Immunogenicity of Stabilized HIV-1 Envelope Trimers with Reduced Exposure of Non-neutralizing Epitopes.

The envelope glycoprotein trimer mediates HIV-1 entry into cells. The trimer is flexible, fluctuating between closed and more open conformations and sometimes sampling the fully open, CD4-bound form. We hypothesized that conformational flexibility and transient exposure of non-neutralizing, immunodominant epitopes could hinder the induction of broadly neutralizing antibodies (bNAbs). We therefore modified soluble Env trimers to stabilize their closed, ground states. The trimer variants were indeed stabilized in the closed conformation, with a reduced ability to undergo receptor-induced conformational changes and a decreased exposure of non-neutralizing V3-directed antibody epitopes. In rabbits, the stabilized trimers induced similar autologous Tier-1B or Tier-2 NAb titers to those elicited by the corresponding wild-type trimers but lower levels of V3-directed Tier-1A NAbs. Stabilized, closed trimers might therefore be useful components of vaccines aimed at inducing bNAbs.

Antibodies Produced in Response to a Live-Attenuated Dengue Vaccine Are Functional in Activating the Complement System.

BACKGROUND: Antibody-driven complement system (CS) activation has been associated with protection against symptomatic dengue virus (DENV) infection. Aggregation, opsonization, lysis, and phagocytosis are mechanisms triggered by antibody-antigen immunocomplexes following fixation of the component 1q (C1q) and activation of the classical pathway. As a result, DENV neutralization and clearance are facilitated, whereas antibody-dependent enhancement of infection is inhibited. We investigated the ability of antibodies produced in response to Takeda's dengue vaccine candidate, TAK-003, to fix C1q and activate CS. METHODS: Serum samples were collected from seronegative and seropositive participants in a phase 2 clinical trial (DEN-203), pre- and postvaccination. Samples were evaluated for the presence of complement-fixing antibodies (CFAs) against DENV using a Luminex multiplex-based immunoassay. RESULTS: TAK-003 elicited production of CFAs against all 4 DENV serotypes, which persisted for 1 year postvaccination, irrespective of baseline serostatus. CFA levels were correlated with neutralizing antibody titers and virus-binding total IgG and IgG1 concentrations. Furthermore, efficiency of CFA fixation was greater in samples with higher polyclonal IgG avidity. CONCLUSIONS: These results indicate that antibodies produced after TAK-003 vaccination are functional in both activating CS and neutralizing virus infection by all DENV serotypes, which may contribute to efficacy of TAK-003. CLINICAL TRIALS REGISTRATION: NCT01511250.

Somatic Hypermutation and Framework Mutations of Variable Region Contribute to Anti-Zika Virus-Specific Monoclonal Antibody Binding and Function.

Zika virus (ZIKV) is a global public health concern due to its ability to cause congenital Zika syndrome and lack of approved vaccine, therapeutic, or other control measures. We discovered eight novel rabbit monoclonal antibodies (MAbs) that bind to distinct ZIKV envelope protein epitopes. The majority of the MAbs were ZIKV specific and targeted the lateral ridge of the envelope (E) protein domain III, while the MAb with the highest neutralizing activity recognized a putative quaternary epitope spanning E protein domains I and III. One of the non-neutralizing MAbs specifically recognized ZIKV precursor membrane protein (prM). Somatic hypermutation of immunoglobulin variable regions increases antibody affinity maturation and triggers antibody class switching. Negative correlations were observed between the somatic hypermutation rate of the immunoglobulin heavy-chain variable region and antibody binding parameters such as equilibrium dissociation constant, dissociation constant, and half-maximal effective concentration value of MAb binding to ZIKV virus-like particles. Complementarity-determining regions recognize the antigen epitopes and are scaffolded by canonical framework regions. Reversion of framework region amino acids to the rabbit germ line sequence decreased anti-ZIKV MAb binding activity of some MAbs. Thus, antibody affinity maturation, including somatic hypermutation and framework region mutations, contributed to the binding and function of these anti-ZIKV MAbs. IMPORTANCE ZIKV is a global health concern against which no vaccine or therapeutics are available. We characterized eight novel rabbit monoclonal antibodies recognizing ZIKV envelope and prM proteins and studied the relationship between somatic hypermutation of complementarity-determining regions, framework regions, mutations, antibody specificity, binding, and neutralizing activity. The results contribute to understanding structural features and somatic mutation pathways by which potent Zika virus-neutralizing antibodies can evolve, including the role of antibody framework regions.

