Developing a Vaccine for Human Rhinoviruses
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Rhinoviruses (RV’s) are extremely common human pathogens of the respiratory tract being the most frequent cause of mild diseases of the upper respiratory tract (common cold) but more importantly they are the major initiator of acute exacerbations of chronic airway diseases. Infections can be life threatening in the latter context however RV-induced common colds have an associated economic cost from loss of productivity due to absence from work or school. There are no appropriate antiviral therapies available and vaccine strategies have failed because of the large number of viral serotypes and the lack of cross-serotype protection generated. Here, approaches past and present for development of a vaccine to these widespread human pathogens are highlighted.
RMcLean G (2014) Developing a Vaccine for Human Rhinoviruses. Ann Vaccines Immunization 1(1): 1004.
Rhinoviruses (RVs) are a species of human pathogens belonging to the genus enterovirus of the picornaviridae family of viruses . RVs are very small viruses about 30nm in diameter that contain a positive sense ssRNA genome of approximately 7500bp that is surrounded by a protein capsid which is composed of 60 copies of 4 protein subunits that assemble an icosahedron. These structural proteins consist of the externally facing VP1, VP2 and VP3 and internal VP4 which lies at the interface of the capsid and RNA genome . RVs infect and cause disease in all humans but most significantly in those individuals with underlying lung diseases such as asthma or chronic obstructive pulmonary disease, where they are the major precipitants of acute exacerbations . The success of RV’s as human pathogens is due not only to their speed of infection and onward transmission but also to their ability to adapt and change, resulting in the existence of numerous antigenically distinct serotypes. The original definition and numbering of serotypes from 1 to 100 was based on antibody neutralisation properties with polyclonal antisera where little or no cross-serotype neutralization is observed . Antibodies are directed against the outer surface of the RV capsid most commonly to exposed areas of VP1, VP2 and VP3 [5,6]. Regions of the capsid sequences display a high degree of heterogeneity amongst serotypes where there are areas with less than 70% homology within the RV polyproteins (Figure 1) . These result in variable surface-exposed immunodominant epitopes that can dictate serotype-specific immune responses. Based on RNA genome sequence analyses, RV’s have now been divided into three groups known as RV types A, B and C and may be further classified by entry receptor usage. Approximately 90% of characterized RV strains (major group) use ICAM-1 as receptors to enter host epithelial cells whereas the minor group exploits members of the Low-Density Lipoprotein (LDL) receptor family. The entry receptor for group C RV’s has yet to be identified due to propagation difficultiesin vitro and it is therefore difficult to pinpoint the exact number of RV serotypes but there may be upwards of 160. The fact that adults experience on average of 2-5 infections and children up to 12 infections per year, when coupled with the lack of cross protective immunity between serotypes ensures that humans can expect a lifetime of RV infections. A broadly cross protective vaccine could alleviate many of these infections and the associated health and economic issues, particularly in those with underlying chronic airways diseases.
Early attempts at RV vaccines
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)[12-17]. This approach was found to provide only minimal protective effects and was abandoned in favour of testing of inactivated multivalent vaccines spanning 10 serotypes . 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 know that it is highly likely that inactivation of RV for vaccine studies is unfavourable for the generation of significant cell mediated immune responses and that antibody responses alone that are often generated in such situations are insufficient for broad protection. Formalin treatment was the most common method for RV inactivation although alternative methods such as heat treatment (pasteurisation), low pH and UV treatment are also effective. 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. 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 however, at that time, Alum was the only approved adjuvant for use in humans whilst there are now several others available.
Experimental studies in immunised animals (rabbits and mice) began to determine some properties of antibody cross reactivity [20-22] briefly encouraging renewed hope for an 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  and others found that the entire VP1 polypeptide had similar effects . 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 an in vivo model for RV infections.
Recent approaches using mouse models of human RV infection
The advent of a small animal model of human RV infection  has permitted new approaches for RV vaccine development where specific virus 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 within the major group entry receptor human ICAM-1and the lack of sustained intracellular viral replication despite minor group RV having the ability to enter via the mouse LDL receptor. 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 . 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, 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 . Although these antibody responses were neutralising in vitro, similar to prior experimentation in humans, very little protective effect was observed in vivofurther 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 a different 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 . Recombinant VP0 derived from RV16 was immunogenic in vivo, inducing immunogen and RV-specific antibodies and crossserotypic systemic cellular immune responses. The use of a Th1-promoting adjuvant in combination with VP0 induced crossserotype cellular T lymphocytes producing the Th1 cytokine IFN and improved Th1-associated RV-specific antibody responses. 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 clearancein 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. 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 . 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 [7,28]. In further experiments both prophylactic antibody administration and maternal immunity transfer to neonates were both protective although heterotypic responses were not evaluated. 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.
Table 1: Summary of early clinical studies investigating efficacy of RV vaccines
|Formalin inactivated RV3, RV7, RV10, RV13, RV14, RV18, RV22, RV42, RV43, RV55 (decavalent)||intramuscular||Minimal homologous and hererologous neutralizing antibody responses||Hamory |
|Formalin inactivated RV13||subcutaneous||Homotypic neutralising antibody generated and reduced viral shedding upon homotypic challenge||Douglas |
|Formalin inactivated RV13||intranasal||Resistance to homotypic challenge||Buscho |
|Formalin inactivated RV13||intranasal||Resistance to homotypic challenge||Perkins |
|Formalin inactivated RV13||Intranasal & intramuscular||Protection to homotypic challenge by intranasal immunization & correlated with level of nasal neutralisaing antibody||Perkins |
|Formalin inactivated single strain||intranasal||Protection to homotypic challenge by intranasal immunization & correlated with level of nasal neutralisaing antibody||Mitchison |
|Live & formalin inactivated single strain||Intranasal & intramuscular||Increased homotypic antibody responses with live intranasal and inactive intramuscular||Doggett |
Table 2: Summary of animal studies investigating RV antibodies after vaccination.
|Immunogen & Animal Model||Administration Route||Findings||Reference|
|Inactivated RV16 Cotton rat||intramuscular||Generation of cross-serotype neutralizing antibody responses following intranasal challenge||Blanco |
|Recombinant VP0 of RV16 Mouse||subcutaneous||Generation of cross-serotype neutralizing antibody responses following intranasal challenge||Blanco |
|Inactivated RV1B Mouse||subcutaneous||Generation of cross-serotype neutralizing antibody responses following intranasal challenge||McLean |
|Recombinant VP1 of RV89 & RV14 Rabbit & mouse||subcutaneous||Generation of cross-serotype neutralizing antibody responses||Edlmayr |
|VP4 peptides of RV14 Rabbit||subcutaneous||Generation of cross-serotype neutralizing antibody responses||Katpally |
|VP1 & VP3 peptides of RV14 Rabbit||subcutaneous||Generation of cross-serotype neutralizing antibody responses||McCray |
|Numerous RV serotypes individually Rabbit||intravenous||Extensive cross-serotype binding antibody responses||Cooney |
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 crossserotype 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 (Table 2). The quest for a RV vaccine now seems somewhat less forlorn than it did a decade ago.