Wataru Akahata

A virus-like particle vaccine for epidemic Chikungunya virus protects nonhuman primates against infection

Chikungunya virus (CHIKV) has infected millions of people in Africa, Europe and Asia since this alphavirus reemerged from Kenya in 2004. The severity of the disease and the spread of this epidemic virus present a serious public health threat in the absence of vaccines or antiviral therapies. Here, we describe a new vaccine that protects against CHIKV infection of nonhuman primates. We show that selective expression of viral structural proteins gives rise to virus-like particles (VLPs) in vitro that resemble replication-competent alphaviruses. Immunization with these VLPs elicited neutralizing antibodies against envelope proteins from alternative CHIKV strains. Monkeys immunized with VLPs produced high-titer neutralizing antibodies that protected against viremia after high-dose challenge. We transferred these antibodies into immunodeficient mice, where they protected against subsequent lethal CHIKV challenge, indicating a humoral mechanism of protection. Immunization with alphavirus VLP vaccines represents a strategy to contain the spread of CHIKV and related pathogenic viruses in humans.

Development of a Highly Protective Combination Monoclonal Antibody Therapy against Chikungunya Virus

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes global epidemics of a debilitating polyarthritis in humans. As there is a pressing need for the development of therapeutic agents, we screened 230 new mouse anti-CHIKV monoclonal antibodies (MAbs) for their ability to inhibit infection of all three CHIKV genotypes. Four of 36 neutralizing MAbs (CHK-102, CHK-152, CHK-166, and CHK-263) provided complete protection against lethality as prophylaxis in highly susceptible immunocompromised mice lacking the type I IFN receptor (Ifnar−/−) and mapped to distinct epitopes on the E1 and E2 structural proteins. CHK-152, the most protective MAb, was humanized, shown to block viral fusion, and require Fc effector function for optimal activity in vivo. In post-exposure therapeutic trials, administration of a single dose of a combination of two neutralizing MAbs (CHK-102+CHK-152 or CHK-166+CHK-152) limited the development of resistance and protected immunocompromised mice against disease when given 24 to 36 hours before CHIKV-induced death. Selected pairs of highly neutralizing MAbs may be a promising treatment option for CHIKV in humans.

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

BACKGROUND 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. METHODS 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. FINDINGS 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. INTERPRETATION 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. FUNDING Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, and National Institutes of Health.

Protein Methyltransferase 2 Inhibits NF-κB Function and Promotes Apoptosis

ABSTRACT The protein arginine methyltransferases (PRMTs) include a family of proteins with related putative methyltransferase domains that modify chromatin and regulate cellular transcription. Although some family members, PRMT1 and PRMT4, have been implicated in transcriptional modulation or intracellular signaling, the roles of other PRMTs in diverse cellular processes have not been fully established. Here, we report that PRMT2 inhibits NF-κB-dependent transcription and promotes apoptosis. PRMT2 exerted this effect by blocking nuclear export of IκB-α through a leptomycin-sensitive pathway, increasing nuclear IκB-α and decreasing NF-κB DNA binding. The highly conserved S-adenosylmethionine-binding domain of PRMT2 mediated this effect. PRMT2 also rendered cells susceptible to apoptosis by cytokines or cytotoxic drugs, likely due to its effects on NF-κB. Mouse embryo fibroblasts from PRMT2 genetic knockouts showed elevated NF-κB activity and decreased susceptibility to apoptosis compared to wild-type or complemented cells. Taken together, these data suggest that PRMT2 inhibits cell activation and promotes programmed cell death through this NF-κB-dependent mechanism.

Structural analyses at pseudo atomic resolution of Chikungunya virus and antibodies show mechanisms of neutralization.

A 5.3 Å resolution, cryo-electron microscopy (cryoEM) map of Chikungunya virus-like particles (VLPs) has been interpreted using the previously published crystal structure of the Chikungunya E1-E2 glycoprotein heterodimer. The heterodimer structure was divided into domains to obtain a good fit to the cryoEM density. Differences in the T = 4 quasi-equivalent heterodimer components show their adaptation to different environments. The spikes on the icosahedral 3-fold axes and those in general positions are significantly different, possibly representing different phases during initial generation of fusogenic E1 trimers. CryoEM maps of neutralizing Fab fragments complexed with VLPs have been interpreted using the crystal structures of the Fab fragments and the VLP structure. Based on these analyses the CHK-152 antibody was shown to stabilize the viral surface, hindering the exposure of the fusion-loop, likely neutralizing infection by blocking fusion. The CHK-9, m10 and m242 antibodies surround the receptor-attachment site, probably inhibiting infection by blocking cell attachment. DOI: http://dx.doi.org/10.7554/eLife.00435.001

A Specific Domain of the Chikungunya Virus E2 Protein Regulates Particle Formation in Human Cells: Implications for Alphavirus Vaccine Design

ABSTRACT Virus-like particles (VLPs) can be generated from Chikungunya virus (CHIKV), but different strains yield variable quantities of particles. Here, we define the genetic basis for these differences and show that amino acid 234 in E2 substantially affects VLP production. This site is located within the acid-sensitive region (ASR) known to initiate a major conformational change in E1/E2. Selected other mutations in the ASR, or changes in pH, also increased VLP yield. These results demonstrate that the ASR of E2 plays an important role in regulating particle generation.

Different Vaccine Vectors Delivering the Same Antigen Elicit CD8+ T Cell Responses with Distinct Clonotype and Epitope Specificity

Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8+ cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8+ T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2Dd better than the native sequences, and clones with distinct specificities were elicited by alternative vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8+ T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.

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

Significance A recent outbreak of chikungunya virus in the Americas has caused more than one million infections in humans. The reemergence of this virus has become a major threat to public health due to a lack of available vaccines and antiviral drugs. We determined the cryo-EM structures of chikungunya virus particles complexed with two of the most potent human antibody fragments described in a previous study. Both antibodies neutralized the virus by stabilizing the position of the viral surface glycoproteins, which blocks the exposure of the glycoprotein fusion loops required to initiate viral entry into the cytoplasm of a target cell. 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.

Comparative Immunogenicity of Human Immunodeficiency Virus Particles and Corresponding Polypeptides in a DNA Vaccine

ABSTRACT The immunogenicity of a plasmid DNA expression vector encoding both Gag and envelope (Env), which produced human immunodeficiency virus (HIV) type 1 virus-like particles (VLP), was compared to vectors expressing Gag and Env individually, which presented the same gene products as polypeptides. Vaccination with plasmids that generated VLP showed cellular immunity comparable to that of Gag and cell-mediated or humoral responses similar to those of Env as immunization with separate vectors. These data suggest that DNA vaccines encoding separated HIV polypeptides generate immune responses similar to those generated by viral particles.

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

ABSTRACT 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. IMPORTANCE 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.