, induced virus-specific antibodies in the serum, nasal wash, and bronchoalveolar lavage fluid of mice relative to a conventional formalin-inactivated vaccine. Furthermore, mice immunized with these PB2-KO viruses were protected from lethal challenges with not only the backbone virus strain but also strains from which their foreign HAs originated, indicating that PB2-KO viruses with antigenically different HAs could serve as bivalent influenza vaccines.     

ICAM-1-Based Rabies Virus Vaccine Shows Increased Infection and Activation of Primary Murine B Cells In Vitro and Enhanced Antibody Titers In-Vivo

We have previously shown that live-attenuated rabies virus (RABV)-based vaccines infect and directly activate murine and human primary B cells in-vitro, which we propose can be exploited to help develop a single-dose RABV-based vaccine. Here we report on a novel approach to utilize the binding of Intracellular Adhesion Molecule-1 (ICAM-1) to its binding partner, Lymphocyte Function-associated Antigen-1 (LFA-1), on B cells to enhance B cell activation and RABV-specific antibody responses. We used a reverse genetics approach to clone, recover, and characterize a live-attenuated recombinant RABV-based vaccine expressing the murine Icam1 gene (rRABV-mICAM-1). We show that the murine ICAM-1 gene product is incorporated into virus particles, potentially exposing ICAM-1 to extracellular binding partners. While rRABV-mICAM-1 showed 10-100-fold decrease in viral titers on baby hamster kidney cells compared to the parental virus (rRABV), rRABV-mICAM-1 infected and activated primary murine B cells in-vitro more efficiently than rRABV, as indicated by significant upregulation of CD69, CD40, and MHCII on the surface of infected B cells. ICAM-1 expression on the virus surface was responsible for enhanced B cell infection since pre-treating rRABV-mICAM-1 with a neutralizing anti-ICAM-1 antibody reduced B cell infection to levels observed with rRABV alone. Furthermore, 100-fold less rRABV-mICAM-1 was needed to induce antibody titers in immunized mice equivalent to antibody titers observed in rRABV-immunized mice. Of note, only 10³ focus forming units (ffu)/mouse of rRABV-mICAM-1 was needed to induce significant anti-RABV antibody titers as early as five days post-immunization. As both speed and potency of antibody responses are important in controlling human RABV infection in a post-exposure setting, these data show that expression of Icam1 from the RABV genome, which is then incorporated into the virus particle, is a promising strategy for the development of a single-dose RABV vaccine that requires only a minimum of virus.     

Phase 1 Evaluation of Intranasal Virosomal Influenza Vaccine with and without Escherichia coli Heat-Labile Toxin in Adult Volunteers

Virosomal vaccines were prepared by extracting hemagglutinin (HA) and neuraminidase from influenza virus and incorporating it in the membranes of liposomes composed of phosphatidylcholine. Two intranasal spray vaccine series were prepared: one series comprised 7.5 micrograms of HA of each of three strains recommended by the World Health Organization and 1 microgram of Escherichia coli heat-labile toxin (HLT), and the other contained the HA without HLT. In addition, a third vaccine preparation contained 15 micrograms of HA and 2 micrograms of HLT. The parenteral virosomal vaccine contained 15 micrograms of HA without additional adjuvant. The immunogenicity of a single spray vaccination (15 micrograms of HA and 2 micrograms of HLT) was compared with that of two vaccinations (7.5 micrograms of HA with or without 1 microgram of HLT) with an interval of 1 week in 60 healthy working adults. Twenty volunteers received one parenteral virosomal vaccine. Two nasal spray vaccinations with HLT-adjuvanted virosomal influenza vaccine induced a humoral immune response which was comparable to that with a single parenteral vaccination. A significantly higher induction of influenza virus-specific immunoglobulin A was noted in the saliva after two nasal applications. The immune response after a single spray vaccination was significantly lower. It could be shown that the use of HLT as a mucosal adjuvant is necessary to obtain a humoral immune response comparable to that with parenteral vaccination. All vaccines were well tolerated.     

Enhanced production of penicillin G acylase from a recombinant Escherichia coli

Penicillin G acylase (pac) gene was cloned into a stable asd ⁺ vector (pYA292) and expressed in Escherichia coli. This recombinant strain produced 1000 units penicillin G acylase g^–1 cell dry wt, which is 23-fold more than that produced by parental Escherichia coli ATCC11105. This enzyme was purified to 16 units mg^–1 protein by a novel two-step process.     

