Chikungunya Virus-like Particles Research Articles

A chikungunya virus-like particle vaccine induces broadly neutralizing and protective antibodies against alphaviruses in humans.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes epidemics of acute and chronic musculoskeletal disease. Here, we analyzed the human B cell response to a CHIKV-like particle-adjuvanted vaccine (PXVX0317) from samples obtained from a phase 2 clinical trial in humans (NCT03483961). Immunization with PXVX0317 induced high levels of neutralizing antibody in serum against CHIKV and circulating antigen-specific B cells up to 6 months after immunization. Monoclonal antibodies (mAbs) generated from peripheral blood B cells of three PXVX0317-vaccinated individuals on day 57 after immunization potently neutralized CHIKV infection, and a subset of these inhibited multiple related arthritogenic alphaviruses. Epitope mapping and cryo-electron microscopy defined two broadly neutralizing mAbs that uniquely bind to the apex of the B domain of the E2 glycoprotein. These results demonstrate the inhibitory breadth and activity of the human B cell response induced by the PXVX0317 vaccine against CHIKV and potentially other related alphaviruses.

Surface decorated reporter-tagged chikungunya virus-like particles for clinical diagnostics and identification of virus entry inhibitors

The ever-evolving and versatile VLP technology is becoming an increasingly popular area of science. This study presents surface decorated reporter-tagged VLPs of CHIKV, an enveloped RNA virus of the genus alphavirus and its applications. Western blot, IFA and live-cell imaging confirm the expression of reporter-tagged CHIK-VLPs from transfected HEK293Ts. CryoEM micrographs reveal particle diameter as ∼67nm and 56–70 nm, respectively, for NLuc CHIK-VLPs and mCherry CHIK-VLPs. Our study demonstrates that by exploiting NLuc CHIK-VLPs as a detector probe, robust ratiometric luminescence signal in CHIKV-positive sera compared to healthy controls can be achieved swiftly. Moreover, the potential activity of the Suramin drug as a CHIKV entry inhibitor has been validated through the reporter-tagged CHIK-VLPs. The results reported in this study open new avenues in the eVLPs domain and offer potential for large-scale screening of clinical samples and antiviral agents targeting entry of CHIKV and other alphaviruses.

Antigenicity and immunogenicity of chikungunya virus-like particles from mosquito cells.

The spread of chikungunya virus (CHIKV) is reaching pandemic levels, and vaccines and antivirals to control CHIKV infection have yet to be approved. Virus-like particles (VLPs), a self-assembled native multi-subunit protein structure, could potentially be used as an antigen for serological detection and vaccine development. In the current study, we describe the production of novel CHIKV VLPs from mosquitoes using a Baculovirus/Mosquito (BacMos) system in a simple Biosafety Level-2 laboratory. Substantial envelope and capsid protein secretions were detected in culture medium. Co-fractionation of CHIKV E2, E1, and capsid proteins via sucrose gradient ultracentrifugation provided evidence of VLP formation. Transmission electron microscopy and dynamic light scattering analysis revealed the formation of VLPs in the form of spherical particles with a diameter of roughly 40nm in transduced cells and culture medium. VLP-based IgM capture ELISA in CHIKV patient sera revealed native epitopes on the VLPs. These non-purified VLPs were shown to act as an antigen in CHIKV-specific IgM capture ELISA. The immunization of CHIKV-VLPs alone in mice induced a balance CHIKV-specific IgG2a/IgG1 antibodies and neutralized antibody responses. The study provides support for the hypothesis that mosquito cell-derived CHIKV VLPs could serve as a novel antigen for serological detection and the development of vaccines against CHIKV infection. KEY POINTS: • CHIKV VLPs secreted from BacMos-CHIKV 26S-transduced mosquito cell. • This CHIKV VLPs potentially serve as an alternative capture antigen for MAC-ELISA. • Unadjuvanted CHIK VLPs induce CHIKV-specific IgG and NT responses in mice.

