Jefferson Santos

The Molecular Determinants of Antibody Recognition and Antigenic Drift in the H3 Hemagglutinin of Swine Influenza A Virus

ABSTRACT Influenza A virus (IAV) of the H3 subtype is an important respiratory pathogen that affects both humans and swine. Vaccination to induce neutralizing antibodies against the surface glycoprotein hemagglutinin (HA) is the primary method used to control disease. However, due to antigenic drift, vaccine strains must be periodically updated. Six of the 7 positions previously identified in human seasonal H3 (positions 145, 155, 156, 158, 159, 189, and 193) were also indicated in swine H3 antigenic evolution. To experimentally test the effect on virus antigenicity of these 7 positions, substitutions were introduced into the HA of an isogenic swine lineage virus. We tested the antigenic effect of these introduced substitutions by using hemagglutination inhibition (HI) data with monovalent swine antisera and antigenic cartography to evaluate the antigenic phenotype of the mutant viruses. Combinations of substitutions within the antigenic motif caused significant changes in antigenicity. One virus mutant that varied at only two positions relative to the wild type had a >4-fold reduction in HI titers compared to homologous antisera. Potential changes in pathogenesis and transmission of the double mutant were evaluated in pigs. Although the double mutant had virus shedding titers and transmissibility comparable to those of the wild type, it caused a significantly lower percentage of lung lesions. Elucidating the antigenic effects of specific amino acid substitutions at these sites in swine H3 IAV has important implications for understanding IAV evolution within pigs as well as for improved vaccine development and control strategies in swine. IMPORTANCE A key component of influenza virus evolution is antigenic drift mediated by the accumulation of amino acid substitutions in the hemagglutinin (HA) protein, resulting in escape from prior immunity generated by natural infection or vaccination. Understanding which amino acid positions of the HA contribute to the ability of the virus to avoid prior immunity is important for understanding antigenic evolution and informs vaccine efficacy predictions based on the genetic sequence data from currently circulating strains. Following our previous work characterizing antigenic phenotypes of contemporary wild-type swine H3 influenza viruses, we experimentally validated that substitutions at 6 amino acid positions in the HA protein have major effects on antigenicity. An improved understanding of the antigenic diversity of swine influenza will facilitate a rational approach for selecting more effective vaccine components to control the circulation of influenza in pigs and reduce the potential for zoonotic viruses to emerge.

All-in-One Bacmids: an Efficient Reverse Genetics Strategy for Influenza A Virus Vaccines

ABSTRACT Vaccination is the first line of defense against influenza virus infection, yet influenza vaccine production methods are slow, antiquated, and expensive as a means to effectively reduce the virus burden during epidemic or pandemic periods. There is a great need for alternative influenza vaccines and vaccination methods with a global scale of impact. We demonstrate here a strategy to generate influenza A virus in vivo by using bacmid DNAs. Compared to the classical reverse genetics system, the “eight-in-one” bacmids (bcmd-RGFlu) showed higher efficiency of virus rescue in various cell types. Using a transfection-based inoculation (TBI) system, intranasal delivery to DBA/2J and BALB/c mice of bcmd-RGFlu plus 293T cells led to the generation of lethal PR8 virus in vivo. A prime-boost intranasal vaccination strategy using TBI in the context of a bcmd-RGFlu carrying a temperature-sensitive H1N1 virus resulted in protection of mice against lethal challenge with the PR8 strain. Taken together, these studies provide proof of principle to highlight the potential of vaccination against influenza virus by using in vivo reverse genetics. IMPORTANCE Vaccination is the first line of defense against influenza virus infections. A major drawback in the preparation of influenza vaccines is that production relies on a heavily time-consuming process of growing the viruses in eggs. We propose a radical change in the way influenza vaccination is approached, in which a recombinant bacmid, a shuttle vector that can be propagated in both Escherichia coli and insect cells, carries an influenza virus infectious clone (bcmd-RGFlu). Using a surrogate cell system, we found that intranasal delivery of bcmd-RGFlu resulted in generation of influenza virus in mice. Furthermore, mice vaccinated with this system were protected against lethal influenza virus challenge. The study serves as a proof of principle of a potentially universal vaccine platform against influenza virus and other pathogens.