Development of a Novel Assay to Assess the Avidity of Dengue Virus-Specific Antibodies Elicited in Response to a Tetravalent Dengue Vaccine.

Antibody affinity maturation is a critical step in development of functional antiviral immunity; however, accurate measurement of affinity maturation of polyclonal serum antibody responses to particulate antigens such as virions is challenging. We describe a novel avidity assay employing biolayer interferometry and dengue virus-like particles. After validation using anti-dengue monoclonal antibodies, the assay was used to assess avidity of antibody responses to a tetravalent dengue vaccine candidate (TAK-003) in children, adolescents, and adults during two phase 2 clinical trials conducted in dengue-endemic regions. Vaccination increased avidity index and avidity remained high through 1 year postvaccination. Neutralizing antibody titers and avidity index did not correlate overall; however, a correlation was observed between neutralizing antibody titer and avidity index in those subjects with the highest degree of antibody affinity maturation. Therefore, vaccination with TAK-003 stimulates polyclonal affinity maturation and functional antibody responses, including neutralizing antibodies. CLINICAL TRIALS REGISTRATION: NCT01511250 and NCT02302066.

Specificity and Breadth of the Neutralizing Antibody Response to a Live-Attenuated Tetravalent Dengue Vaccine.

BACKGROUND: An effective dengue vaccine should ideally induce broadly neutralizing antibody (nAb) responses against all 4 dengue virus (DENV) serotypes. METHODS: We characterized the specificity and breadth of the nAb response to TAK-003, a live-attenuated tetravalent dengue vaccine, in serum samples from phase 2 and 3 clinical trials. RESULTS: Microneutralization tests using postvaccination serum showed comparable neutralization against diverse DENV-1-4 genotypes. Reporter virus particle neutralization assays after depletion of anti-DENV-2 nAbs demonstrated that the nAb response to DENV-1, -3, and -4 comprises both type-specific (TS) and cross-reactive (CR) nAbs. CONCLUSIONS: Therefore, TAK-003 induces broad tetravalent TS and CR nAb responses.

Characterization of the Type-Specific and Cross-Reactive B-Cell Responses Elicited by a Live-Attenuated Tetravalent Dengue Vaccine.

BACKGROUND: Dengue is caused by 4 antigenically distinct serotypes of dengue virus (DENV1-4). Takeda's live attenuated tetravalent dengue vaccine (TAK-003) candidate is composed of an attenuated DENV2 and chimeric viruses containing prM/E of DENV1, 3 and 4 on the DENV2 backbone. The multicolor FluoroSpot (MCF) assay enables quantitation of serotype-specific and cross-reactive individual memory B cells (MBCs) secreting DENV-specific antibodies in a polyclonal mixture. METHODS: Using the MCF assay, we determined the type-specific and cross-reactive MBC response in peripheral blood mononuclear cells collected pre- and postvaccination from 7 macaques and 15 randomly selected individuals who received TAK-003 (8 DENV seronegative and 7 DENV seropositive) in a phase 2 clinical trial in Singapore (DEN-205 study). RESULTS: Preexisting DENV-specific MBC responses were detected only in seropositive vaccine recipients at day 0. Following vaccination, both type-specific and cross-reactive MBCs to all 4 DENV serotypes were observed in all macaques and clinical trial participants. The proportion of type-specific MBCs was higher than cross-reactive MBCs and remained stable between day 30 and 360 post vaccination. CONCLUSIONS: These results demonstrate that, unlike primary or secondary natural DENV infection, tetravalent vaccination elicits tetravalent type-specific MBCs, and thus all 4 components of TAK-003 contribute to the DENV-specific MBC response following vaccination. CLINICAL TRIALS REGISTRATION: NCT02425098.

Primary infection with dengue or Zika virus does not affect the severity of heterologous secondary infection in macaques.

