FACTORES INTERNOS

Cell culture systems have proved invaluable for virus characterisation and early evaluation of RSV treatments. However, these models lack an immune system and are thus inappropriate for vaccine efficacy studies. Potential vaccine candidates for RSV have been developed (Section 1.7), but these studies evaluated vaccine efficacy in an inappropriate in vivo model, the mouse. This is because mice are at best only semi-permissive for RSV infection. They can be infected but do not display outward signs of disease. Thus, efficacy must be measured through viral titre reduction, which does not correlate with clinical signs (Easton et al., 2004). Neither can RSV vaccine candidates be studied in children. Thus, an appropriate in vivo model for RSV needs to be developed.

1.5.4.1 RSV in vivo models.

The main advantage of in vivo models is that the disease process of a pathogen as a whole can be investigated, including toxicity and immune responses. In addition, the impact of genetic differences within a population on these processes can be investigated which would be problematic to study in cell culture.

Several species have been used to study RSV pathogenesis and also to evaluate vaccine candidates including chimpanzee and other primates, bovine, cotton rat,

guinea pig and mouse models (BALB/c, C3H and C57BL). Each model has its own advantages and disadvantages (Table 1.2). The main argument for the reduction in the use of animal models is that the findings within an animal do not necessarily reflect the situation in humans. Chimpanzees can be infected with RSV and exhibit upper respiratory tract illness but these are the most expensive and ethically problematic animal system; other primates also support RSV replication but do not demonstrate signs of disease (Belshe, 1977). The advantage of the chimpanzee model is that the immune responses of these animals is similar to humans, as opposed to rodents which do not always have the same immune genes (Moore & Peebles, 2006). Other primates can be used, but again, the immunological reagents available are extremely limited when compared to the rodent or chimpanzee model. Such animals also require a high inoculum of RSV in order to ensure that infection and viral replication occurs, which does not reflect the situation in humans (Simoes, 1999).

The cotton rat model for RSV infection at present remains the best rodent model for studying RSV. The main problem with this system is the lack of availability of reagents and genetic strains, for which the mouse model is superior. Cotton rats are susceptible to both upper and lower respiratory tract infection with RSV, similar to that in humans (Prince, 1978). In addition, the immune genes of the cotton rat are more similar to those of humans than those within the mouse model. The cotton rat genome encodes Mx genes which humans also have and in addition, they also show diminished immune responses with increased age (Boukhvalova et al., 2009).

The most commonly used animal model for RSV is the mouse, mainly due to its low cost, small size, availability of several genetic strains and wide range of available immunological reagents (Table 1.2). These animals are often used as the first stage of vaccine development. For some vaccines, successful evaluation in rodent models is sufficient to justify a move to human clinical trials. However, the knowledge of RSV immunosuppressive and sensitisation functions on the human immune system requires that potential candidates are thoroughly tested to ensure safety before use in humans. This often involves the use of rodents followed by primates before the studies are advanced to human Phase 1 clinical trials. However, despite the stringent evaluation of novel therapeutics before human trials, occasionally, the results from animals models do not reflect the effects within a human.

Type of model Advantages Disadvantages Chimpanzee Highly related to humans

Immunological reagents available Similar RSV pathogenesis

Vaccine enhanced disease observed Genetically varied

Expensive

Limited availability of animals and facilities Genetically varied

Require high inoculums, 104-106 p.f.u. Expensive, ethically problematic Bovine (cow) Use of natural pathogen (bRSV)

Similar disease pathogenesis Bronchiolitis develops

Expensive, ethically problematic

Limited immunological reagents available Bacterial superinfection common and can complicate findings

No inbred strains available RSV requires 106 p.f.u. inoculum Cotton rat Inbred strains available

Greater susceptibility to RSV than mice Vaccine enhanced disease observed

Mx genes (these encode proteins which block the replication of some viruses)

Limited immunological reagents available Non-permissive for viral replication No knockout strains available

Pneumonia disease rather than bronchiolitis RSV requires 105 p.f.u. inoculum

Guinea pigs Juveniles develop bronchiolitis Inbred strains available

Large size allows repeated measurements Suitable for asthma studies

Similar cost to mice

Limited immunological reagents available No knockout strains available

Pneumonia disease rather than bronchiolitis Mouse Multiple strains available: inbred, transgenic, knockout

Large range of immunological reagents Low cost and easily bred

Easy to manipulate large colonies Vaccine enhanced pathology Well defined immunology

Limited viral replication Large dose of RSV required

No disease observed, only histopathology RSV requires 106 p.f.u. inoculums

PVM Mouse model As above for mouse model Use of natural pathogen (PVM) Bronchiolitis disease develops

Low dose of PVM is required to cause a fatal disease Extensive replication of PVM is observed

Requires 10-60 p.f.u. inoculum

Regular screening of the colony required to ensure PVM- free

Difficulties in using the virus in PVM-free environments (may require CAT3 conditions)

Limited PVM-specific reagents available

Table 1.2. Comparison of in vivo models for evaluation of RSV treatments, including the PVM mouse model (adapted from (Moore & Peebles, 2006, Stark, 2006).

The immune response towards RSV has been extensively researched in mice, of which the BALB/c strain was determined to be the most susceptible (Jafri, 2004, Moore & Peebles, 2006, Prince, 1979). However, there are several disadvantages. A high dose of RSV is required to achieve infection, which results in only limited viral replication as the mouse is at best only semi-permissive to RSV (Bonville et al., 2006a, Jafri, 2004). In addition, the disease profile of RSV infection in mice is inappropriate for pathogenesis studies, as no overt signs of disease develop. More importantly for vaccine development, the enhanced disease observed in human infants is not replicated in the mouse model (Connors et al., 1992b).