Cross-Reactive Neutralizing Antibodies Directed against Pandemic H1N1 2009 Virus Are Protective in a Highly Sensitive DBA/2 Mouse Influenza Model

Our ability to rapidly respond to an emerging influenza pandemic is hampered somewhat by the lack of a susceptible small-animal model. To develop a more sensitive model, we pathotyped 18 low-pathogenic non-mouse-adapted influenza A viruses of human and avian origin in DBA/2 and C57BL/6 mice. The majority of the isolates (13/18) induced severe morbidity and mortality in DBA/2 mice upon intranasal challenge with 1 million infectious doses. Also, at a 100-fold-lower dose, more than 50% of the viruses induced severe weight loss, and mice succumbed to the infection. In contrast, only two virus strains were pathogenic for C57BL/6 mice upon high-dose inoculation. Therefore, DBA/2 mice are a suitable model to validate influenza A virus vaccines and antiviral therapies without the need for extensive viral adaptation. Correspondingly, we used the DBA/2 model to assess the level of protection afforded by preexisting pandemic H1N1 2009 virus (H1N1pdm) cross-reactive human antibodies detected by a hemagglutination inhibition assay. Passive transfer of these antibodies prior to infection protected mice from H1N1pdm-induced pathogenicity, demonstrating the effectiveness of these cross-reactive neutralizing antibodies in vivo.Respiratory tract infections are the third leading cause of mortality in the world (27). Influenza, a disease of the airways caused by influenza viruses, is responsible for approximately half a million deaths and 3 to 5 million hospitalizations per year (28). In addition to the annual disease burden, influenza A virus is more notoriously known for its ability to cause pandemics. Three pandemics have been reported in the twentieth century: the first that occurred in 1918 (Spanish influenza) killed 20 to 50 million individuals (15); the other two in 1957 and 1968, although less lethal, killed millions due to the lack of preexisting immunity. In April 2009, two cases of febrile illness were confirmed to be caused by swine-origin influenza A virus (H1N1) (4, 8). Continuous spread within North America and other parts of the world has signaled the first influenza pandemic of this century.To study the pathogenicity of influenza A viruses, including the current pandemic A (H1N1) 2009 virus (H1N1pdm), in mammalian hosts and to determine the effectiveness of pharmaceutical interventions, it is essential to have a sensitive animal model. Although influenza has some important differences in mice and humans, a murine model is the only animal model thus far described that allows for relatively high group numbers and any relatively high throughput. Unfortunately, only a few strains of influenza A virus—almost exclusively belonging to the highly pathogenic avian influenza virus isolates of the H5 and H7 subtypes—are pathogenic in most commonly used mouse strains without adaptation through serial passaging. The hemagglutinin (HA) proteins of these H5 and H7 viruses contain a basic amino acid cleavage site, allowing them to spread systemically (12, 19, 26). Most other subtypes of influenza virus, including H1N1 and H3N2, either do not infect or cause very mild disease in mice. The requirement for adaptation of a pandemic virus to commonly used mouse strains can lead to a delay in the gathering of important data to help guide public health control strategies. As such, the lack of a sensitive small-animal model to study the infection dynamics of various subtypes of avian influenza viruses severely hampers the rapid and effective response required during a pandemic or prepandemic situation.This study was designed to demonstrate the utility of DBA/2 mice, previously reported to be susceptible to highly pathogenic influenza viruses (1), to study infections caused by several influenza A virus subtypes isolated from birds or humans without the need for prior adaptation. To assess the utility of the model to respond to emerging strains, we used DBA/2 mice to examine the functional activity of sera from individuals previously shown to have preexisting cross-reactive H1N1pdm antibodies. It is hypothesized that these individuals may be partially protected from infection because of the presence of cross-reactive neutralizing antibodies produced after infection with a different but related H1N1 virus. This hypothesis is supported by in vitro microneutralization and hemagglutination inhibition (HI) assays (2, 10); however, it is not yet known whether these antibodies are also functional in vivo.     