Zika virus (ZIKV) and dengue virus (DENV) are genetically and antigenically related flaviviruses that now co-circulate in much of the tropical and subtropical world. The rapid emergence of ZIKV in the Americas in 2015 and 2016, and its recent associations with Guillain-Barré syndrome, birth defects, and fetal loss have led to the hypothesis that DENV infection induces cross-reactive antibodies that influence the severity of secondary ZIKV infections. It has also been proposed that pre-existing ZIKV immunity could affect DENV pathogenesis. We examined outcomes of secondary ZIKV infections in three rhesus and fifteen cynomolgus macaques, as well as secondary DENV-2 infections in three additional rhesus macaques up to a year post-primary ZIKV infection. Although cross-binding antibodies were detected prior to secondary infection for all animals and cross-neutralizing antibodies were detected for some animals, previous DENV or ZIKV infection had no apparent effect on the clinical course of heterotypic secondary infections in these animals. All animals had asymptomatic infections and, when compared to controls, did not have significantly perturbed hematological parameters. Rhesus macaques infected with DENV-2 approximately one year after primary ZIKV infection had higher vRNA loads in plasma when compared with serum vRNA loads from ZIKV-naive animals infected with DENV-2, but a differential effect of sample type could not be ruled out. In cynomolgus macaques, the serotype of primary DENV infection did not affect the outcome of secondary ZIKV infection.

Development and Characterization of a Multiplex Assay to Quantify Complement-Fixing Antibodies against Dengue Virus.

Antibodies capable of activating the complement system (CS) when bound with antigen are referred to as "complement-fixing antibodies" and are involved in protection against Flaviviruses. A complement-fixing antibody test has been used in the past to measure the ability of dengue virus (DENV)-specific serum antibodies to activate the CS. As originally developed, the test is time-consuming, cumbersome, and has limited sensitivity for DENV diagnosis. Here, we developed and characterized a novel multiplex anti-DENV complement-fixing assay based on the Luminex platform to quantitate serum antibodies against all four serotypes (DENV1-4) that activate the CS based on their ability to fix the complement component 1q (C1q). The assay demonstrated good reproducibility and showed equivalent performance to a DENV microneutralization assay that has been used to determine DENV serostatus. In non-human primates, antibodies produced in response to primary DENV1-4 infection induced C1q fixation on homologous and heterologous serotypes. Inter-serotype cross-reactivity was associated with homology of the envelope protein. Interestingly, the antibodies produced following vaccination against Zika virus fixed C1q on DENV. The anti-DENV complement fixing antibody assay represents an alternative approach to determine the quality of functional antibodies produced following DENV natural infection or vaccination and a biomarker for dengue serostatus, while providing insights about immunological cross-reactivity among different Flaviviruses.

Magnitude and Functionality of the NS1-Specific Antibody Response Elicited by a Live-Attenuated Tetravalent Dengue Vaccine Candidate.

BACKGROUND: Dengue virus (DENV) can cause life-threatening disease characterized by endothelial dysfunction and vascular leakage. DENV nonstructural protein 1 (NS1) induces human endothelial hyperpermeability and vascular leak in mice, and NS1 vaccination confers antibody-mediated protective immunity. We evaluated the magnitude, cross-reactivity, and functionality of NS1-specific IgG antibody responses in sera from a phase 2 clinical trial of Takeda's live-attenuated tetravalent dengue vaccine candidate (TAK-003). METHODS: We developed an enzyme-linked immunosorbent assay to measure anti-DENV NS1 IgG in sera from DENV-naive or preimmune subjects pre- and postvaccination with TAK-003 and evaluated the functionality of this response using in vitro models of endothelial permeability. RESULTS: TAK-003 significantly increased DENV-2 NS1-specific IgG in naive individuals, which cross-reacted with DENV-1, -3, and -4 NS1 to varying extents. NS1-induced endothelial hyperpermeability was unaffected by prevaccination serum from naive subjects but was variably inhibited by serum from preimmune subjects. After TAK-003 vaccination, all samples from naive and preimmune vaccinees completely abrogated DENV-2 NS1-induced hyperpermeability and cross-inhibited hyperpermeability induced by DENV-1, -3, and -4 NS1. Inhibition of NS1-induced hyperpermeability correlated with NS1-specific IgG concentrations. Postvaccination sera also prevented NS1-induced degradation of endothelial glycocalyx components. CONCLUSION: We provide evidence for functional NS1-specific IgG responses elicited by a candidate dengue vaccine. CLINICAL TRIALS REGISTRATION: NCT01511250.

