Controlling swine influenza viruses through assessments of a live attenuated vaccine strain and their relative zoonotic risk

Influenza A viruses (IAVs) are a major cause of respiratory disease in swine as well as in humans. In swine, multiple distinct lineages of IAVs circulate, and currently available vaccines fail to protect against all variants. Therefore, more broadly protective influenza vaccines for swine are needed. Most IAVs of swine are derived from influenza viruses that circulated in humans in the past. Due to a separate IAV evolution in both species, contemporary human influenza viruses genetically and antigenically differ from those in swine. IAVs of birds and horses are also distinct from the current human influenza viruses. Because of these differences, immunity against human influenza viruses is unlikely to protect against infection with an animal influenza virus. IAVs sometimes cross the species barrier from animals to humans. Should an animal influenza virus adapt to efficient replication and transmission in the human population and population immunity is lacking, a pandemic is possible.In chapter 1, we give a general introduction to influenza virus classification, as well as a description of IAV structure, replication, and evolution. We also discuss implications of different aspects of the influenza A virus replication for their host range. Then, we summarize the epidemiology of IAVs in wild and domestic birds, horses, dogs, swine, and humans, with emphasis on swine and humans. We also describe cross-species transmissions and the pandemic potential of IAVs. Finally, we give an overview of the immune responses to influenza viruses in humans and swine. We describe innate and adaptive immune mechanisms upon influenza virus infection as well as current and alternative vaccination strategies.Chapter 2 outlines the aims of the thesis. The first aim was to investigate the safety and efficacy of a live attenuated swine influenza vaccine strain of the H3N2 subtype. The second aim was to assess the public health risk of different animal influenza viruses of subtypes H3 and H1.In chapter 3, we characterized a live attenuated influenza vaccine strain for swine, lvTX98. It belongs to the H3N2 subtype and was included in the first live attenuated swine influenza vaccine, which reached the US market in 2017. In chapter 3.1, we evaluated the level of attenuation of lvTX98 in pigs. We compared its excretion and pathogenesis with that of the wild type virus, from which the vaccine strain is derived by a deletion in the NS1 gene. Vaccine strain lvTX98 was partially attenuated in pigs. After intranasal inoculation, nasal shedding was lower than for the wild type virus but still substantial. After intratracheal inoculation, vaccine virus replication in the swine respiratory tract was reduced at early timepoints after infection, but lvTX98 could replicate to similar titers as the wild type virus by day 3. The vaccine strain caused significantly milder clinical signs as compared to the wild type virus. However, it caused comparable levels of macroscopic and microscopic lung lesions, neutrophil infiltration, and pro-inflammatory cytokine levels in the lungs. Based on findings in vitro, attenuation of lvTX98 was believed to result from the induction of elevated levels of type I interferon. These pro-inflammatory cytokines are important to induce an antiviral response in the host. However, we could not associate attenuation of the vaccine strain in pigs in vivo with higher type I interferon levels in the lungs as compared with the wild type virus. We conclude that influenza vaccine strain lvTX98 is not sufficiently attenuated to ensure its safety. We also conclude that its attenuation is due to the loss of viral NS1 functions other than interference with type I interferon-mediated antiviral host response. In chapter 3.2, we determined the efficacy of a single intranasal vaccination of pigs with lvTX98 against divergent swine influenza viruses of the H3 subtype. The vaccination offered complete protection against the homologous wild type virus and partial protection against divergent H3 viruses that represent major swine IAV lineages circulating in North America and Europe. These lineages included North American cluster IV, North American novel human-like, and European swine H3 influenza viruses. We could not detect virus neutralizing antibodies against thevariable HA protein of these heterologous H3 swine viruses in serum of vaccinated pigs. Therefore, partial protection likely resulted from mucosal and cellular immune responses against conserved parts of the swine influenza virus proteins. We conclude that one vaccination with lvTX98 was not sufficient to provide the desired broad protection. However, this live attenuated influenza vaccine strain could be useful in combination with a second vaccine in a prime-boost regimen.Chapter 4 deals with the public health risk of different animal influenza A viruses of subtypes H3 and H1. Chapter 4.1 is about H3 influenza viruses of swine, birds, and horses. We selected viruses representative of the major contemporary H3 lineages in these species. We estimated human population immunity against these animal IAVs by testing serum of persons of different age groups for protective antibody levels. We also assessed the replication potential of these animal IAVs in human airway tissues. Population immunity was high for North American swine H3 IAVs: more than 50% of the test persons had protective antibody levels. We found intermediate population immunity against European swine H3 IAVs, with protective antibodies in 7%U+201337% of the persons. Less than 13% of the persons had protective serum antibody levels against H3 IAVs of birds and horses, suggesting that population immunity against these viruses is minimal. Antibody responses against H3 IAVs of swine, which are all derived from past human H3 IAVs, showed age-dependent trends that seemed to reflect exposure to related human influenza viruses. This indicates slow antigenic evolution of swine H3 influenza viruses and supports the role of swine as a reservoir for past human H3 influenza viruses. Antibody levels for avian and equine H3 IAVs were minimal in all age groups. Virus replication in human airway tissues was efficient for swine H3 IAVs and intermediate for a H3 IAV from horses and one from poultry. H3 IAVs from wild birds could not replicate in the human airway tissues. We conclude that among the H3 influenza viruses of different species, those of swine pose the highest threat to public health. In chapter 4.2, we used the same approach as in chapter 4.1 to estimate human population immunity against all major H1 influenza virus lineages of swine and their human ancestor IAVs. We found that at least 50% of the persons had protective serum antibody levels against viruses of 2 lineages called the European human-like and the classical swine H1 lineage. Population immunity was intermediate for viruses of 2 other lineages called the Asian avian-like and the North American human-like U+03B41a swine H1 lineage. At least 24% of the test persons had protective serum antibody levels against these IAVs. For viruses of the last 2 lineages designated the European avian-like and the North American human-like U+03B41b swine H1 lineage, antibody levels were minimal, with not more than 10% of the persons having protective antibody levels. As for swine H3 IAVs, human antibody responses against swine H1 IAVs showed age-dependent trends. Antibody levels against human-derived swine IAVs showed different extents of correlation with those against the presumed human ancestor IAV. Our results suggest that among the different H1 IAVs of swine, those belonging to the North American human-like U+03B41b and European avian-like swine H1 influenza virus lineages pose the highest public health risk.In chapter 5, we discuss the overall findings of all experimental studies performed during this doctoral research. Our pathobiology studies with live attenuated swine influenza vaccine strain lvTX98 suggest safety concerns. We discuss possible strategies to improve lvTX98 attenuation and safety. One vaccination with lvTX98 offered partial protection against a range of distinct swine H3 IAVs. We speculate about different applications of lvTX98 and similar live attenuated vaccine strains in heterologous prime-boost strategies that could provide broad protection against IAVs of one or multiple subtypes. We found that humans have varying levels of antibodies against animal H3 and H1 influenza viruses. We discuss the correlation between antibody levels against the animal IAVs and those against related human IAVs. Since animal IAVs might jump to humans, we compare the public health risk of different animal H3 and H1 influenza viruses based on serum antibody levels in humans and the replication potential of these animal viruses in human tissues. We also mention other attributes of influenza A viruses and humans that influence the public health risk of animal H3 and H1 influenza viruses.

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