Dengue Virus Vaccine Research Articles

Elaboration of tetravalent antibody responses against dengue viruses using a subunit vaccine comprised of a single consensus dengue envelope sequence.

Dengue viruses (DENVs) are re-emerging pathogens transmitted by mosquitoes mainly in tropical and subtropical regions. Each year, they are estimated to infect 390 million people globally. The major challenge confronting dengue vaccine development is the need to induce balanced, long lasting tetravalent immune responses against four co-circulating virus serotypes (DENV-I, -II, -III, -IV), because primary infection by any one of which may predispose infected individuals to more severe diseases during a heterotypic secondary infection. Another difficulty is to select representative strains in vaccine design to provide cross-protection against most circulating virus strains. In this study, aimed at developing a tetravalent subunit vaccine with a representative single protein, we designed two vaccines (named cE80(D4) and cE80(max)) based on the consensus sequences of the ectodomain of envelope protein of 3127 DENV strains, and then expressed them in the baculovirus expression system. Both vaccines were capable of eliciting specific antibodies against all four DENV serotypes, and the predominant IgG subtype elicited by the two vaccines was IgG1. Moreover, these vaccines activated both type I and type II antigen-specific helper T cells that secreted IFN-γ and IL-4, respectively. This proof-of-concept study has set foundation for further optimization of a single protein-based tetravalent DENV vaccine.

Infections of the Central Nervous System in Returning Travelers and Immigrants.

This review highlights current knowledge in travel-related neuroinfectious diseases, providing insight on approaches to prevention, diagnosis, and treatment of infections of the central nervous system (CNS) in travelers and immigrants. Updates on travel vaccine recommendations including vaccine-specific interactions with immunosuppressive agents, advances in Zika virus and dengue virus vaccine development, new diagnostic criteria for neurocysticercosis, updates on treatment approaches for tuberculosis meningitis. Increasing rates of travel are leading to the spread of known infectious diseases and the emergence of new diseases in travel medicine. Among these infections, neuroinfectious diseases carry significant morbidity and mortality. To reduce the effect of travel-related illness, appropriate pre-travel measures and up-to-date diagnostic and treatment strategies are essential for optimal outcomes. This review highlights important travel information relevant to neuroinfectious diseases for several populations including immunocompetent, immunocompromised, pregnant, and infant/children travelers. It also outlines the travel risk, clinical presentation, diagnosis, and management of a select list of neuroinfectious diseases by region, including neurocysticercosis, Zika virus, tuberculosis meningitis, rabies, and tick-borne encephalitis.

Nine year trends of dengue virus infection in Mumbai, Western India.

INTRODUCTION:Dengue virus (DENV) causes a wide range of diseases in humans, from acute febrile illness Dengue fever (DF) to life-threatening Dengue hemorrhagic fever (DHF) or Dengue shock syndrome (DSS). Factors believed to be responsible for spread of Dengue virus infection include explosive population growth, unplanned urban overpopulation with inadequate public health systems, poor standing water and vector control, climate changes and increased international recreational, business, military travel to endemic areas. All of these factors must be addressed to control the spread of Dengue and other mosquito-borne infections. The detection of Dengue virus RNA by reverse transcriptase PCR (RT-PCR) in human serum or plasma samples is highly indicative of acute Dengue fever. Moreover, the method is able to identify the Dengue virus serotype by demonstrating defined sequence homologies in the viral genomic RNA.METHODS AND RESULTS:During the nine year period of this study analysis, 6767 strongly suspected cases were tested by RT-PCR. 1685 (24.9%) were Dengue PCR positive and confirmed as Dengue cases. Observations on the seasonality were based on the nine year's data as the intensity of sampling was at its maximum during monsoon season. Dengue typing was done on 100 positive samples after storage of Dengue RNA at – 80°C. Dengue serotypes were detected in 69 samples of which Dengue 2 was most predominant. 576 samples were processed for NS1 antigen and PCR simultaneously. 19/576 were positive (3.3 %) for NS1 as well as by PCR. 23/576 samples were negative for NS1 antigen, but were positive by RT-PCR. The remaining 534 samples which were negative for NS1 antigen were also negative by Dengue RT-PCR.CONCLUSION:In this study we sought to standardize rapid, sensitive, and specific fluorogenic probe-based RT-PCR assay to screen and serotype a representative range of Dengue viruses that are found in and around Mumbai. Qualitative Dengue virus TaqMan assays could have tremendous utility for the epidemiological investigation of Dengue illness and especially for the study of the viremic response with candidate live-attenuated dengue virus vaccines.