Analyzing the Human Serum Antibody Responses to a Live Attenuated Tetravalent Dengue Vaccine Candidate.

Background: Dengue virus serotypes 1-4 (DENV-1-4) are the most common vector-borne viral pathogens of humans and the etiological agents of dengue fever and dengue hemorrhagic syndrome. A live-attenuated tetravalent dengue vaccine (TDV) developed by Takeda Vaccines has recently progressed to phase 3 safety and efficacy evaluation. Methods: We analyzed the qualitative features of the neutralizing antibody (nAb) response induced in naive and DENV-immune individuals after TDV administration. Using DENV-specific human monoclonal antibodies (mAbs) and recombinant DENV displaying different serotype-specific Ab epitopes, we mapped the specificity of TDV-induced nAbs against DENV-1-3. Results: Nearly all subjects had high levels of DENV-2-specific nAbs directed to epitopes centered on domain III of the envelope protein. In some individuals, the vaccine induced nAbs that tracked with a DENV-1-specific neutralizing epitope centered on domain I of the envelope protein. The vaccine induced binding Abs directed to a DENV-3 type-specific neutralizing epitope, but findings of mapping of DENV-3 type-specific nAbs were inconclusive. Conclusion: Here we provide qualitative measures of the magnitude and epitope specificity of the nAb responses to TDV. This information will be useful for understanding the performance of TDV in clinical trials and for identifying correlates of protective immunity.

Identification and quantitation of multiple variants in RNA virus genomes.

The goal of the study was to identify and characterize RNA virus variants containing mutations spread over genomic distances >5 kb. As proof of concept, high-quality viral RNA of the Dengue 2 component of Takeda's tetravalent dengue vaccine candidate (TDV-2) was used to develop a reverse transcription-polymerase chain reaction protocol to amplify a ∼5.3 kb cDNA segment that contains the three genetic determinants of TDV-2 attenuation. Unique molecular identifiers were incorporated into each viral cDNA molecule for PacBio library preparation to improve the quantitative precision of the observed variants at the attenuation loci. Following assay optimization, PacBio long-read sequencing was validated with multiple clone-derived TDV-2 revertant variants and four complex revertant mixtures containing various compositions of TDV-2 and revertant viruses. PacBio sequencing analysis correctly identified and quantified variant composition in all tested samples, demonstrating that TDV-2 revertants could be identified and characterized and supporting the use of this method in the differentiation and quantification of complex variants of other RNA viruses. Long-read sequencing can identify complex RNA virus variants containing multiple mutations on a single-genome molecule, which is useful for in-depth genetic stability and revertant detection of live-attenuated viral vaccines, as well as research in virus evolution to reveal mechanisms of immune evasion and host cell adaption.

Impact of prior flavivirus vaccination on immunogenicity and efficacy of an inactivated Zika vaccine in Indian rhesus macaques.

Flaviviruses are antigenically related. We evaluated the immunogenicity and efficacy of Takeda's purified inactivated Zika vaccine (PIZV) candidate in macaques previously vaccinated with several commercially available heterologous flavivirus vaccines. Heterologous flavivirus vaccination did not elicit Zika virus (ZIKV) neutralizing antibodies and did not impact neutralizing antibody titers after one dose of PIZV. After a second PIZV dose previous vaccination with flavivirus vaccines had variable impact on ZIKV neutralizing antibody titers. However, all macaques were protected against viremia after Zika virus challenge 8-12 months post-PIZV vaccination. Therefore, vaccine-induced immunity against heterologous flavivirus vaccines does not impact PIZV efficacy in macaques.

Correction: HIV-1-neutralizing antibody induced by simian adenovirus- and poxvirus MVA-vectored BG505 native-like envelope trimers.

[This corrects the article DOI: 10.1371/journal.pone.0181886.].

Enhanced control of pathogenic Simian immunodeficiency virus SIVmac239 replication in macaques immunized with an interleukin-12 plasmid and a DNA prime-viral vector boost vaccine regimen.

