Resumen de Towards a universal influenza vaccine: generation and evaluation of vaccine formulations based on ha-epitopes in different influenza hosts
Influenza viruses (IAVs) have been implicated in five pandemics and are the cause of seasonal epidemics year after year. They are characterized for their high zoonotic potential, affecting a wide range of hosts. Wild aquatic birds are their main reservoirs that transmit the virus to domesticated birds and may spread further to pigs and humans. Moreover, pigs are considered intermediate hosts, susceptible to infection for both humans and avian IAVs. The most effective countermeasure against IVs are the vaccines. Most of them are inactivated vaccines containing strains of the most common circulating subtypes of IVs. However, they present several inconveniences like limiting cross-protection capacity. These limitations require reformulation of vaccines matched to the genetic changes of the virus and demand continuous vigilance for possible pandemics. To overcome these hurdles, current research focuses on seeking for a universal influenza vaccine, implementing several vaccine strategies by using different immunomodulators and highly-conserved IV epitopes in vaccine formulations. This doctoral thesis evaluates distinct approaches both in improving vaccine formulation and its application/evaluation to different IV natural hosts and has been divided into 3 parts: In the Part I, the general introduction (Chapter I) and the general and specific objectives (Chapter II), are described. The introduction explains (i) influenza viruses (ii) the viruses of the genus A (iii) host's immune response against IVs; (iv) the commercial vaccines available against influenza in human, avian and porcine (v) approaches to obtain a universal vaccine and the (vi) ISM, an in silico tool which has been used in the thesis to predict conserved peptides. Chapter II describes the objective of this thesis. Overall, it is intended to create a universal prototype vaccine against different subtypes of IVs using techniques that incorporate in silico predicted conserved HA-epitopes introduced in plasmid or soluble form along with different adjuvants.
In Part II (Chapters III-V), three studies that are either published/submitted to international peer-reviewed scientific journals, are included. The design of vaccine prototypes with possible multivalent character and its subsequent application in pigs and birds is evaluated. In the first study, one conserved HA-epitope NF-34, predicted by ISM, was modified and formulated in a plasmid with CTLA-4 (pCMV-CTLA4-Ig-NG34), promoter of the adaptive response. The vaccine approach was used in swine influenza seronegative and seropositive pigs and challenged against heterologous H3N2. Vaccinated pigs secreted fewer viruses, cleared the virus in the respiratory airways, presented humoral response to the most relevant circulating subtypes and elicited neutralizing antibodies. However, there were no differences in the degree of pulmonary lesions and clinical signs. Maternal antibodies did not interfere with the effect of the vaccine. In the second study, a cocktail of HA-epitopes combined with flagellin (VC-4 flagellin), a promoter of the innate response, was used in SIV seronegative and seropositive pigs and were challenged with homologous and heterologous IAV strains. Vaccinated pigs reduced virus excretion, produced humoral response to subtypes H1 and H3, and neutralizing antibodies against both viruses. Maternal antibodies were not an obstacle but, clinical signs and pathology in the lungs were not reduced. Finally, in the third study, baculovirus expression vector system (BEVS) was used to obtain protein extracts containing HA-peptides linked with flagellin. The formulation was used to immunize chickens that were challenged with H7N1, a highly pathogenic virus. Vaccinated animals survived without showing any clinical sign and fewer or no virus secretion. The protection mechanism is under investigation.
The general discussion is described in part III (Chapters VI-VII) with possible significance of the results obtained and the relation of conclusions drawn from each study. The bibliographical references (Chapter VIII) and appendices are also included.