Neuraminidase-Inhibiting Antibody Is a Correlate of Cross-Protection against Lethal H5N1 Influenza Virus in Ferrets Immunized with Seasonal Influenza Vaccine

In preparing for the threat of a pandemic of avian H5N1 influenza virus, we need to consider the significant delay (4 to 6 months) necessary to produce a strain-matched vaccine. As some degree of cross-reactivity between seasonal influenza vaccines and H5N1 virus has been reported, this was further explored in the ferret model to determine the targets of protective immunity. Ferrets were vaccinated with two intramuscular inoculations of trivalent inactivated split influenza vaccine or subcomponent vaccines, with and without adjuvant, and later challenged with a lethal dose of A/Vietnam/1203/2004 (H5N1) influenza virus. We confirmed that vaccination with seasonal influenza vaccine afforded partial protection against lethal H5N1 challenge and showed that use of either AlPO₄ or Iscomatrix adjuvant with the vaccine resulted in complete protection against disease and death. The protection was due exclusively to the H1N1 vaccine component, and although the hemagglutinin contributed to protection, the dominant protective response was targeted toward the neuraminidase (NA) and correlated with sialic acid cleavage-inhibiting antibody titers. Purified heterologous NA formulated with Iscomatrix adjuvant was also protective. These results suggest that adjuvanted seasonal trivalent vaccine could be used as an interim measure to decrease morbidity and mortality from H5N1 prior to the availability of a specific vaccine. The data also highlight that an inducer of cross-protective immunity is the NA, a protein whose levels are not normally monitored in vaccines and whose capacity to induce immunity in recipients is not normally assessed.     

Neutralizing Epitopes of Influenza Virus Hemagglutinin: Target for the Development of a Universal Vaccine against H5N1 Lineages

The nature of influenza virus to randomly mutate and evolve into new types with diverse antigenic determinants is an important challenge in the control of influenza infection. Particularly, variations within the amino acid sequences of major neutralizing epitopes of influenza virus hemagglutinin (HA) hindered the development of universal vaccines against H5N1 lineages. Based on distribution analyses of the identified major neutralizing epitopes of hemagglutinin, we selected three vaccine strains that cover the entire variants in the neutralizing epitopes among the H5N1 lineages. HA proteins of selected vaccine strains were expressed on the baculovirus surface (BacHA), and the preclinical efficacy of the vaccine formulations was evaluated in a mouse model. The combination of three selected vaccine strains could effectively neutralize viruses from clades 1, 2.1, 2.2, 4, 7, and 8 of influenza H5N1 viruses. In contrast, a vaccine formulation containing only adjuvanted monovalent BacHA (mono-BacHA) or a single strain of inactivated whole viral vaccine was able to neutralize only clade 1 (homologous), clade 2.1, and clade 8.0 viruses. Also, the trivalent BacHA vaccine was able to protect 100% of the mice against challenge with three different clades (clade 1.0, clade 2.1, and clade 7.0) of H5N1 strains compared to mono-BacHA or inactivated whole viral vaccine. The present findings provide a rationale for the development of a universal vaccine against H5N1 lineages. Furthermore, baculoviruses displaying HA will serve as an ideal choice for a vaccine in prepandemic or pandemic situations and expedite vaccine technology without the requirement of high-level-biocontainment facilities or tedious protein purification processes.The nature of influenza virus to randomly mutate and evolve into new types with diverse antigenic determinants is an important challenge in the control of influenza infection (20). This has been evidently recognized by the recent outbreaks of H5N1 avian influenza virus infection and the current pandemic situation with H1N1 swine-origin influenza A virus (S-OIV). In fact, it has been well documented in the literature that H5N1 had acquired the ability to infect human tissues due mainly to the occurrence of mutation events (1). Highly pathogenic avian influenza (HPAI) H5N1 viruses are antigenically distinguishable owing to differences in hemagglutinin (HA) sequences, the principal determinant of immunity to influenza virus, resulting in different lineages or clades of H5N1 (13, 33). The control of infection with current H5N1 vaccines does not appear to be effective against heterologous strains or phylogenetically variant clades of H5N1 in part due to variations in the HA sequences, particularly within the neutralizing epitope region. Since present vaccines are based solely on the induction of neutralizing antibodies against these epitopes, differences in these sequences may render current vaccines unqualified for the prevention of influenza globally (15, 28, 31). To overcome such limitations and to completely realize the potential of vaccines worldwide, the concept of universal vaccines based on conserved viral proteins has recently been proposed. The highly conserved ion channel protein (M2) and the nucleoprotein (NP) of influenza virus have been evaluated for the induction of cross-protective cellular immunity and viral clearance (2, 35). Antibodies generated against these conserved proteins may reduce viral spread and accelerate recovery from influenza (14). However, antibodies specific to these proteins are poorly immunogenic and were found previously to be infection permissive (5-7, 13). Thus, the development of a vaccine based on influenza virus hemagglutinin appears to be the only viable option to prevent infections by HPAI viruses such as H5N1 viruses. Nevertheless, amino acid variations within the major antigenic neutralizing epitope regions among H5 subtypes restrict the development of such universal vaccines against different H5N1 lineages.The development of a universal vaccine based entirely on HA of influenza virus is still feasible, if the variation or conservation of neutralizing epitopes among the several HPAI H5N1 virus clades can be identified. An understanding of the distribution pattern of such neutralizing epitopes could help in the design of future vaccines by incorporating two or more ideal H5N1 strains in the vaccine composition. The neutralizing epitopes of the selected viral strains should cover the variations among most H5 subtypes in order to acquire broad-range protective immunity against most H5N1 subtypes. Previous attempts to identify amino acid substitutions within HA sequences of variants that escaped from neutralization by monoclonal antibodies (MAbs) revealed the neutralizing epitope sites of HA (9, 10). Along with previous findings, we report here the identification of other major neutralizing epitopes of H5N1 by mapping their amino acid sequences using neutralizing monoclonal antibodies (n-MAbs). Analysis of the distribution of all identified neutralizing epitopes among H5 subtypes revealed variations within the antigenic determinants of H5N1 subtypes from both human and avian sources. Based on these results, we have selected three vaccine strains comprising the major neutralizing epitopes of HA to cover the entire variants within H5N1 lineages. In order to test our hypothesis in vivo, HA proteins of selected vaccine strains were expressed on the baculovirus surface (BacHA), and the efficacy of the vaccine formulations was evaluated with a mouse model challenged with phylogenetically variant H5N1 strains.     