DNA priming has previously been shown to elicit augmented immune responses when administered by electroporation (EP) or codelivered with a plasmid encoding interleukin-12 (pIL-12). We hypothesized that the efficacy of a DNA prime and recombinant adenovirus 5 boost vaccination regimen (DNA/rAd5) would be improved when incorporating these vaccination strategies into the DNA priming phase, as determined by pathogenic simian immunodeficiency virus SIVmac239 challenge outcome. The whole SIVmac239 proteome was delivered in 5 separate DNA plasmids (pDNA-SIV) by EP with or without pIL-12, followed by boosting 4 months later with corresponding rAd5-SIV vaccine vectors. Remarkably, after repeated low-dose SIVmac239 mucosal challenge, we demonstrate 2.6 and 4.4 log reductions of the median SIV peak and set point viral loads in rhesus macaques (RMs) that received pDNA-SIV by EP with pIL-12 compared to the median peak and set point viral loads in mock-immunized controls (P < 0.01). In 5 out of 6 infected RMs, strong suppression of viremia was observed, with intermittent "blips" in virus replication. In 2 RMs, we could not detect the presence of SIV RNA in tissue and lymph nodes, even after 13 viral challenges. RMs immunized without pIL-12 demonstrated a typical maximum of 1.5 log reduction in virus load. There was no significant difference in the overall magnitude of SIV-specific antibodies or CD8 T-cell responses between groups; however, pDNA delivery by EP with pIL-12 induced a greater magnitude of SIV-specific CD4 T cells that produced multiple cytokines. This vaccine strategy is relevant for existing vaccine candidates entering clinical evaluation, and this model may provide insights into control of retrovirus replication.

Characterization of the cell-mediated immune response to Takeda's live-attenuated tetravalent dengue vaccine in adolescents participating in a phase 2 randomized controlled trial conducted in a dengue-endemic setting.

BACKGROUND: As robust dengue-specific CD4+ and CD8+ T cell responses are essential for protective immunity, we assessed cell-mediated immune (CMI) responses to a DENV-2-based dengue tetravalent vaccine candidate (TAK-003) in adolescents living in Panama, a dengue-endemic country. METHODS: Peripheral blood mononuclear cells were collected from a subset of 67 participants ≥ 10 years old included in a phase 2 clinical trial of TAK-003 (Clinicaltrials.gov: NCT02302066). Following stimulation with dengue peptides, the frequency, magnitude, and cross-reactivity of the CD8+ and CD4+ T cell IFN-γ, TNF-α and IL-2 responses were assessed by flow cytometry. RESULTS: Intracellular cytokine staining identified NS1, NS3, and NS5 as the most common non-structural (NS) targets of the CD4+ T-cell response (IFN-γ+); NS3 and NS5 were the main NS targets of the CD8+ T cell response (IFN-γ+). Both CD4+ and CD8+ T-cell responses were multi-functional (IFN-γ + TNF-α + IL-2+) and cross-reactive against DENV-1, -3, and -4 serotypes. Similar responses were seen in all CMI assessments irrespective of participant baseline status for dengue neutralizing antibodies and T cells. CONCLUSIONS: TAK-003 elicited cross-reactive, multi-functional CD4+ and CD8+ T-cell responses, irrespective of dengue pre-exposure.

Neutralization fingerprinting technology for characterizing polyclonal antibody responses to dengue vaccines.

Dengue is a major public health threat. There are four dengue virus (DENV) serotypes; therefore, efforts are focused on developing safe and effective tetravalent DENV vaccines. While neutralizing antibodies contribute to protective immunity, there are still important gaps in understanding of immune responses elicited by dengue infection and vaccination. To that end, here, we develop a computational modeling framework based on the concept of antibody-virus neutralization fingerprints in order to characterize samples from clinical studies of TAK-003, a tetravalent vaccine candidate currently in phase 3 trials. Our results suggest a similarity of neutralizing antibody specificities in baseline-seronegative individuals. In contrast, amplification of pre-existing neutralizing antibody specificities is predicted for baseline-seropositive individuals, thus quantifying the role of immunologic imprinting in driving antibody responses to DENV vaccines. The neutralization fingerprinting analysis framework presented here can contribute to understanding dengue immune correlates of protection and help guide further vaccine development and optimization.

TAK - 021, an inactivated Enterovirus 71 vaccine candidate, provides cross-protection against heterologous sub-genogroups in human scavenger receptor B2 transgenic mice.