Biodistribution and safety of a live attenuated tetravalent dengue vaccine in the cynomolgus monkey.

The first licensed dengue vaccine is a recombinant, live, attenuated, tetravalent dengue virus vaccine (CYD-TDV; Sanofi Pasteur). This study assessed the biodistribution, shedding, and toxicity of CYD-TDV in a non-human primate model as part of the nonclinical safety assessment program for the vaccine. Cynomolgus monkeys were given one subcutaneous injection of either one human dose (5log10 CCID50/serotype) of CYD-TDV or saline control. Study endpoints included clinical observations, body temperature, body weight, food consumption, clinical pathology, immunogenicity, and post-mortem examinations including histopathology. Viral load, distribution, persistence, and shedding in tissues and body fluids were evaluated by quantitative reverse transcriptase polymerase chain reaction. The subcutaneous administration of CYD-TDV was well tolerated. There were no toxicological findings other than expected minor local reactions at the injection site. A transient low level of CYD-TDV viral RNA was detected in blood and the viral genome was identified primarily at the injection site and in the draining lymph nodes following immunization. These results, together with other data from repeat-dose toxicity and neurovirulence studies, confirm the absence of toxicological concern with CYD-TDV and corroborate clinical study observations.

Which Dengue Vaccine Approach Is the Most Promising, and Should We Be Concerned about Enhanced Disease after Vaccination? The Challenges of a Dengue Vaccine.

A dengue vaccine has been pursued for more than 50 years and, unlike other flaviviral vaccines such as that against yellow fever, progress has been slow. In this review, we describe progress toward the first licensed dengue vaccine Dengvaxia, which does not give complete protection against disease. The antibody response to the dengue virion is reviewed, highlighting immunodominant yet poorly neutralizing responses in the context of a highly dynamic structurally flexible dengue virus particle. Finally, we review recent evidence for cross-reactivity between antibody responses to Zika and dengue viruses, which may further complicate the development of broadly protective dengue virus vaccines.

T-cell Responses in Individuals Infected with Zika Virus and in Those Vaccinated Against Dengue Virus.

Background:The outbreak of Zika virus (ZIKV) infection in Brazil has raised concerns that infection during pregnancy could cause microcephaly and other severe neurodevelopmental malformations in the fetus. The mechanisms by which ZIKV causes fetal abnormalities are largely unknown. The importance of pre-infection with dengue virus (DENV), or other flaviviruses endemic to Brazil, remains to be investigated. It has been reported that antibodies directed against DENV can increase ZIKV infectivity by antibody dependent enhancement (ADE), suggesting that a history of prior DENV infection might worsen the outcome of ZIKV infection.Methods:We used bioinformatics tools to design 18 peptides from the ZIKV envelope containing predicted HLA-I T-cell epitopes and investigated T-cell cross-reactivity between ZIKV-infected individuals and DENV-vaccinated subjects by IFNγ ELISPOT.Results:Three peptides induced IFNγ production in both ZIKV-infected subjects and in DENV-vaccinated individuals. Flow cytometry indicated that 1 ZIKV peptide induced a CD4+ T-cell response in DENV-vaccinated subjects.Conclusions:We demonstrated that vaccination against DENV induced a T-cell response against ZIKV and identified one such CD4+ T-cell epitope. The ZIKV-reactive CD4+ T cells induced by DENV vaccination and identified in this study could contribute to the appearance of cross-reactive antibodies mediating ADE.

