A study of the Immune responses to Puumala virus glycoproteins using recombinant vesicular stomatitis virus

Puumala orthohantavirus (PUUV) is an enveloped, negative-stranded RNA virus with a trisegmented genome, of the genus Orthohantavirus in the order Bunyavirales. Bank vole-borne, PUUV spreads through inhalation of aerosolized, virus-laden urine, saliva or faeces from infected bank vole. Incidences of human infections have been documented since the 1950s and have a 3–4-year cycle which coincides with peak years of bank vole abundance. The outcome of PUUV infections could be subclinical or acute causing a form of multiphasic haemorrhagic fever with renal syndrome (HFRS) called Nephropathia epidemica (NE) with varying severities. According to the European centre for disease control and prevention (ECDC), from 2014 to 2018, there were an average of 3000 (ranging from 1826 to 4249) reported cases of NE annually with a case fatality rate of about 0.1%, with Finland, Sweden, and Germany making up 83% of the reported cases (Finland alone accounted for 53% of all cases). The pathogenesis of NE is one of a hyper stimulated immune response to viral infection of the renal/glomerular endothelial cells and includes: increased vascular/capillary leakage which could be caused by a combination of cytokine activity, vascular endothelial growth factor, the complement system, and immune cell mediated cytotoxicity; thrombocytopenia which is a major cause of all bleeding symptoms associated with NE; and acute kidney injury (AKI) which, despite the lack of sufficient supporting evidence, seems to be caused by immune complex deposition in the kidneys. Diagnosis of NE is mainly serological since the low-level viremia in patients is usually short-lived disappearing almost by the time symptoms start, so, although reverse transcriptase polymerase chain reactions (RT- PCR) may be useful diagnostic tool, serological tests like immunofluorescent assay (IFA) and enzyme-linked immunosorbent assay (ELISA) are more helpful. Virus neutralization assays have also been applied for serotyping. Treatment of NE is mainly supportive and a variety of therapeutic drugs which treat symptoms, antivirals, vaccines and immune treatments have been either trialled or approved for the treatment and management of NE with varying levels of success. Vesicular Stomatitis Indiana Virus, VSIV, (family Rhabdoviridae, genus Vesiculovirus), is widely used in research, to study the replication and assembly of enveloped RNA viruses. It is a non-segmented negative- stranded (NNS) bullet shaped virus with a genome that codes for 5 proteins: a nucleoprotein (N), a phosphoprotein (P), matrix protein (M), a glycoprotein (G) and an RNA dependent RNA polymerase (L) in that order. VSIV replication involves a stop-start mechanism controlled by cis-acting signals in the intergenic regions of the viral genome. Virus entry into cells is through interaction with the ubiquitous low density lipoprotein receptor and replication leads to production of particles which are promiscuous by virtue of their ability to assemble and package using any suitable glycoprotein available as its envelope in a process known as phenotypic mixing or pseudotyping. The establishment of a VSIV reverse genetics system together with the knowledge of VSIV replication and pseudotyping has led to a wide variety of clinical and research applications including the production and use of a recombinant, replication defective VSIV in which the glycoprotein gene is replaced with eGFP (rVSVΔG*eGFP) for use in producing pseudotype VSIV bearing foreign glycoproteins. In this work, we set out to establish a rescue system for rVSVΔG*eGFP and use it in the production and testing of VSVIV pseudotypes bearing PUUV Gn and Gc. Using site directed mutagenesis on PUUV Gn and Gc we produced pseudotypes for use to map the neutralizing epitopes of three NAbs against Gn and Gc. Finally, we sought to ascertain if high early neutralizing antibodies are a prognostic factor in NE. To accomplish this, rVSVΔG*eGFP was rescued using a vaccinia virus free protocol that involved EMCV enabled IRES rescue plasmids and BSRT7/5 cells. The rescued particles were used to produce pseudotype particles bearing PUUV Gn and Gc. The pseudotypes were then used in a pseudotype focus reduction neutralization test (pFRNT) with patient serum and the results were compared to results from orthodox focus reduction neutralization test (oFRNT) done with the same patient serum and the results showed strong positive correlation (rs = 0.82) between the methods. While developing the system we identified three amino acids which were mutated in the Vero E6 cell culture adapted PUUV prototype Sotkamo strain sequence, and changing these residues was critical for expression and neutralizing antibody binding of PUUV glycoproteins. The neutralizing epitopes for three earlier produced NAbs were previously mapped using pepscan and phage display techniques. Selected amino acids(aa) residues were mutated on the PUUV M segment plasmid and the mutated plasmids were used to produce pseudotypes. These pseudotypes were then used in a neutralization assay. Replacement of cysteine residues with alanines resulted in pseudotype particles with diminished (16 to 18 %) neutralization titres; double Cys→Ala mutants, as well as mutants with bulky aromatic and charged residues replaced with alanines, have shown even stronger reduction in neutralization titres (from 25 % to the escape phenotype). In silico modelling of the neutralization epitopes supported the hypothesis that these critical residues are located on the surface of viral glycoprotein molecules and thus can be recognized by the antibodies indeed. A similar pattern was observed in experiments with mutant pseudotypes, and sera collected from patients suggesting that these neutralization epitopes are utilized in a course of human PUUV infection. These data will help understanding the mechanisms of hantavirus neutralization and assist construction of vaccine candidates. Viral load, PUUV nucleocapsid protein IgG antibodies and neutralizing antibody titres were determined in an extensively characterized NE patient cohort (n = 116) form Tampere university. Analysis of the results showed that upon hospitalization, most patients already had considerable neutralizing and anti-PUUV-N IgG antibody levels. However, contrary to expectations, neutralizing antibody titres from the first day of hospitalization did not appear to protect from AKI or correlate with more favourable disease outcomes. This indicates that further studies are needed to investigate the applicability of neutralizing antibodies as a therapy for hospitalized NE patients. Finally, this thesis focused on creating innovative tools for the study of PUUV glycoproteins and NE. The tools created here can and has been applied in numerous situations using a wide variety of viruses and could be modified to meet other needs including safe and efficient vaccine development.