Early attempts at RV vaccinesTop

During the late 1960s and early 1970s clinical trials were performed to investigate a common cold vaccine, largely through administration of a formalin inactivated single RV serotype (RV13) [16, 17, 18, 19, 20, 21]. This approach was found to provide only minimal protective effects and was abandoned in favour of testing of inactivated multivalent vaccines spanning 10 serotypes [22]. Although these vaccines attempted to address the issue of weak cross-serotype protection induced by monovalent vaccination, they also lost popularity when surprisingly they failed to induce significant cross protection amongst RV serotypes. Table 1 summarises these studies that have been performed in humans using inactivated RV preparations as vaccines. We now suspect that inactivation of RV for vaccine studies is unfavourable for the generation of significant cell mediated immune responses and that the antibody responses alone that are often generated in such situations are insufficient for broad protection [23]. Formalin treatment was the most common method for RV inactivation [16, 17, 18, 19, 20, 21] although alternative methods such as heat treatment (pasteurisation), low pH and UV treatment are also effective [24, 25, 26]. These methods, whilst largely safe for human application, are likely to destroy many epitopes required for optimal immune responses and therefore can impact negatively on vaccine efficacy by reducing preparation immunogenicity [27]. In addition, another potential reason as to why these prior studies displayed limited success is that there was no evidence indicating that an adjuvant was used to amplify immune responses. The use of adjuvants would most likely have improved vaccine efficacy significantly [28] however, at that time, Alum was the only approved adjuvant for use in humans whilst there are now several others available [29].

Following the human trials, experimental studies in immunised animals (rabbits and mice) began to determine some properties of antibody cross reactivity [30, 31, 32] briefly encouraging renewed hope for a RV vaccine as cross-serotype neutralising antibodies were convincingly demonstrated (Table 2). Despite these positive steps, RV vaccine research studies in the scientific literature then virtually disappeared for over 20 years before further studies in immunised animals with recombinant RV capsid protein subunits and synthetic peptides again proposed possibilities for cross-serotype protective antibodies generation. Here, short conserved regions at the N-terminus of the capsid protein VP4 were identified that elicit cross-serotype protective antibodies [33] and others found that the entire VP1 polypeptide had similar effects [34]. Despite these encouraging studies and the application of modern molecular analyses, the formal demonstration of protective vaccine responses to RV’s in in vivo settings remained elusive largely because of the absence of a small animal in vivo model of RV infections.

Recent approaches using mouse models of human RV infection
The advent of a mouse model of human RV infection [26] has permitted new approaches for RV vaccine development where specific RV challenge following immunisation can be addressed. Previously, infection of mouse cells and indeed live mice with human RV’s was not thought possible due to significant sequence differences between the major group entry receptor human ICAM-1 and the mouse counterpart [35]. Furthermore, there was a lack of sustained intracellular viral replication in mouse cells despite minor group RV having the ability to enter via the mouse LDL receptor [36]. Mice transgenic for human ICAM-1, and improved methods for generating high titre RV inoculum, have now allowed the intranasal infection of mice with RV’s [26]. Here, RV was shown to replicate and cause acute lung inflammation as well as activating innate immune responses and initiating adaptive immune responses. Immunisation and challenge strategies have subsequently been investigated in this model system [7, 23], providing a basis for evaluating the immunological correlates of protection to RV’s in vivo. Hyper-immunisation of mice with inactivated RV1B, followed by homologous intranasal challenge, generated strong cross-serotype neutralising humoral immune responses which were directed at the capsid protein VP1 [23]. Although these antibody responses were neutralising in vitro, similar to prior experimentation in humans, very little protective effect was observed in vivo further confirming that the use of inactivated RV preparations as immunogens does not provide the appropriate immunological stimulation that can result in broad RV protection.