T-cell Responses in Individuals Infected with Zika Virus and in Those Vaccinated Against Dengue Virus

Background: The outbreak of Zika virus (ZIKV) infection in Brazil has raised concerns that infection during pregnancy could cause microcephaly and other severe neurodevelopmental malformations in the fetus. The mechanisms by which ZIKV causes fetal abnormalities are largely unknown. The importance of pre-infection with dengue virus (DENV), or other flaviviruses endemic to Brazil, remains to be investigated. It has been reported that antibodies directed against DENV can increase ZIKV infectivity by antibody dependent enhancement (ADE), suggesting that a history of prior DENV infection might worsen the outcome of ZIKV infection.Methods: We used bioinformatics tools to design 18 peptides from the ZIKV envelope containing predicted HLA-I T-cell epitopes and investigated T-cell cross-reactivity between ZIKV-infected individuals and DENV-vaccinated subjects by IFNg ELISPOT.Results: Three peptides induced IFNg production in both ZIKV-infected subjects and in DENV-vaccinated individuals. Flow cytometry indicated that 1 ZIKV peptide induced a CD4+ T-cell response in DENV-vaccinated subjects.Conclusions: We demonstrated that vaccination against DENV induced a T-cell response against ZIKV and identified one such CD4+ T-cell epitope. The ZIKV-reactive CD4+ T cells induced by DENV vaccination and identified in this study could contribute to the appearance of cross-reactive antibodies mediating ADE.

Seroprevalence of antibodies to dengue and chikungunya viruses in Thailand.

The abundance of Aedes mosquito species enabled widespread transmission of mosquito-borne chikungunya virus (CHIKV) and dengue virus (DENV) in Southeast Asia. Periodic seroprevalence surveys are therefore necessary to assess the viral burden in the population and the effectiveness of public health interventions. Since the current seroprevalence for CHIKV and DENV in Thailand are unknown, we evaluated evidence of past infection among Thais. Eight-hundred and thirty-five serum samples obtained from individuals living in central and southern Thailand were assessed for anti-CHIKV and anti-DENV IgG antibodies using commercial enzyme-linked immunosorbent assays. Overall, 26.8% (224/835) of individuals were seropositive for CHIKV, the majority of whom were also DENV-seropositive (91.1%, 204/224). Approximately half of all adults in their fifth decade of life had attained CHIKV seropositivity. Children under 15 years of age in southern Thailand were significantly more likely to be CHIKV-seropositive compared to those residing in central Thailand. In contrast, 79.2% (661/835) of Thais were DENV-seropositive, 30.9% (204/661) of whom also had antibodies to CHIKV. CHIKV/DENV dual seropositivity among Thais was 24.4% (204/835). The age-standardized seroprevalence for DENV was three times that of CHIKV (80.5% vs. 27.2%). Relatively high CHIKV seroprevalence among adults living in central Thailand revealed an under-recognized CHIKV burden in the region, while the low-to-moderate transmission intensity of DENV (seroprevalence <50% at 9 years) is expected to reduce the impact of DENV vaccination in Thailand. This most recent seroprevalence data provide serological baselines for two of the most common mosquito-borne viruses in this region.

Update From the Advisory Committee on Immunization Practices

The Advisory Committee on Immunization Practices (ACIP), a group of medical and public health experts, meets 3 times per year to develop recommendations for vaccine use in the United States. The group has 15 voting members, and each member's term is 4 years. ACIP members and Centers for Disease Control and Prevention (CDC) staff discuss the epidemiology of vaccine-preventable diseases and vaccine research, effectiveness, safety data, and clinical trial results. Representatives from the American Academy of Pediatrics (C. L. B. and Y. A. M.) and the Pediatric Infectious Diseases Society (S. T. O.) are present as liaisons to the ACIP. The ACIP met February 22 and 23, 2017, to discuss proposed recommendations regarding vaccination for unprotected infants born to hepatitis B surface antigen (HBsAg)-positive mothers; topics included cost analysis, influenza surveillance, influenza vaccine effectiveness, herpes zoster vaccine, and considerations for meningococcal serogroup B booster doses in groups at increased risk. Updates on mumps epidemiology, Dengue virus vaccines, Zika virus vaccines, adult immunization, and yellow fever vaccine were also provided.

Cost-Effectiveness of Dengue Vaccination Programs in Brazil.