Safety and immunogenicity of PXVX0317, an aluminium hydroxide-adjuvanted chikungunya virus-like particle vaccine: a randomised, double-blind, parallel-group, phase 2 trial

Chikungunya virus (CHIKV) disease is an ongoing public health threat. We aimed to evaluate the safety and immunogenicity of PXVX0317, an aluminium hydroxide-adjuvanted formulation of a CHIKV virus-like particle (VLP) vaccine. This randomised, double-blind, parallel-group, phase 2 trial was conducted at three clinical trial centres in the USA. Eligible participants were healthy CHIKV-naïve adults aged 18-45 years. Participants were stratified by site and randomly assigned (1:1:1:1:1:1:1:1) to one of the eight vaccination groups using a block size of 16. Group 1 received two doses of unadjuvanted PXVX0317 28 days apart (2 × 20 μg; standard); all other groups received adjuvanted PXVX0317: groups 2-4 received two doses 28 days apart (2 × 6 μg [group 2], 2 × 10 μg [group 3], or 2 × 20 μg [group 4]; standard); group 4 also received a booster dose 18 months after the first active injection (40 μg; standard plus booster); groups 5-7 received two doses 14 days apart (2 × 6 μg [group 5], 2 × 10 μg [group 6], or 2 × 20 μg [group 7]; accelerated); and group 8 received one dose (1 × 40 μg; single). The primary endpoint was the geometric mean titre of anti-CHIKV neutralising antibody on day 57 (28 days after the last vaccination), assessed in the immunogenicity-evaluable population. Additionally, we assessed safety. This trial is registered at ClinicalTrials.gov, NCT03483961. This trial was conducted from April 18, 2018, to Sept 21, 2020; 468 participants were assessed for eligibility. Of these, 415 participants were randomly assigned to eight groups (n=53 in groups 1, 5, and 6; n=52 in groups 2 and 8; n=51 in groups 3 and 7; and n=50 in group 4) and 373 were evaluable for immunogenicity. On day 57, serum neutralising antibody geometric mean titres were 2057·0 (95% CI 1584·8-2670·0) in group 1, 1116·2 (852·5-1461·4; p=0·0015 vs group 1 used as a reference) in group 2, 1465·3 (1119·1-1918·4; p=0·076) in group 3, 2023·8 (1550·5-2641·7; p=0·93) in group 4, 920·1 (710·9-1190·9; p<0·0001) in group 5, 1206·9 (932·4-1562·2; p=0·0045) in group 6, 1562·8 (1204·1-2028·3; p=0·14) in group 7, and 1712·5 (1330·0-2205·0; p=0·32) in group 8. In group 4, a booster dose increased serum neutralising antibody geometric mean titres from 215·7 (95% CI 160·9-289·1) on day 547 to 10 941·1 (7378·0-16 225·1) on day 575. Durability of the immune response (evaluated in groups 1, 4, and 8) was shown up to 2 years. The most common solicited adverse event was pain at the injection site, reported in 12 (23%) of 53 participants who received the unadjuvanted vaccine (group 1) and 111 (31%) of 356 who received the adjuvanted vaccine. No vaccine-related serious adverse events were reported. PXVX0317 was well tolerated and induced a robust and durable serum neutralising antibody immune response against CHIKV up to 2 years. A single 40 μg injection of adjuvanted PXVX0317 is being further investigated in phase 3 clinical trials (NCT05072080 and NCT05349617). Emergent BioSolutions.

A virus like particle approach to study the Chikungunya virus envelope protein mutations.

Virus like particles (VLPs) are used as a tool to study the mutations in the structural genes that influence the virus assembly and entry process. We observed that Chikungunya VLP with the E1:V291I mutation produced more fluorescence-positive cells in Vero cells than the other mutant VLPs (E1:A226V, D284E, and E2:V264A) and wild-type VLP tested in this study. According to the findings, the V291I mutation may aid the virus's ability to enter the cells more efficiently than wild-type VLPs. The study concludes that VLP is a useful model for studying the virus entry process in cells.

Design of Alphavirus Virus-Like Particles Presenting Circumsporozoite Junctional Epitopes That Elicit Protection against Malaria.

