Puumala hantavirus : immune responses and vaccines

Abstract

Hantaviruses (family Bunyaviridae) is a group of closely related zoonotic rodent-borne viruses that are transmitted to humans through inhalation of infected rodent excreta. They cause hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS) with varying degree of severity depending on the causative virus. Hantavirus infections are found worldwide, but the majority of cases occur in Asia and northern Europe. As hantaviruses cause diseases with high morbidity and mortality, and as to date there is no specific treatment, efforts are concentrated on the development of vaccines.

The aim of this thesis was to study immune responses to Puumala virus (PUUV), which causes a milder form of HFRS, known as nephropathia epidemica (NE), and to evaluate the use of PUUV nucleocapsid (N) protein as a potential vaccine candidate.

Selection of a human combinatorial antibody phage-display library, generated from splenocytes of a PUUV immune individual, against native PUUV antigens immobilized by monoclonal antibodies (MAbs) was shown to be a suitable method to obtain glycoprotein 2 (G2)-specific PUUV neutralizing recombinant Fab fragments. A new epitope, designated G2a3, partially overlapping the neutralizing epitope of the human MAb 1C9, was identified.

PUUV-specific serum lgA responses in NE patients were characterized regarding kinetics, antigen specificity and neutralizing activity. Elevated levels of total IgA were observed in acute-phase samples and the majority of patients had N- and G2-specific IgA1 antibodies, while responses against G1 were low. PUUV neutralizing IgA1 was detected in acute-phase NE sera and PUUV-specific lgA was detected in late convalescent (>10-year) samples. lgA was shown to be a useful complement to IgM detection in the diagnosis of PUUV infection.

The role of the immune response in protection, as well as in the pathogenesis of hantavirus infection, is not clear. Immune responses against the N protein were studied in inbred mice immunized with PUUV recombinant N (rN) protein. PUUV-specific IgG of all subclasses was induced, and epitope mapping showed that B-cell sites were mainly located in the aminoterminal part of N. Several T-cell recognition sites, spanning amino acids 6-27, 96-117, 211 232 and 256-277 of N, were identified by proliferation assays and rN restimulated T-helper (Th)-cells mainly produced interleukin (M)-2 and interferon-gamma, together with low levels of IL-4 and IL-6, indicating a mixed Thl/Th2 response.

As hantaviruses are closely related the characteristics of cross-protective responses are interesting. Bank voles (Clethrionomys glareolus), the natural host of PUUV, were immunized with PUUV, Andes, Dobrava and Topografov virus rN proteins and subsequently challenged with PUUV. The rN proteins elicited cross-protection against challenge, and analysis of the elicited immune responses indicated a more important role for the cellular immune response compared to the humoral in cross-protection elicited by the N protein.

Since hantaviruses are transmitted to humans mainly through inhalation, intranasal vaccination could be an interesting alternative vaccine strategy. This was evaluated in a transnasal/respiratory challenge model in bank voles, after immunization with PUUV rN protein formulated with the lipid based Eurocine® adjuvant (mono-olein/oleic acidlsoy bean oil). Protection was observed in 5/7 animals immunized three times intranasally, while 319 animals, immunized once subcutaneously and twice intranasally, were protected. In contrast, immunization with rN without Eurocine® gave rise to protection in only 2/9 bank voles. High titer IgG responses were detected in serum after intranasal vaccination, showing that a strong systemic antibody response could be elicited.