The first approved dengue vaccine, CYD-TDV, a chimeric, live-attenuated, tetravalent dengue virus vaccine, was recently licensed in 13 countries, including Brazil. In light of recent vaccine approval, we modeled the cost-effectiveness of potential vaccination policies mathematically based on data from recent vaccine efficacy trials that indicated that vaccine efficacy was lower in seronegative individuals than in seropositive individuals. In our analysis, we investigated several vaccination programs, including routine vaccination, with various vaccine coverage levels and those with and without large catch-up campaigns. As it is unclear whether the vaccine protects against infection or just against disease, our model incorporated both direct and indirect effects of vaccination. We found that in the presence of vaccine-induced indirect protection, the cost-effectiveness of dengue vaccination decreased with increasing vaccine coverage levels because the marginal returns of herd immunity decreases with vaccine coverage. All routine dengue vaccination programs that we considered were cost-effective, reducing dengue incidence significantly. Specifically, a routine dengue vaccination of 9-year-olds would be cost-effective when the cost of vaccination per individual is less than $262. Furthermore, the combination of routine vaccination and large catch-up campaigns resulted in a greater reduction of dengue burden (by up to 93%) than routine vaccination alone, making it a cost-effective intervention as long as the cost per course of vaccination is $255 or less. Our results show that dengue vaccination would be cost-effective in Brazil even with a relatively low vaccine efficacy in seronegative individuals.

Zika and Dengue Virus Specific and Cross-reactive Memory B Cell Responses

Abstract Zika virus (ZIKV) belongs to the Flaviviridae family and is transmitted to humans by Aedes mosquitoes. Since 2015, ZIKV has spread rapidly to 70 countries, most notably in the Americas, where it co-circulates with other flaviviruses that also cause human disease, such as dengue virus (DENV). DENV is comprised of four serotypes that are conserved 65–70% in amino acid sequence of the envelope protein among the DENV serotypes and conserved 54–59% with ZIKV. The antigenic similarity of ZIKV and DENV has raised important biological concerns regarding the effect of immunological cross-reactivity on protection and potential enhancement of ZIKV and DENV infections and vaccines. To date, the cross-reactivity of ZIKV and DENV B cell responses is not fully understood in the context of natural human infections. Here, we use a novel ELISPOT-based assay designated Quad-Color Fluorospot that allows investigation of the DENV serotype specificity vs. cross-reactivity of the memory B cell (MBC) population at a single-cell level, adding a fifth color to include ZIKV. We analyzed a unique set of peripheral blood mononuclear cells from the Nicaraguan Pediatric Dengue Cohort Study, a prospective study of dengue, chikungunya, and Zika now in its 13th year. Samples were collected ~2 weeks and several months after RT-PCR-confirmed ZIKV infection from children who were previously DENV-immune or DENV-naïve. We also included a set of dengue patients who were ZIKV-naïve. Preliminary results show that despite the antigenic similarity between DENV and ZIKV, MBCs from ZIKV-infected subjects were highly specific to ZIKV, with lesser cross-reactivity to DENV. These studies provide much-needed information on how the human immune response reacts to ZIKV and DENV over time

Evaluation in Mice of the Immunogenicity of a Tetravalent Subunit Vaccine Candidate Against Dengue Virus Using Mucosal and Parenteral Immunization Routes.

Our group has developed a subunit vaccine candidate against Dengue virus (DENV) based on two different viral regions, the domain III of the envelope protein and the capsid protein. The chimeric proteins for each serotype (DIIIC1-4), aggregated with the oligodeoxynucleotide 39 M, form the tetravalent formulation named Tetra DIIIC. Tetra DIIIC induces a protective immune response in mice when it is inoculated by intraperitoneal route. However, if children are the main targets for a DENV vaccine, then a needle-free route of administration should be attractive and advantageous. In this study, we evaluated for the first time, in vivo, a vaccine candidate against DENV based on recombinant proteins using the intranasal route. After three doses of Tetra DIIIC in mice, we measured the humoral immune response against the four DENV serotypes and the corresponding recombinant proteins. Moreover, the functionality of these antibodies was evaluated through a plaque reduction neutralization test. Finally, to assess the cellular immune response induced, we measured the IFN-γ-levels secreted by spleen cells after in vitro stimulation with DENV. The results presented in this study indicate that the intranasal immunization with Tetra DIIIC favors the generation of DENV-specific cell-mediated immunity. On the other hand, the immunization using intraperitoneal and intranasal routes, simultaneously, generate functional antibodies (anti-DIIIC and anti-DENV) and an in vitro response of IFN-γ secretion.

