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► This Phase 1 trial evaluated the safety and immunogenicity of an RSV (F) nanoparticle vaccine. ► The vaccine was well-tolerated and no related SAEs were observed. ► Both RSV A and B microneutralizating antibodies were increased in vaccinees versus placebo. ► There was a 7- to 19-fold increase in anti-F IgG and a 7- to 24-fold increase in palivizumab-like antibodies. ► The levels of immunity are similar to those seen in other settings with decreased hospitalizations. We performed a Phase 1 randomized, observer-blinded, placebo-controlled trial to evaluate the safety and immunogenicity of a recombinant respiratory syncytial virus (RSV) fusion (F) protein nanoparticle vaccine. Six formulations with (5, 15, 30 and 60μg) and without (30 and 60μg) aluminum phosphate (AdjuPhos) were administered intramuscularly on day 0 and 30 in a dose escalating fashion to healthy adults 18–49 years of age. Solicited and unsolicited events were collected through day 210. Immunogenicity measures taken at day 0, 30 and 60 included RSV A and B microneutralization, anti-F IgG, antigenic site II peptide and palivizumab competitive antibodies. The vaccine was well-tolerated, with no evident dose-related toxicity or attributable SAEs. At day 60 both RSV A and B microneutralization was significantly increased in vaccinees versus placebo. Across all vaccinees there was a 7- to 19-fold increase in the anti-F IgG and a 7- to 24-fold increase in the antigenic site II binding and palivizumab competitive antibodies. The RSV F nanoparticle vaccine candidate was well tolerated without dose-related increases in adverse events. Measures of immunity indicate that neutralization, anti-RSV F IgG titers and palivizumab competing antibodies were induced at levels that have been associated with decreased risk of hospitalization. NCT01290419.

► A norovirus GII.4 Consensus virus-like particle was engineered for development as a vaccine candidate. ► Intramuscular immunization elicited strong antibody responses in rabbits. ► Antibodies developed against the Consensus GII.4 VLPs cross-reacted with a panel of VLPs representing different GII.4 variants. ► A bivalent formulation of GI.1 and GII.4 VLPs was shown to effectively induce heterotypic responses. Noroviruses, a major cause of acute gastroenteritis worldwide, present antigenic diversity that must be considered for the development of an effective vaccine. In this study, we explored approaches to increase the broad reactivity of virus-like particle (VLP) norovirus vaccine candidates. The immunogenicity of a GII.4 “Consensus” VLP that was engineered from sequences of three genetically distinct naturally occurring GII.4 strains was examined for its ability to induce cross-reactive immune responses against different clusters of GII.4 noroviruses. Rabbits immunized with GII.4 Consensus VLPs developed high serum antibody titers against VLPs derived from a number of distinct wild-type GII.4 viruses, including some that had been circulating over 30 years ago. Because the sera exhibited low cross-reactivity with antigenically distinct GI norovirus strains, we investigated the serum antibody response to a bivalent vaccine formulation containing GI.1 (Norwalk virus) and GII.4 Consensus VLPs that was administered to animals under varying conditions. In these studies, the highest homologous and heterologous antibody titers to the bivalent vaccine were elicited following immunization of animals by the intramuscular route using Alhydrogel (Al(OH)3) as adjuvant. Our data indicate that the use of both genetically engineered norovirus VLPs that incorporate relevant epitopes from multiple strains and multivalent vaccine formulations increase the breadth of the immune response to diverse variants within a genotype and, thus, prove helpful in the rational design of VLP-based vaccines against human noroviruses.

► A rapid method for VLP vaccine preparation comprising bacterial expression and in vitro self assembly is reported. ► Chimeric VLPs and capsomeres displaying antigen from Streptococcus and influenza are evaluated. ► VLPs induce high antibody response with no significant improvement due to adjuvant. ► Capsomeres are immunogenic and response is improved with adjuvant. Studies on a platform technology able to deliver low-cost viral capsomeres and virus-like particles are described. The technology involves expression of the VP1 structural protein from murine polyomavirus (MuPyV) in Escherichia coli, followed by purification using scaleable units and optional cell-free VLP assembly. Two insertion sites on the surface of MuPyV VP1 are exploited for the presentation of the M2e antigen from influenza and the J8 peptide from Group A Streptococcus (GAS). Results from testing on mice following subcutaneous administration demonstrate that VLPs are self adjuvating, that adding adjuvant to VLPs provides no significant benefit in terms of antibody titre, and that adjuvanted capsomeres induce an antibody titre comparable to VLPs but superior to unadjuvanted capsomere formulations. Antibodies raised against GAS J8 peptide following immunization with chimeric J8-VP1 VLPs are bactericidal against a GAS reference strain. E. coli is easily and widely cultivated, and well understood, and delivers unparalleled volumetric productivity in industrial bioreactors. Indeed, recent results demonstrate that MuPyV VP1 can be produced in bioreactors at multi-gram-per-litre levels. The platform technology described here therefore has the potential to deliver safe and efficacious vaccine, quickly and cost effectively, at distributed manufacturing sites including those in less developed countries. Additionally, the unique advantages of VLPs including their stability on freeze drying, and the potential for intradermal and intranasal administration, suggest this technology may be suited to numerous diseases where adequate response requires large-scale and low-cost vaccine manufacture, in a way that is rapidly adaptable to temporal or geographical variation in pathogen molecular composition.