Thus an alternate approach was initiated that focussed on the induction of broadly reactive T cell immunity. Here, a conserved region (VP0) of the RV polyprotein amongst type A and type B strains was identified (Figure 1), the recombinant protein was produced in E. coli and used as an immunogen in mice [7]. In this study, recombinant VP0 derived from RV16 was immunogenic in vivo, inducing immunogen and RV-specific antibodies and cross-serotypic systemic cellular immune responses. Furthermore, the use of a Th1-promoting adjuvant in combination with VP0 induced cross-serotype cellular T lymphocytes producing the Th1 cytokine IFNγ and improved Th1-associated RV-specific antibody responses. It was also shown that immunised mice challenged with heterologous RV strains displayed enhanced cross-reactive cellular, increased memory CD4 T cell numbers and stronger humoral immune responses suggesting broad cross-serotype reactivity was obtained with this strategy. Most importantly, VP0 immunisation followed by live RV challenge improved the generation of neutralising antibodies to a variety of RV serotypes and also caused more rapid virus clearance in vivo. VP0 therefore represents a useful candidate for a subunit RV vaccine and may function by generating significant cross-reactive Th1 cells that upon heterologous RV challenge quickly stimulates additional protective immune responses [7]. Further experimentation in this model system of RV infection and translation to humans awaits.

Very recently it has been shown that cotton rats are permissive for RV16 infection and display characteristics similar to the mouse RV infection model [37]. Interestingly, in this model, prior immunisation with inactivated RV via the intramuscular route but not by the intranasal route produced significant neutralising antibody responses and reduced the viral load in the lungs upon homotypic challenge, confirming findings in the mouse model [7, 23]. In further experiments both prophylactic antibody administration and maternal immunity transfer to neonates were both protective although heterotypic responses were not evaluated [37]. The use of this model system in addition to the mouse model will complement human studies and hopefully aid in the identification and development of RV vaccines.

Public health challenges of RV vaccine delivery to humans
There is a large unmet medical need resulting from RV infections that would be corrected by a safe and effective vaccine. The major target population of an RV vaccine would be those suffering from chronic lung diseases such as asthma and chronic obstructive pulmonary disease where infections with RV are a major precipitant of life threatening acute exacerbations that can require hospitalisation [3]. What would be the risk that a RV vaccine might exacerbate airway inflammation in such chronic lung diseases Since it is known that RV infection in asthma induces Th2 responses that are linked to lower airway disease [38] it is of concern that a RV vaccine might exacerbate this response, particularly following natural RV exposure. Thus, the favoured RV vaccine approach should promote Th1 responses which is hypothesised to reset the unbalanced immune responses observed in asthmatics. Such an approach as outlined above has been demonstrated already in mice [7]. Whether the vaccine induced enhancement of Th1 cell responses to RV will prove a safe strategy for preventing RV-induced disease awaits confirmation in a clinical trial.

A secondary population that would benefit from a RV vaccine are healthy individuals. Here a broadly protective RV vaccine could reduce the burden of the common cold. Clearly any population receiving a RV vaccine would require initial safety and efficacy testing in healthy individuals with subsequent careful monitoring of airways inflammation following both natural and experimental RV exposure. This would be necessary to eliminate the possibility of an undesirable disease augmentation occurring following challenge as had occurred previously when testing a formalin inactivated RSV vaccine in infants during the 1960s [39].

ConclusionTop

Attempts to produce a protective vaccine to RV’s have failed due to the large number of antigenically distinct serotypes and the lack of a suitable small animal model of infection to test candidates in. The recent discovery of a previously unrecognised clade of RV’s has complicated this further. Nevertheless, studies in immunised animals have demonstrated that significant cross-serotype protection is possible. With the advent of small animal immunisation and challenge models, suitable vaccine candidates can now be evaluated thoroughly before translation to humans. The quest for a RV vaccine now seems somewhat less forlorn than it did a decade ago.

Acknowledgements
Supported in part by Serendipity Award SA29/0513 from the Dunhill Medical Trust.

Conflict of interest
The author is listed as an inventor on International Patent Application No. EP2014/052349 (Induction of Cross-Reactive Cellular Response Against Rhinovirus Antigens).

AbbreviationsTop

RV = rhinovirus, VP = virus protein, ICAM-1 = intercellular adhesion molecule-1, IFNγ = interferon gamma, Th1 = T cell helper type 1, Th2 = T cell helper type 2.