Evaluation of an inactivated Ross River virus vaccine in active and passive mouse immunization models and establishment of a correlate of protection
Introduction
Since 1928, Australia has reported epidemics of a disease characterized by arthritis particularly of the small joints of the hands and feet, often associated with fever and rash [1], [2], [3]. The arthritic symptoms typically last from six weeks to six months, and occasionally for as long as a year, with a correspondingly significant burden on the public health system [4]. Ross River virus (RRV), the causative agent of this syndrome known as epidemic polyarthritis or also RRV disease, is a mosquito-transmitted alphavirus belonging to the Semliki Forest antigenic complex that also Chikungunya (CHIKV), O’nyong-nyong (ONNV) and Barmah Forest viruses (BFV) [5] Although experimental inactivated RRV vaccines have been described previously [6], [7], [8], at present, no licensed vaccine against RRV is available.
In adult, immune-competent mice, RRV challenge does not cause clinical signs, however, a short viremia is induced. Therefore, protection from RRV infection following challenge with the prototype strain T48 was previously evaluated by the absence of viremia [7]. Protection from viremia, however, is not sufficient to define the protective effects of a vaccine in humans, and more specific models are needed for vaccine characterization, particularly when animal models are used to define a correlate of human protection [9].
Recently, an infection model using mice of younger age showing clinical features of arthritic disease has been developed [10], [11]. The model has been extensively characterized with regard to pathogenesis and showed a number of signs similar to those observed in humans such as inflammation and damage of muscle tissue, and prolonged persistence of virus in joint-associated muscle tissue. Since immune-competent mice are susceptible to RRV-induced disease only in the first two weeks of life, the young mouse disease model is useful to study virus-induced pathology and for passive immunization experiments. Moreover, alphaviruses are known as strong inducers of the type-I interferon response [12], and symptomatic and in part lethal infection in interferon-α/β receptor knock-out (IFN-α/βR−/−) mice has been shown for CHIKV and Sindbis virus [13], [14]. Because IFN-α/βR−/− mice show no overt anomalies, but are unable to cope with certain viral infections, despite otherwise normal immune responses [15], [16], they provide a model to investigate the adaptive immune response and perform active protection studies under stringent, frequently lethal conditions.
We have now investigated the immunogenicity and efficacy of an inactivated whole virus RRV vaccine in several mouse models. The vaccine used was an improved version of a previously published vaccine produced in Vero cells [7] now comprising an additional RNA purification step of the primary seed virus and a double virus inactivation by formalin and UV and a further purification step. The evaluation described here was performed in the traditional adult mouse active immunization model [7] and additionally, by active immunization in the IFN-α/βR−/− mouse model. Since immunity induced by inactivated virus vaccines is mainly conferred by neutralizing antibodies, human vaccinee sera were investigated in two different models of passive protection, the adult mouse viremia and the young mouse disease model. In the latter model, we present for the first time animal data on passive protection from human arthritis-like disease signs, and analyze the effect of antibodies on virus persistence and distribution. Further, a correlate of protection was established.
Finally, the phenomenon of antibody-dependent enhancement (ADE) of virus infection in vivo was addressed. ADE has been initially described to be caused by the presence of cross-reactive, yet not cross-neutralizing antibodies to a related virus [17], or by subneutralizing concentrations of antibodies to the same virus [18]. Despite rather limited evidence for its relevance to humans beyond the Dengue viruses [19], [20], ADE by inactivated virus vaccines remains a theoretical concern, recently also raised for RRV [21]. For RRV, it has been possible to enhance infection of cells with RRV by the addition of small amounts of antibody in vitro [22]. This evidence for the potential occurrence of ADE in both of the mouse models was carefully evaluated. The vaccine did neither enhance infection by RRV nor by the related alphavirus Chikungunya virus.
Section snippets
Ethics statement
All animal experiments were reviewed by the Baxter Vienna/Orth Institutional Animal Care and Use Committee (IACUC) and approved by the Austrian regulatory authorities. All animal experiments were conducted in accordance with Austrian laws on animal experimentation and guidelines set out by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Animals were housed in facilities accredited by the AAALAC.
Viruses and cell lines
The mouse virulent RRV prototype strain T48 [23], [24] was
Protection of adult mice from viremia after active immunization
After vaccination of adult mice, twice within 28 days with vaccine doses between 10 μg and down to 0.0025 μg (Table 1), mice developed RRV antibodies as quantified by ELISA, in a clearly dose-dependent fashion. Immunization with 0.039 μg resulted in titers approximately 10-fold over the limit of detection (LOD < 100) and doses of 0.625 μg or higher drove ELISA titers to more than 100-fold the LOD. Similarly, while a vaccine dose of only 0.0025 μg did not afford protection of mice from RRV viremia,
Discussion
Immunogenicity of the RRV vaccine was first studied by active immunization of mice and subsequent challenge to demonstrate protection. The vaccine elicited a similarly strong antibody response both in immune-competent mice (Table 1) and IFN-α/βR−/− mice (Table 2), and fully protected challenged mice from viremia (Fig. 1), or from death and disease, in case of the knock-out mice (Fig. 2). The latter model has been used because alphaviruses are usually efficiently cleared in immune competent mice
Acknowledgments
We thank Elisabeth Hitter for animal work in the active immunization viremia mouse model, and Reinhard Ilk for performing statistical analyses.
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These authors have equal contribution.