BACKGROUND: Enterovirus 71 (EV71) is a major cause of outbreaks of hand, foot and mouth disease, most frequently in children, and is a public health concern in the Asia-Pacific region. Takeda is developing TAK-021, an inactivated EV71 vaccine candidate based on sub-genogroup B2 strain MS87. In a phase I clinical trial, TAK-021 was safe, well tolerated, and immunogenic in healthy adults and elicited cross-neutralizing antibodies against heterologous EV71 sub-genogroup viruses. TAK-021 confers protection from lethal challenge with a mouse-adapted homologous strain in AG129 mice. However, it has not been determined whether TAK-021 can provide cross-protection against heterologous EV71 sub-genogroups. METHODS: We examined the efficacy of TAK-021 against challenge with EV71 sub-genogroups B4, B5, C1, C2, and C4 on day 42 (short-term) and sub-genogroups B5 and C4 on day 120 (long-term) after immunization of human scavenger receptor B2 transgenic (hSCARB2-tg) mice with TAK-021 on days 0 and 28. Antibody titers were monitored over 120 days using plaque reduction neutralization test of the homologous vaccine virus. RESULTS: TAK-021 elicited neutralizing antibody (nAb) in greater than 90% of the mice and nAb persisted through day 120. Challenge of control animals led to weight loss and death, as well as virus detection in various organs and histopathological lesions in the brain. All mice that received two doses of TAK-021 developed nAb and survived a short-term challenge given on day 42, while more than 80% survived a long-term challenge given on day 120. EV71 was detected less frequently and at lower levels in organs of immunized mice compared to non-immunized control mice. CONCLUSIONS: The results show that TAK-021 can confer protection in mice against the EV71 sub-genogroups tested.

Transcriptome-wide changes in gene expression, splicing, and lncRNAs in response to a live attenuated dengue virus vaccine.

The tetravalent dengue vaccine candidate, TAK-003, induces a functional antibody response, but the titers of antibodies against the four serotypes of the dengue virus (DENV) can vary. Here, through a transcriptomic analysis on whole blood collected from recipients of a two-dose schedule of TAK-003, we examine gene expression, splicing, and transcript isoform-level changes for both protein-coding and noncoding genes to broaden our understanding of the immune response. Our analysis reveals a dynamic pattern of vaccine-associated regulation of long noncoding RNAs (lncRNAs), differential splicing of interferon-stimulated gene exons, and gene expression changes related to multiple signaling pathways that detect viral infection. Co-expression networks isolate immune cell-type-related and interferon-response modules that represent specific biological processes that correlate with more robust antibody responses. These data provide insights into the early determinants of the variable immune response to the vaccine, highlighting the significance of splicing and isoform-level gene regulatory mechanisms in defining vaccine immunogenicity.

A high throughput reporter virus particle microneutralization assay for quantitation of Zika virus neutralizing antibodies in multiple species.

Zika virus is a Flavivirus, transmitted via Aedes mosquitos, that causes a range of symptoms including Zika congenital syndrome. Zika has posed a challenging situation for health, public and economic sectors of affected countries. To quantitate Zika virus neutralizing antibody titers in serum samples, we developed a high throughput plate based Zika virus reporter virus particle (RVP) assay that uses an infective, non-replicating particle encoding Zika virus surface proteins and capsid (CprME) and a reporter gene (Renilla luciferase). This is the first characterization of a Zika virus RVP assay in 384-well format using a Dengue replicon Renilla reporter construct. Serially diluted test sera were incubated with RVPs, followed by incubation with Vero cells. RVPs that have not been neutralized by antibodies in the test sera entered the cells and expressed Renilla luciferase. Quantitative measurements of neutralizing activity were determined using a plate-based assay and commercially available substrate. The principle of limiting the infection to a single round increases the precision of the assay measurements. RVP log10EC50 titers correlated closely with titers determined using a plaque reduction neutralization test (PRNT) (R2>95%). The plate-based Zika virus RVP assay also demonstrated high levels of precision, reproducibility and throughput. The assay employs identical reagents for human, rhesus macaque and mouse serum matrices. Spiking studies indicated that the assay performs equally well in different species, producing comparable titers irrespective of the serum species. The assay is conducted in 384-well plates and can be automated to simultaneously achieve high throughput and high reproducibility.