The most advanced malaria vaccine, RTS,S, includes the central repeat and C-terminal domains of the Plasmodium falciparum circumsporozoite protein (PfCSP). We have recently isolated human antibodies that target the junctional region between the N-terminal and repeat domains that are not included in RTS,S. Due to the fact that these antibodies protect against malaria challenge in mice, their epitopes could be effective vaccine targets. Here, we developed immunogens displaying PfCSP junctional epitopes by genetic fusion to either the N-terminus or B domain loop of the E2 protein from chikungunya (CHIK) alphavirus and produced CHIK virus-like particles (CHIK-VLPs). The structural integrity of these junctional-epitope–CHIK-VLP immunogens was confirmed by negative-stain electron microscopy. Immunization of these CHIK-VLP immunogens reduced parasite liver load by up to 95% in a mouse model of malaria infection and elicited better protection than when displayed on keyhole limpet hemocyanin, a commonly used immunogenic carrier. Protection correlated with PfCSP serum titer. Of note, different junctional sequences elicited qualitatively different reactivities to overlapping PfCSP peptides. Overall, these results show that the junctional epitopes of PfCSP can induce protective responses when displayed on CHIK-VLP immunogens and provide a basis for the development of a next generation malaria vaccine to expand the breadth of anti-PfCSP immunity.

Relocation of the attTn7 Transgene Insertion Site in Bacmid DNA Enhances Baculovirus Genome Stability and Recombinant Protein Expression in Insect Cells.

Baculovirus expression vectors are successfully used for the commercial production of complex (glyco)proteins in eukaryotic cells. The genome engineering of single-copy baculovirus infectious clones (bacmids) in E. coli has been valuable in the study of baculovirus biology, but bacmids are not yet widely applied as expression vectors. An important limitation of first-generation bacmids for large-scale protein production is the rapid loss of gene of interest (GOI) expression. The instability is caused by the mini-F replicon in the bacmid backbone, which is non-essential for baculovirus replication in insect cells, and carries the adjacent GOI in between attTn7 transposition sites. In this paper, we test the hypothesis that relocation of the attTn7 transgene insertion site away from the mini-F replicon prevents deletion of the GOI, thereby resulting in higher and prolonged recombinant protein expression levels. We applied lambda red genome engineering combined with SacB counterselection to generate a series of bacmids with relocated attTn7 sites and tested their performance by comparing the relative expression levels of different GOIs. We conclude that GOI expression from the odv-e56 (pif-5) locus results in higher overall expression levels and is more stable over serial passages compared to the original bacmid. Finally, we evaluated this improved next-generation bacmid during a bioreactor scale-up of Sf9 insect cells in suspension to produce enveloped chikungunya virus-like particles as a model vaccine.

The transient expression of CHIKV VLP in large stirred tank bioreactors.

Transient gene expression (TGE) bioprocesses have been difficult to scale up in large stirred tank bioreactors with volumes of more than 1.5L. Low production levels are often observed, but the causes have not been investigated (Gutierrez-Granados et al. in Crit Rev Biotechnol 38:918-940, 2018). Chikungunya Virus-like particle (VLP), expressed by DNA-PEI transient transfection, is a representative case study for these difficulties. Clinical materials were produced in shake flasks, but the process suffered when transferred to large stirred tank bioreactors. The resulting process was not operationally friendly nor cost effective. In this study, a systematic approach was used to investigate the root causes of the poor scale up performance. The transfection conditions were first screened in ambr® 15 high throughput mini bioreactors then examined in 3L stirred-tank systems. The studies found that production level was negatively correlated with inoculum cell growth status (P < 0.05). The pH range, DNA and PEI levels, order of the reagent addition, and gas-sparging systems were also studied and found to affect process performance. Further hydromechanical characterizations (Re, energy dissipation rates, and P/V, etc.) of shake flasks, ambr® 15, and 3-L stirred tank systems were performed. Overall, the study discovered that the shear stress (caused by a microsparger) and PEI toxicity together were the root causes of scale-up failure. Once the microsparger was replaced by a macrosparger, the scale-up was successful.