Widespread Virus Replication in Alveoli Drives Acute Respiratory Distress Syndrome in Aerosolized H5N1 Influenza Infection of Macaques

Human infections with highly pathogenic avian influenza A (H5N1) virus are frequently fatal but the mechanisms of disease remain ill-defined. H5N1 infection is associated with intense production of proinflammatory cytokines, but whether this cytokine storm is the main cause of fatality or is a consequence of extensive virus replication that itself drives disease remains controversial. Conventional intratracheal inoculation of a liquid suspension of H5N1 influenza virus in nonhuman primates likely results in efficient clearance of virus within the upper respiratory tract and rarely produces severe disease. We reasoned that small particle aerosols of virus would penetrate the lower respiratory tract and blanket alveoli where target cells reside. We show that inhalation of aerosolized H5N1 influenza virus in cynomolgus macaques results in fulminant pneumonia that rapidly progresses to acute respiratory distress syndrome with a fatal outcome reminiscent of human disease. Molecular imaging revealed intense lung inflammation coincident with massive increases in proinflammatory proteins and IFN-α in distal airways. Aerosolized H5N1 exposure decimated alveolar macrophages, which were widely infected and caused marked influx of interstitial macrophages and neutrophils. Extensive infection of alveolar epithelial cells caused apoptosis and leakage of albumin into airways, reflecting loss of epithelial barrier function. These data establish inhalation of aerosolized virus as a critical source of exposure for fatal human infection and reveal that direct viral effects in alveoli mediate H5N1 disease. This new nonhuman primate model will advance vaccine and therapeutic approaches to prevent and treat human disease caused by highly pathogenic avian influenza viruses.

Oral Fluids as a Live-Animal Sample Source for Evaluating Cross-Reactivity and Cross-Protection following Intranasal Influenza A Virus Vaccination in Pigs

ABSTRACT In North American swine, there are numerous antigenically distinct H1 influenza A virus (IAV) variants currently circulating, making vaccine development difficult due to the inability to formulate a vaccine that provides broad cross-protection. Experimentally, live-attenuated influenza virus (LAIV) vaccines demonstrate increased cross-protection compared to inactivated vaccines. However, there is no standardized assay to predict cross-protection following LAIV vaccination. Hemagglutination-inhibiting (HI) antibody in serum is the gold standard correlate of protection following IAV vaccination. LAIV vaccination does not induce a robust serum HI antibody titer; however, a local mucosal antibody response is elicited. Thus, a live-animal sample source that could be used to evaluate LAIV immunogenicity and cross-protection is needed. Here, we evaluated the use of oral fluids (OF) and nasal wash (NW) collected after IAV inoculation as a live-animal sample source in an enzyme-linked immunosorbent assay (ELISA) to predict cross-protection in comparison to traditional serology. Both live-virus exposure and LAIV vaccination provided heterologous protection, though protection was greatest against more closely phylogenetically related viruses. IAV-specific IgA was detected in NW and OF samples and was cross-reactive to representative IAV from each H1 cluster. Endpoint titers of cross-reactive IgA in OF from pigs exposed to live virus was associated with heterologous protection. While LAIV vaccination provided significant protection, LAIV immunogenicity was reduced compared to live-virus exposure. These data suggest that OF from pigs inoculated with wild-type IAV, with surface genes that match the LAIV seed strain, could be used in an ELISA to assess cross-protection and the antigenic relatedness of circulating and emerging IAV in swine.

Replication and transmission of mammalian-adapted H9 subtype influenza virus in pigs and quail.

