RSV-derived F and G proteins and their derivatives bearing neutralizing epitopes have undergone preclinical and clinical assessments as non-replicating protein subunit vaccines. The first of such preclinical studies utilized RSV F and G proteins that were purified from RSV-infected cell cultures and F and F/G chimeric proteins produced in baculovirus-infected insect cells or transfected mammalian cell lines39, 40
Rodents immunized with these preparations generated antibodies similar to those observed for FI-RSV in that despite recognition of RSV antigen in ELISAs, the resulting antibodies were poorly neutralizing41, 42
. In addition, rodents immunized with such RSV-encoded proteins and subsequently challenged 3–6 months later developed lung pathology similar to those observed for FI-RSV42
. To circumvent the potential deleterious consequences of subunit vaccination, several groups have utilized various adjuvants, including those specific for various Toll-like receptors (TLRs) (e.g. monophosphoryl lipid A (MPL) for TLR-4, CpG oligonucleotides for TLR-9) and have reported that in some cases, the use of adjuvants can significantly alter the immunogenicity and adverse effects of subunit vaccines43, 44
. Of relevance is the recent observation that TLR agonists may play a significant role in the affinity maturation of antibodies against RSV-encoded proteins45
. Thus, in principle, appropriate TLR stimulation during vaccination with RSV subunit preparations may generate high-affinity anti-RSV antibodies that recognize appropriate epitopes in non-denatured configurations.
With respect to clinical evaluation of RSV subunit vaccine candidates, several preparations of RSV-encoded proteins have been tested. Such preparations include: PFP (purified F protein) derivatives (PFP-1, -2, and -3); FG chimeric protein; a co-purified formulation of F, G, and M proteins, and a bacterially derived RSV G derivative (BBG2Na). Such efforts are briefly described below.
The PFP series have been purified from RSV-infected Vero cells and successive versions are comprised of higher purity of RSV F (i.e. 90–95% F for PFP-1 vs. > 98% for PFP-2 and -3)46, 47
. PFP-1 has been tested in RSV-seropositive children in two clinical trials involving 24–48 month old and 18–36 month old, RSV-seropositive children47, 48
. In these studies, no obvious adverse events were evident and ≥ 4-fold increase in RSV-neutralizing antibody titer was observed in a majority of subjects. A single priming intramuscular dose of PFP-2 (50 µg) was administered to 1–12 year old children with bronchopulmonary dysplasia (n = 10) or 1–8 year olds with cystic fibrosis (n = 17); in this study, the majority of participants exhibited ≥ 4 fold increase in RSV-neutralizing antibody titers49, 50
. PFP-3 has been tested as a single 30 µintramuscular vaccination dose in a phase II study among 143 1–12 year old children with cystic fibrosis51
. As in the case of PFP-2, PFP-3 also elicited a ≥ 4-fold increase in RSV neutralizing titers. However, the rate of RSV infection after vaccination was not significantly different between the vaccinated and control groups. In two studies involving elderly adults, a single 50 µg dose of PFP-2 was also immunogenic in 61% (out of 33 subjects > 60 years old) and 47% (of 36 frail elderly adults > 65 years old)52, 53
. Lastly, a single 20 µg dose of PFP-2 administered to 20 pregnant women was well tolerated54
. Although the vaccine led to a ≥4-fold increase in anti-F antibodies in the vast majority of vaccinees and their infants up to 6 months following birth, only 10% of the study subjects had a corresponding ≥ 4-fold increase in RSV neutralizing antibody titers.
BBG2Na, a bacterially derived protein bearing aa 130–230 of subgroup A RSV G protein fused to the albumin-binding domain of streptococcal protein G, has also undergone preclinical and clinical evaluation. In rodent models, BBG2Na conferred protection against RSV even though it generated modest levels of RSV-neutralizing antibodies55
. However, in limited primate studies, this antigen was associated with limited viral challenge protection and also with Th2-biased immune response56
. Among healthy young adults and elderly adults, this antigen was also well tolerated in phase II studies19
. However, in a phase III study involving elderly adults, anti-RSV serum antibody titers declined significantly within four weeks after vaccination and limited number of unanticipated adverse events have been reported.
With respect to other clinical trials with RSV subunit/protein-derived vaccine candidates, limited data are available. For FG chimeric protein testing, phase I and II studies involving its administration with various adjuvants have been started but not formally reported19
. In one study, a co-purified formulation of RSV F, G, and M proteins were emulsified in one of two adjuvants (aluminum hydroxide and poly(di[carboxylatophenoxy]phosphazene; PCPP) and administered to healthy adults (total n = 70)57
. In this study, both vaccine formulations induced similar levels of ≥ 4-fold increase in RSV neutralizing antibody titers and collectively in > 80% of the study population. In a large-scale phase II study in which 1169 elderly adults with cardiopulmonary conditions, the RSV F, G and M-containing vaccine was immunogenic; a greater proportion of subjects immunized with non-adjuvanted vaccine had ≥ 4-fold increase in RSV neutralizing antibody titers as compared to that in the group challenged with alum-containing vaccine candidate (168/383 (44%) vs. 129/400 (33%))58
Short peptides derived from RSV F and G proteins have been tested in animal models. In the case of short, RSV F-derived peptides, limited immunogenicity was seen59
. Recent data suggest that in mice, administration of bacterially derived short fragments of RSV F (including aa 255–278 and 412–524) fused to the ctxA(2)B cholera holotoxin led to anti-F, RSV-neutralizing antibody response and a modest protective efficacy in mouse RSV challenge studies60, 61
. For RSV G, the aa sequence (174–187) derived from the central core of RSV G has been administered to mice; in such experiments, protections against RSV infection was noted despite inefficient RSV neutralizing response62, 63
An alternative platform for RSV subunit vaccines is virus-like particles (VLPs), which are non-replicating, non-infectious particles derived from virus-encoded proteins64
. In the case of human papillomavirus (HPV) VLP-based licensed vaccine, the HPV L1 capsid protein self-assembles into particles that appear morphologically indistinguishable from native HPV virions65
. The resulting VLPs elicit strong humoral and cellular immune responses and provide durable protection against HPV. Other virus-encoded proteins, such as the influenza hemaglutinin and the hepatitis B surface antigen (HBSAg), can also form VLPs and in the latter case, have been engineered to contain an RSV-encoded CTL epitope66
. Most recently, nanoparticles derived from bacterially derived RSV nucleocapsid proteins have been shown to confer a moderate degree of protection against RSV challenge in rodent models67
. Because of their potent immunogenicity, VLPs may be an alternative platform for RSV subunit vaccine provided that appropriate epitopes can be successfully presented.