A Phase 2 Open-label Study to Assess the Safety and Immunogenicity of an Alum-adjuvanted Chikungunya Virus-like Particle Vaccine (PXVX0317) in Prior Recipients of Other Alphavirus Vaccines

A Phase 2 Open-label Study to Assess the Safety and Immunogenicity of an Alum-adjuvanted Chikungunya Virus-like Particle Vaccine (PXVX0317) in Prior Recipients of Other Alphavirus Vaccines

Cryo-EM Structure of Chikungunya Virus in Complex with the Mxra8 Receptor

Mxra8 is a receptor for multiple arthritogenic alphaviruses that cause debilitating acute and chronic musculoskeletal disease in humans. Herein, we present a 2.2Å resolution X-ray crystal structure of Mxra8 and 4 to 5Å resolution cryo-electron microscopy reconstructions of Mxra8 bound to chikungunya (CHIKV) virus-like particles and infectious virus. The Mxra8 ectodomain contains two strand-swapped Ig-like domains oriented in a unique disulfide-linked head-to-head arrangement. Mxra8 binds by wedging into a cleft created by two adjacent CHIKV E2-E1 heterodimers in one trimeric spike and engaging a neighboring spike. Two binding modes are observed with the fully mature VLP, with one Mxra8 binding with unique contacts. Only the high-affinity binding mode was observed in the complex with infectious CHIKV, as viral maturation and E3 occupancy appear to influence receptor binding-site usage. Our studies provide insight into how Mxra8 binds CHIKV and creates a path for developing alphavirus entry inhibitors.

Vaccination with a chikungunya virus-like particle vaccine exacerbates disease in aged mice.

IntroductionChikungunya virus (CHIKV) is a re-emerging pathogen responsible for causing outbreaks of febrile disease accompanied with debilitating joint pain. Symptoms typically persist for two weeks, but more severe and chronic chikungunya illnesses have been reported, especially in the elderly. Currently, there are no licensed vaccines or antivirals against CHIKV available. In this study, we combined a CHIK virus-like particle (VLP) vaccine with different adjuvants to enhance immunogenicity and protection in both, adult and aged mice.MethodsCHIK VLP-based vaccines were tested in 6-8-week-old (adult) and 18-24-month-old (aged) female C57BL/6J mice. Formulations contained CHIK VLP alone or adjuvants: QuilA, R848, or Imject Alum. Mice were vaccinated three times via intramuscular injections. CHIKV-specific antibody responses were characterized by IgG subclass using ELISA, and by microneutralization assays. In addition, CHIKV infections were characterized in vaccinated and non-vaccinated adult mice and compared to aged mice.ResultsIn adult mice, CHIKV infection of the right hind foot induced significant swelling, which peaked by day 7 post-infection at approximately 170% of initial size. Viral titers peaked at 2.53 × 1010 CCID50/ml on day 2 post-infection. Mice vaccinated with CHIK VLP-based vaccines developed robust anti-CHIKV-specific IgG antibody responses that were capable of neutralizing CHIKV in vitro. CHIK VLP alone or CHIK plus QuilA administered by IM injections protected 100% of mice against CHIKV. In contrast, the antibody responses elicited by the VLP-based vaccines were attenuated in aged mice, with negligible neutralizing antibody titers detected. Unvaccinated, aged mice were resistant to CHIKV infection, while vaccination with CHIKV VLPs exacerbated disease.ConclusionsUnadjuvanted CHIK VLP vaccination elicits immune responses that protect 100% of adult mice against CHIKV infection. However, an improved vaccine/adjuvant combination is still necessary to enhance the protective immunity against CHIKV in the aged.

Assessment of Immunogenicity and Neutralisation Efficacy of Viral-Vectored Vaccines Against Chikungunya Virus.