Influenza A virus is a major pathogen of birds, swine and humans. Strains can jump between species in a process often requiring mutations and reassortment, resulting in outbreaks and, potentially, pandemics. H9N2 avian influenza is predominant in poultry across Asia and occasionally infects humans and swine. Pandemic H1N1 (H1N1pdm) is endemic in humans and swine and has a history of reassortment in pigs. Previous studies have shown the compatibility of H9N2 and H1N1pdm for reassortment in ferrets, a model for human infection and transmission. Here, the effects of ferret adaptation of H9 surface gene segments on the infectivity and transmission in at-risk natural hosts, specifically swine and quail, were analysed. Reassortant H9N1 and H9N2 viruses, carrying seven or six gene segments from H1N1pdm, showed infectivity and transmissibility in swine, unlike the wholly avian H9N2 virus with ferret-adapted surface genes. In quail, only the reassortant H9N2 with the six internal gene segments from the H1N1pdm strain was able to infect and transmit, although less efficiently than the wholly avian H9N2 virus with ferret-adapted surface genes. These results highlight that ferret-adapted mutations on the haemagglutinin of H9 subtype virus do not restrict the ability of the virus to infect swine and quail, and that the ability to transmit in these species depends on the context of the whole virus. As such, this study emphasizes the threat that H9N2 reassortant viruses pose to humans and agricultural species and the importance of the genetic constellation of the virus to its ability to replicate and transmit in natural hosts of influenza.

Short- and long-term protective efficacy against clade 2.3.4.4 H5N2 highly pathogenic avian influenza virus following prime-boost vaccination in turkeys

Highly pathogenic avian influenza virus (HPAIV) infections are frequently associated with systemic disease and high mortality in domestic poultry, particularly in chickens and turkeys. Clade 2.3.4.4 represents a genetic cluster within the Asian HPAIV H5 Goose/Guangdong lineage that has transmitted through migratory birds and spread throughout the world. In 2014, clade 2.3.4.4 strains entered the U.S. via the Pacific flyway, reassorted with local strains of the North American lineage, and produced novel HPAIV strains of the H5N1, H5N2, and H5N8 subtypes. By 2015, the H5N2 HPAIVs disseminated eastwards within the continental U.S. and Canada and infected commercial poultry, causing the largest animal health outbreak in recent history in the U.S. The outbreak was controlled by traditional mass depopulation methods, but the outbreak was of such magnitude that it led to the consideration of alternative control measures, including vaccination. In this regard, little information is available on the long-term protection of turkeys vaccinated against avian influenza. In this report, a vaccination study was carried out in turkeys using 3 prime-boost approaches with a combination of 2 different vaccines, an alphavirus-based replicon vaccine and an adjuvanted-inactivated reverse genetics vaccine. Vaccine efficacy was assessed at 6 and 16weeks of age following challenge with a prototypic novel clade 2.3.4.4 H5N2 HPAIV. All three vaccines protocols were protective with significantly reduced virus shedding and mortality after challenge at 6weeks of age. In contrast, significant variations were seen in 16-week old turkeys after challenge: priming with the alphavirus-based replicon followed by boost with the adjuvanted-inactivated vaccine conferred the best protection, whereas the alphavirus-based replicon vaccine given twice provided the least protection. Our study highlights the importance of studying not only different vaccine platforms but also vaccination strategies to maximize protection against HPAIV especially with regards to the longevity of vaccine-induced immune response.

Plasmid-Based Reverse Genetics of Influenza A Virus

Influenza A viruses have broad host range with a recognized natural reservoir in wild aquatic birds. From this reservoir, novel strains occasionally emerge with the potential to establish stable lineages in other avian and mammalian species, including humans. Understanding the molecular changes that allow influenza A viruses to change host range is essential to better assess their animal and public health risks. Reverse genetics systems have transformed the ability to manipulate and study negative strand RNA viruses. In the particular case of influenza A viruses, plasmid-based reverse genetics approaches have allowed for a better understanding of, among others, virulence, transmission, mechanisms of antiviral resistance, and the development of alternative vaccines and vaccination strategies. In this chapter we describe the cloning of cDNA copies of viral RNA segments derived from a type A influenza virus into reverse genetics plasmid vectors and the experimental procedures for the successful generation of recombinant influenza A viruses.

