Caracterización antigénica e inmunogénica de la proteína F del Metaneumovirus humano y comparación con la del virus respiratorio sincitial humano

Abstract

Human metapneumovirus (hMPV) and human respiratory syncytial virus (hRSV) are the main etiological agents of acute lower respiratory tract infections in infants and very young children (ARI). ARI is an important cause of childhood morbidity and mortality worldwide. hMPV and hRSV are members of the Pneumovirinae subfamily within the Paramyxoviridae family. They are enveloped, non-segmented viruses, with negative-sense single stranded RNA genomes. hRSV is the best characterized virus of this group. hMPV was first reported in 2001as the infectious agent of so far etiologically uncharacterized respiratory samples. hMPV was found to be structurally similar to hRSV in genomic organization, viral structure, antigenicity and clinical symptoms. There are no licensed hRSV or hMPV vaccines currently available. Most present efforts to control these pathogens are focused on the prophylactic use of neutralizing antibodies against the fusion protein (F) and the development of efficacious vaccines. In humans, high titers of serum neutralizing antibodies against the F protein correlate with protection of adult volunteers against a hRSV challenge and lower incidence of severe hRSV infection in children. The F protein promotes fusion of the virus and cell membranes during virus entry and also fusion of the membranes of infected cells with those of surrounding cells to form syncytia. During membrane fusion, the F protein transits from a metastable pre-fusion conformation to a highly stable post-fusion conformation. Previous studies in the laboratory found that antibodies specific of the pre-fusion form of the hRSV F protein (hRSV_F) accounted for most of the neutralizing activity found in human immunoglobulin (Ig) preparations. Similar antibodies could be induced in rabbits inoculated with recombinant vaccinia viruses expressing full-length forms of hRSV_F. These findings preceded the isolation of human pre-fusion specific MAbs which exhibited the highest neutralizing activity so far reported. Further developments led to the design of soluble hRSV_F constructs stabilized in the pre-fusion conformation which induced higher titers of neutralizing antibodies in animal models than soluble post-fusion hRSV_F. The main objective of this Thesis was to compare the antigenic and immunogenic properties of hMPV_F with those of hRSV_F. In order to characterize conformation-specific antibody responses to hMPV, a soluble form of the hMPV_F protein stabilized in the post-fusion conformation (hMPV_FTM-_Δ103-111_KKRKRR_B1) was initially isolated. After depletion of antibodies that bound to hMPV_FTM-_Δ103-111_KKRKRR_B1, those specific for the pre-fusion form of hMNV_F could be uncovered. A vaccinia virus recombinant was used to express a soluble trimeric form of the hMPV_F ectodomain, stabilized in the post-fusion conformation by i) substitution of the natural

2 cleavage site for the second cleavage site of hRSV_F, ii) deletion of the first 9 amino acids of the fusion peptide and iii) addition of a fibritin trimerization domain at the C-terminus of the hMPV_F ectodomain. Unlike it was observed previously with hRSV_F, no neutralizing activity could be detected after depletion of rabbit or human Ig preparations with hMPV_FTM-_Δ103- 111_KKRKRR_B1. This result indicated that the neutralizing antibodies raised against hMPV_F protein are conformation independent; i.e., the majority of these antibodies recognize epitopes shared by the pre-fusion and post-fusion conformations of hMPV_F and probably inhibited virus infectivity by binding to the pre-fusion conformation of the F protein and blocking its refolding. Finally, screening of several MAbs specific for antigenic site IV of hRSV_F identified an antibody, 101F that was able to bind to hMPV_F and inhibited infectivity of hMPV. This finding substantiates other reports about cross-neutralization of hMNV and hRSV by a limited number of monoclonal antibodies. However, polyclonal antibody preparations obtained in rabbits inoculated with vaccinia virus recombinants expressing the full-length or anchorless forms of either hMPV_F or hRSV_F did not show assessable cross-neutralization, indicating that crossneutralization of hMPV and hRSV by antibodies is very limited. Collectively, the results reported here represent a major step forward in our understanding of the antigenic an immunogenic properties of hMNV_F, in comparison with the highly related F protein of hRSV.