Chikungunya virus (CHIKV) has caused extensive outbreaks in several countries within the Americas, Asia, Oceanic/Pacific Islands, and Europe. In humans, CHIKV infections cause a debilitating disease with acute febrile illness and long-term polyarthralgia. Acute and chronic symptoms impose a major economic burden to health systems and contribute to poverty in affected countries. An efficacious vaccine would be an important step towards decreasing the disease burden caused by CHIKV infection. Despite no licensed vaccine is yet available for CHIKV, there is strong evidence of effective asymptomatic viral clearance due to neutralising antibodies against the viral structural proteins. We have designed viral-vectored vaccines to express the structural proteins of CHIKV, using the replication-deficient chimpanzee adenoviral platform, ChAdOx1. Expression of the CHIKV antigens results in the formation of chikungunya virus-like particles. Our vaccines induce high frequencies of anti-chikungunya specific T-cell responses as well as high titres of anti-CHIKV E2 antibodies with high capacity for in vitro neutralisation. Our results indicate the potential for further clinical development of the ChAdOx1 vaccine platform in CHIKV vaccinology.

Comparative study of chikungunya Virus-Like Particles and Pseudotyped-Particles used for serological detection of specific immunoglobulin M

The incidence of chikungunya virus (CHIKV) infection has increased dramatically in recent decades. Effective diagnostic methods must be available to optimize patient management. IgM-capture Enzyme-Linked Immunosorbent Assay (MAC-ELISA) is routinely used for the detection of specific CHIKV IgM. This method requires inactivated CHIKV viral lysate (VL). The use of viral bioparticles such as Virus-Like Particles (VLPs) and Pseudotyped-Particles (PPs) could represent an alternative to VL.Bioparticles performances were established by MAC-ELISA; physico-chemical characterizations were performed by field-flow fractionation (HF5) and confirmed by electron microscopy.Non-purified PPs give a detection signal higher than for VL. Results suggested that the signal difference observed in MAC-ELISA was probably due to the intrinsic antigenic properties of particles.The use of CHIKV bioparticles such as VLPs and PPs represents an attractive alternative to VL. Compared to VL and VLPs, non-purified PPs have proven to be more powerful antigens for specific IgM capture.

Expression and Characterization of Yeast Derived Chikungunya Virus Like Particles (CHIK-VLPs) and Its Evaluation as a Potential Vaccine Candidate.

Chikungunya virus (CHIKV) has emerged as a global health concern due to its recent spread in both old and new world. So far, no CHIKV specific drug or vaccine is licensed for human use. In this study, we report production of Chikungunya virus like particles (CHIK-VLPs) using novel yeast expression system (Pichia pastoris) and its evaluation as vaccine candidate. The gene encoding structural polyprotein of CHIKV from a recent epidemic strain was cloned into yeast expression system. The multicopy integrants were processed for expression of CHIK-VLPs. The VLPs were purified and confirmed through electron microscopic analysis for their morphological identity with CHIKV. The in vitro and in vivo evaluation of CHIK-VLPs as vaccine candidate was determined in Balb/c mice. Induction of both humoral and cellular immune response was observed with different doses of CHIK-VLPs. The humoral immune response was studied through different techniques like enzyme linked immunosorbent assay, IgG Isotyping and plaque reduction neutralization test. CHIK-VLPs were found to elicit high titer of antibodies that are able to recognize native CHIKV. Higher level of IgG2a and IgG1 subtypes was identified suggestive of balanced Th1/Th2 response. Both in vitro and in vivo neutralization activity of CHIK-VLPs antibodies was observed even with low concentration, which shows its high specificity and neutralizing activity against two different CHIKV strains. Neonatal mice receiving anti-CHIK-VLPs antibodies were protected from CHIKV challenge. Induction of cellular immune response was confirmed through higher level of TNF-α, IL-10 and substantial level of IL-2, IL-4 and IFN-γ indicating a balanced response. This is the first report, where CHIK-VLPs has been expressed by Pichia pastoris and evaluated for neutralizing activity against CHIKV. These promising results indicate the utility of CHIK-VLPs as a promising vaccine candidate against emerging CHIKV.