Virus-like particles (VLPs) derived from enteric pathogens like Norwalk virus (NV) are well suited to study oral immunization. We previously described stable transgenic plants that accumulate recombinant NV-like particles (rNVs) that were orally immunogenic in mice and humans. The transgenic approach suffers from long generation time and modest level of antigen accumulation. We now overcome these constraints with an efficient tobacco mosaic virus (TMV)-derived transient expression system using leaves of Nicotiana benthamiana. We produced properly assembled rNV at 0.8 mg/g leaf 12 days post-infection (dpi). Oral immunization of CD1 mice with 100 or 250 μg/dose of partially purified rNV elicited systemic and mucosal immune responses. We conclude that the plant viral transient expression system provides a robust research tool to generate abundant quantities of rNV as enriched, concentrated VLP preparations that are orally immunogenic.

SARS-CoV was the cause of the global pandemic in 2003 that infected over 8000 people in 8 months. Vaccines against SARS are still not available. We developed a novel method to produce high levels of a recombinant SARS virus-like particles (VLPs) vaccine containing the SARS spike (S) protein and the influenza M1 protein using the baculovirus insect cell expression system. These chimeric SARS VLPs have a similar size and morphology to the wild type SARS-CoV. We tested the immunogenicity and protective efficacy of purified chimeric SARS VLPs and full length SARS S protein vaccines in a mouse lethal challenge model. The SARS VLP vaccine, containing 0.8μg of SARS S protein, completely protected mice from death when administered intramuscular (IM) or intranasal (IN) routes in the absence of an adjuvant. Likewise, the SARS VLP vaccine, containing 4μg of S protein without adjuvant, reduced lung virus titer to below detectable level, protected mice from weight loss, and elicited a high level of neutralizing antibodies against SARS-CoV. Sf9 cell-produced full length purified SARS S protein was also an effective vaccine against SARS-CoV but only when co-administered IM with aluminum hydroxide. SARS-CoV VLPs are highly immunogenic and induce neutralizing antibodies and provide protection against lethal challenge. Sf9 cell-based VLP vaccines are a potential tool to provide protection against novel pandemic agents.

An optimized VP2 gene from the current prevalent CPV strain (new CPV-2a) in China was expressed in a baculovirus expression system. It was found that the VP2 proteins assembled into virus-like particles (VLPs) with antigenic properties similar to those of natural CPV and with an especially high hemagglutination (HA) titer (1:2²⁰). Dogs intramuscularly or orally immunized with VLPs produced antibodies against CPV with >1:80 hemagglutination inhibition (HI) units for at least 3 months. The CPV VLPs could be considered for use as a vaccine against CPV or as a platform for research on chimeric VLP vaccines against other diseases.

► A P particle-M2e chimeric vaccine was generated by insertion of human influenza A M2e epitope into norovirus P particle. ► Mice developed significantly increased IgG against M2e after immunization with this chimeric vaccine and 100% survived from a lethal challenge with influenza virus. ► Antibodies induced by the chimeric vaccine blocked norovirus VLP and P particle binding to HBGA receptors, suggesting an opportunity to develop a dual vaccine against both influenza and noroviruses. The extracellular domain of the matrix protein 2 (M2e) of influenza viruses is highly conserved among all influenza A subtypes, making it a suitable target for a universal influenza vaccine. In this study, we demonstrated an enhanced immune response and protection of a chimeric M2e vaccine against influenza A viruses using our newly developed vaccine platform, the norovirus P particle, to present the M2e peptide. The 23-amino acid peptide was inserted into one of the surface loops of the P protein, resulting in 24 copies of M2e presented on each P particle. Significantly (P<0.001) increased antibody responses to M2e were observed in mice immunized with the P particle-M2e chimera compared with those immunized with the free M2e peptides. Mice immunized with the P particle-M2e vaccine were fully protected (100% survived) against lethal challenge of a mouse adapted human influenza virus PR8 (H1N1), while only low survival rates (<12.5%) were found in mice immunized with the free M2e peptides or wild type P particle. In addition, the mouse sera collected after immunization with the P particle-M2e vaccine were able to block the binding of norovirus virus-like particle and P particle to histo-blood group antigen receptors. These results suggest that the P particle-M2e chimera can be used as dual vaccine against both noroviruses and influenza viruses.