Development of an Alternative Modified Live Influenza B Virus Vaccine

ABSTRACT Influenza B virus (IBV) is considered a major human pathogen, responsible for seasonal epidemics of acute respiratory illness. Two antigenically distinct IBV hemagglutinin (HA) lineages cocirculate worldwide with little cross-reactivity. Live attenuated influenza virus (LAIV) vaccines have been shown to provide better cross-protective immune responses than inactivated vaccines by eliciting local mucosal immunity and systemic B cell- and T cell-mediated memory responses. We have shown previously that incorporation of temperature-sensitive (ts) mutations into the PB1 and PB2 subunits along with a modified HA epitope tag in the C terminus of PB1 resulted in influenza A viruses (IAV) that are safe and effective as modified live attenuated (att) virus vaccines (IAV att). We explored whether analogous mutations in the IBV polymerase subunits would result in a stable virus with an att phenotype. The PB1 subunit of the influenza B/Brisbane/60/2008 strain was used to incorporate ts mutations and a C-terminal HA tag. Such modifications resulted in a B/Bris att strain with ts characteristics in vitro and an att phenotype in vivo. Vaccination studies in mice showed that a single dose of the B/Bris att candidate stimulated sterilizing immunity against lethal homologous challenge and complete protection against heterologous challenge. These studies show the potential of an alternative LAIV platform for the development of IBV vaccines. IMPORTANCE A number of issues with regard to the effectiveness of the LAIV vaccine licensed in the United States (FluMist) have arisen over the past three seasons (2013–2014, 2014–2015, and 2015–2016). While the reasons for the limited robustness of the vaccine-elicited immune response remain controversial, this problem highlights the critical importance of continued investment in LAIV development and creates an opportunity to improve current strategies so as to develop more efficacious vaccines. Our laboratory has developed an alternative strategy, the incorporation of 2 amino acid mutations and a modified HA tag at the C terminus of PB1, which is sufficient to attenuate the IBV. As a LAIV, this novel vaccine provides complete protection against IBV strains. The availability of attenuated IAV and IBV backbones based on contemporary strains offers alternative platforms for the development of LAIVs that may overcome current limitations.

Development and characterization of a packaging cell line for pseudo-infectious yellow fever virus particle generation

INTRODUCTION Pseudo-infectious yellow fever viral particles (YFV-PIVs) have been used to study vaccines and viral packaging. Here, we report the development of a packaging cell line, which expresses the YFV prM/E proteins. METHODS HEK293 cells were transfected with YFV prM/E and C (84 nt) genes to generate HEK293-YFV-PrM/E-opt. The cells were evaluated for their ability to express the heterologous proteins and to package the replicon repYFV-17D-LucIRES, generating YFV-PIVs. RESULTS The expression of prM/E proteins was confirmed, and the cell line trans-packaged the replicon for recovery of a reporter for the YFV-PIVs. CONCLUSIONS HEK293-YFV-prM/E-opt trans-packaging capacity demonstrates its possible biotechnology application.

Reverse Genetics of Influenza B Viruses

Annual influenza epidemics are caused not only by influenza A viruses but also by influenza B viruses. Initially established for the generation of recombinant influenza A viruses, plasmid-based reverse genetics techniques have allowed researchers the generation of wild type and mutant viruses from full-length cDNA copies of the influenza viral genome. These reverse genetics approaches have allowed researchers to answer important questions on the biology of influenza viruses by genetically engineering infectious recombinant viruses. This has resulted in a better understanding of the molecular biology of influenza viruses, including both viral and host factors required for genome replication and transcription. With the ability to generate recombinant viruses containing specific mutations in the viral genome, these reverse genetics tools have also allowed the identification of viral and host factors involved in influenza pathogenesis, transmissibility, host-range interactions and restrictions, and virulence. Likewise, reverse genetics techniques have been used for the implementation of inactivated or live-attenuated influenza vaccines and the identification of anti-influenza drugs and their mechanism of antiviral activity. In 2002, these reverse genetics approaches allowed also the recovery of recombinant influenza B viruses entirely from plasmid DNA. In this chapter we describe the cloning of influenza B/Brisbane/60/2008 viral RNAs into the ambisense pDP-2002 plasmid and the experimental procedures for the successful generation of recombinant influenza B viruses.