Characterization of N-glycosylation profiles from mammalian and insect cell derived chikungunya VLP.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes severe arthralgia. The envelope of CHIKV is composed of 240 copies of two glycoproteins: E1 and E2. In this work, we have characterized the N-glycosylation patterns of CHIKV virus-like particles (VLPs), containing both E1 and E2 proteins, derived from mammalian and insect cells using hydrophilic interaction liquid chromatography (HILIC) with fluorescence (FL) and mass spectrometry (MS) detection. While HEK293 derived CHIKV VLPs contain oligomannose, hybrid and complex glycans, VLPs derived from SfBasic predominantly contain oligomannose glycans. This strong host dependence of N-glycosylation pattern resembles other alphaviruses such as SINV. The VLPs from HEK293 and SfBasic, with significantly different N-glycosylation profiles, are valuable reagents enabling future in-depth correlation studies between immunogenicity and glycosylation. In addition, the characterization tools presented here allow one to monitor glycosylation during vaccine process development and ensure process consistency.

Neutralizing Monoclonal Antibodies Block Chikungunya Virus Entry and Release by Targeting an Epitope Critical to Viral Pathogenesis.

We evaluated the mechanism by which neutralizing human monoclonal antibodies inhibit chikungunya virus (CHIKV) infection. Potently neutralizing antibodies (NAbs) blocked infection at multiple steps of the virus life cycle, including entry and release. Cryo-electron microscopy structures of Fab fragments of two human NAbs and chikungunya virus-like particles showed a binding footprint that spanned independent domains on neighboring E2 subunits within one viral spike, suggesting a mechanism for inhibiting low-pH-dependent membrane fusion. Detailed epitope mapping identified amino acid E2-W64 as a critical interaction residue. An escape mutation (E2-W64G) at this residue rendered CHIKV attenuated in mice. Consistent with these data, CHIKV-E2-W64G failed to emerge in vivo under the selection pressure of one of the NAbs, IM-CKV063. As our study suggests that antibodies engaging the residue E2-W64 can potently inhibit CHIKV at multiple stages of infection, antibody-based therapies or immunogens that target this region might have protective value.

Structural Studies of Chikungunya Virus-Like Particles Complexed with Human Antibodies: Neutralization and Cell-to-Cell Transmission.

Chikungunya virus is a positive-stranded RNA alphavirus. Structures of chikungunya virus-like particles in complex with strongly neutralizing antibody Fab fragments (8B10 and 5F10) were determined using cryo-electron microscopy and X-ray crystallography. By fitting the crystallographically determined structures of these Fab fragments into the cryo-electron density maps, we show that Fab fragments of antibody 8B10 extend radially from the viral surface and block receptor binding on the E2 glycoprotein. In contrast, Fab fragments of antibody 5F10 bind the tip of the E2 B domain and lie tangentially on the viral surface. Fab 5F10 fixes the B domain rigidly to the surface of the virus, blocking exposure of the fusion loop on glycoprotein E1 and therefore preventing the virus from becoming fusogenic. Although Fab 5F10 can neutralize the wild-type virus, it can also bind to a mutant virus without inhibiting fusion or attachment. Although the mutant virus is no longer able to propagate by extracellular budding, it can, however, enter the next cell by traveling through junctional complexes without being intercepted by a neutralizing antibody to the wild-type virus, thus clarifying how cell-to-cell transmission can occur. Alphaviral infections are transmitted mainly by mosquitoes. Chikungunya virus (CHIKV), which belongs to the Alphavirus genus, has a wide distribution in the Old World that has expanded in recent years into the Americas. There are currently no vaccines or drugs against alphaviral infections. Therefore, a better understanding of CHIKV and its associated neutralizing antibodies will aid in the development of effective treatments.