Enterovirus 71 (EV71) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes outbreaks with significant mortality among young children. To develop the vaccine, we have produced and purified the EV71 virus-like particle (VLP) that resembles the authentic virus in appearance, capsid structure and protein composition. In this study, we further evaluated the potential of VLP as a vaccine by comparing the humoral and cellular immune responses elicited by the purified VLP, denatured VLP and heat-inactivated EV71 virus. After immunization of BALB/c mice, EV71 VLP induced potent and long-lasting humoral immune responses as evidenced by the high total IgG titer and neutralization titer. The splenocytes collected from the VLP-immunized mice exhibited significant cell proliferation and produced high levels of IFN-γ, IL-2 and IL-4 after stimulation, indicating the induction of Th1 and Th2 immune responses by VLP immunization. More importantly, the VLP immunization of mother mice conferred protection (survival rate up to 89%) to neonatal mice against the lethal (1000 LD 50) viral challenge. Compared with the VLP immunization, immunization with denatured VLP and heat-inactivated EV71 elicited lower neutralization titers and conferred less effective protection to newborn mice, although they induced comparable levels of total IgG and cellular immune responses. These data collectively indicate the importance of the preservation of VLP structure and implicate the potential of VLP as a vaccine to prevent EV71 infection.

Noroviruses (NoV) cause the great majority of epidemic nonbacterial gastroenteritis in humans. Expression of the capsid protein in recombinant systems, including insect and plant cells, yields assembly of virus-like particles (VLPs) that mimic the antigenic structure of authentic virions, and are relatively acid- and heat-stable. Norwalk virus (NV), the prototype NoV, has been studied extensively, and Norwalk virus-like particles (NVLPs) produced in insect cells and plants are immunogenic in mice and humans when delivered orally, stimulating the production of systemic and mucosal anti-NV antibodies. NVLPs are also highly immunogenic when delivered intranasally, provoking antibodies at levels similar to orally delivered VLP at much lower doses. Oral and nasal delivery of NVLPs efficiently produces antibodies at distal mucosal sites, which suggests that NVLPs could be used to deliver heterologous peptide antigens by production of genetic fusion chimeric capsid proteins. Examination of norovirus VLP surface structures and receptor binding motifs facilitates identification of potential sites for insertion of foreign peptides that will minimally affect the efficiency of VLP assembly and receptor binding. Thus, there is strong potential to use norovirus VLPs as vaccine-delivery vehicles.

► Comparison of BTV VLPs (VP2, VP3, VP5 and VP7) and CLPs (VP3 and VP7) in sheep. ► Investigated the importance of geographical lineages in vaccine development. ► VLPs prevent disease and viraemia, while CLPs only mitigated severity. ► Outer capsid (VP2 and VP5) was essential for complete protection. ► Lineage was not critical for development of a serotype specific vaccine. There have been multiple separate outbreaks of Bluetongue (BT) disease of ruminants in Europe since 1998, often entering via the Mediterranean countries of Italy, Spain and Greece. BT is caused by an orbivirus, Bluetongue virus (BTV), a member of the family Reoviridae. BTV is a non-enveloped double-capsid virus, which encodes 7 structural proteins (VP1-VP7) and several non-structural proteins (NS1, NS2, NS3/3a and NS4) from ten double-stranded RNA segments of the genome. In this report, we have prepared BTV virus-like particles (VLPs, composed of VP2, VP3, VP5 and VP7) and sub-viral, inner core-like particles (CLPs, VP3 and VP7) using a recombinant baculovirus expression system. We compared the protective efficacy of VLPs and CLPs in sheep and investigated the importance of geographical lineages of BTV in the development of vaccines. The Greek crossbred Karagouniko sheep, which display mild to sub-clinical BT, were vaccinated with VLPs or CLPs of BTV-1, derived from western lineage and were challenged with virulent BTV-1 from an eastern lineage. All VLP-vaccinated animals developed a neutralising antibody response to BTV-1 from both lineages prior to challenge. Moreover, post-challenged animals had no clinical manifestation or viraemia and the challenged virus replication was completely inhibited. In contrast, CLP-vaccinated animals did not induce any neutralising antibody response but developed the group specific VP7 antibodies. CLPs also failed to prevent the clinical manifestation and virus replication, but in comparison to controls, the severity of disease manifestation and viraemia was mitigated. The data demonstrated that the outer capsid was essential for complete protection, while the geographical origin of the BTV was not critical for development of a serotype specific vaccine.