Protective Efficacy of a Human Endogenous Retrovirus Envelope-Coated,Nonreplicable, Baculovirus-Based Hemagglutin Vaccine against Pandemic Influenza H1N1 2009

Despite the advantages of DNA vaccines, overcoming their lower efficacy relative to that of conventional vaccines remains a challenge. Here, we constructed a human endogenous retrovirus (HERV) envelope-coated, nonreplicable, baculovirus-based HA vaccine against swine influenza A/California/04/2009(H1N1) hemagglutin (HA) (AcHERV-sH1N1-HA) as an alternative to conventional vaccines and evaluated its efficacy in two strains of mice, BALB/c and C57BL/6. A commercially available, killed virus vaccine was used as a positive control. Mice were intramuscularly administered AcHERV-sH1N1-HA or the commercial vaccine and subsequently given two booster injections. Compared with the commercial vaccine, AcHERV-sH1N1-HA induced significantly higher levels of cellular immune responses in both BALB/c and C57BL/6 mice. Unlike cellular immune responses, humoral immune responses depended on the strain of mice. Following immunization with AcHERV-sH1N1-HA, C57BL/6 mice showed HA-specific IgG titers 10- to 100-fold lower than those of BALB/c mice. In line with the different levels of humoral immune responses, the survival of immunized mice after intranasal challenge with sH1N1 virus (A/California/04/2009) depended on the strain. After challenge with 10-times the median lethal dose (MLD₅₀) of sH1N1 virus, 100% of BALB/c mice immunized with the commercial vaccine or AcHERV-sH1N1-HA survived. In contrast, C57BL/6 mice immunized with AcHERV-sH1N1-HA or the commercial vaccine showed 60% and 70% survival respectively, after challenge with sH1N1 virus. In all mice, virus titers and results of histological analyses of lung tissues were consistent with the survival data. Our results indicate the importance of humoral immune response as a major defense system against influenza viral infection. Moreover, the complete survival of BALB/c mice immunized with AcHERV-sH1N1-HA after challenge with sH1N1 virus suggests the potential of baculoviral vector-based vaccines to achieve an efficacy comparable to that of killed virus vaccines.