Cryo-EM structures elucidate neutralizing mechanisms of anti-chikungunya human monoclonal antibodies with therapeutic activity

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes severe acute and chronic disease in humans. Although highly inhibitory murine and human monoclonal antibodies (mAbs) have been generated, the structural basis of their neutralizing activity remains poorly characterized. Here, we determined the cryo-EM structures of chikungunya virus-like particles complexed with antibody fragments (Fab) of two highly protective human mAbs, 4J21 and 5M16, that block virus fusion with host membranes. Both mAbs bind primarily to sites within the A and B domains, as well as to the B domain's β-ribbon connector of the viral glycoprotein E2. The footprints of these antibodies on the viral surface were consistent with results from loss-of-binding studies using an alanine scanning mutagenesis-based epitope mapping approach. The Fab fragments stabilized the position of the B domain relative to the virus, particularly for the complex with 5M16. This finding is consistent with a mechanism of neutralization in which anti-CHIKV mAbs that bridge the A and B domains impede movement of the B domain away from the underlying fusion loop on the E1 glycoprotein and therefore block the requisite pH-dependent fusion of viral and host membranes.

Safety and tolerability of chikungunya virus-like particle vaccine in healthy adults: a phase 1 dose-escalation trial

Chikungunya virus--a mosquito-borne alphavirus--is endemic in Africa and south and southeast Asia and has recently emerged in the Caribbean. No drugs or vaccines are available for treatment or prevention. We aimed to assess the safety, tolerability, and immunogenicity of a new candidate vaccine. VRC 311 was a phase 1, dose-escalation, open-label clinical trial of a virus-like particle (VLP) chikungunya virus vaccine, VRC-CHKVLP059-00-VP, in healthy adults aged 18-50 years who were enrolled at the National Institutes of Health Clinical Center (Bethesda, MD, USA). Participants were assigned to sequential dose level groups to receive vaccinations at 10 μg, 20 μg, or 40 μg on weeks 0, 4, and 20, with follow-up for 44 weeks after enrolment. The primary endpoints were safety and tolerability of the vaccine. Secondary endpoints were chikungunya virus-specific immune responses assessed by ELISA and neutralising antibody assays. This trial is registered with ClinicalTrials.gov, NCT01489358. 25 participants were enrolled from Dec 12, 2011, to March 22, 2012, into the three dosage groups: 10 μg (n=5), 20 μg (n=10), and 40 μg (n=10). The protocol was completed by all five participants at the 10 μg dose, all ten participants at the 20 μg dose, and eight of ten participants at the 40 μg dose; non-completions were for personal circumstances unrelated to adverse events. 73 vaccinations were administered. All injections were well tolerated, with no serious adverse events reported. Neutralising antibodies were detected in all dose groups after the second vaccination (geometric mean titres of the half maximum inhibitory concentration: 2688 in the 10 μg group, 1775 in the 20 μg group, and 7246 in the 40 μg group), and a significant boost occurred after the third vaccination in all dose groups (10 μg group p=0·0197, 20 μg group p<0·0001, and 40 μg group p<0·0001). 4 weeks after the third vaccination, the geometric mean titres of the half maximum inhibitory concentration were 8745 for the 10 μg group, 4525 for the 20 μg group, and 5390 for the 40 μg group. The chikungunya VLP vaccine was immunogenic, safe, and well tolerated. This study represents an important step in vaccine development to combat this rapidly emerging pathogen. Further studies should be done in a larger number of participants and in more diverse populations. Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, and National Institutes of Health.

Development and application of a reversed-phase high-performance liquid chromatographic method for quantitation and characterization of a Chikungunya virus-like particle vaccine

To effectively support the development of a Chikungunya (CHIKV) virus-like particle (VLP) vaccine, a sensitive and robust high-performance liquid chromatography (HPLC) method that can quantitate CHIKV VLPs and monitor product purity throughout the manufacturing process is needed. We developed a sensitive reversed-phase HPLC (RP-HPLC) method that separates capsid, E1, and E2 proteins in CHIKV VLP vaccine with good resolution. Each protein component was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS). The post-translational modifications on the viral glycoproteins E1 and E2 were further identified by intact protein mass measurements with liquid chromatography–mass spectrometry (LC–MS). The RP-HPLC method has a linear range of 0.51–12μg protein, an accuracy of 96–106% and a precision of 12% RSD, suitable for vaccine product release testing. In addition, we demonstrated that the RP-HPLC method is useful for characterizing viral glycoprotein post-translational modifications, monitoring product purity during process development and assessing product stability during formulation development.