Antiviral activity of Carbenoxolone disodium against dengue virus infection.

As one of the most important mosquito‐borne viral diseases, dengue infection is now becoming a global concern due to its rapid spread and rise in incidence. Currently, there is no approved vaccine or effective antiviral drug for dengue virus (DENV) infection. Glycyrrhetinic acid (GNa) and its related derivatives have been reported to inhibit a broad spectrum of viruses. However, it is unknown whether Carbenoxolone disodium (CBX), one of the GNa derivatives, affects DENV infection. Here, we found that the production of infectious DENV particles was significantly decreased by CBX treatment in DENV‐permissive cells, while the viral RNA and viral protein synthesis were not affected. Moreover, results from time‐of‐addition study showed that the inhibitory effect of CBX on DENV was exhibited by targeting the virus itself, not the host cells. Directly incubating DENV with CBX resulted in a remarkable reduction of virus titer and virus infectivity. Furthermore, DENV RNA from progeny virions in the supernatants was significantly decreased by CBX treatment in a dose‐dependent manner. Taken together, these data indicate that the antiviral activity of CBX against DENV may be mainly due to a virucidal effect exerted by the compound itself. Our work, for the first time, demonstrates that CBX has antiviral activity against DENV infection, providing useful information for development of potential therapeutic interventions against dengue. J. Med. Virol. 89:571–581, 2017 . © 2016 Wiley Periodicals, Inc.

Induction of multiple cytotoxic T lymphocyte responses in mice by a multiepitope DNA vaccine against dengue virus serotype 1.

Dengue virus (DENV) is still a major threat to human health in most tropical and subtropical countries and regions. In the present study, a multi-epitope DNA vaccine that encodes 15 immunogenic and conserved HLA-A*0201-, HLA-A*1101-, HLA-A*2402-restricted CTL epitopes from DENV serotype 1 (DENV-1) was constructed based on the eukaryotic expressing plasmid pcDNATM 3.1/myc-His(-) A. Immunization of HLA-A*0201, HLA-A*1101 and HLA-A*2402 transgenic mice with the recombinant plasmid pcDNATM 3.1/myc-His(-) A-DENV-1-Meg resulted in significantly greater IFN-γ-secreting T-cell responses against most (14/15) CTL epitopes than occurred in mice immunized with the empty plasmid pcDNATM 3.1/myc-His(-) A. Additionally, the epitope-specific T cells directed to some epitopes secreted not only IFN-γ but also IL-6 and/or TNF-α. Finally, the induced epitope-specific T cells also efficiently lysed epitope-pulsed splenocytes and DENV-1-infected splenic monocytes. The present study confirms the immunogenicity of multi-epitope DENV vaccine, suggesting that it may contribute to the development of a universal DENV vaccine.

Dengue virus antibodies enhance Zika virus infection.

For decades, human infections with Zika virus (ZIKV), a mosquito-transmitted flavivirus, were sporadic, associated with mild disease, and went underreported since symptoms were similar to other acute febrile diseases. Recent reports of severe disease associated with ZIKV have greatly heightened awareness. It is anticipated that ZIKV will continue to spread in the Americas and globally where competent Aedes mosquito vectors are found. Dengue virus (DENV), the most common mosquito-transmitted human flavivirus, is both well-established and the source of outbreaks in areas of recent ZIKV introduction. DENV and ZIKV are closely related, resulting in substantial antigenic overlap. Through antibody-dependent enhancement (ADE), anti-DENV antibodies can enhance the infectivity of DENV for certain classes of immune cells, causing increased viral production that correlates with severe disease outcomes. Similarly, ZIKV has been shown to undergo ADE in response to antibodies generated by other flaviviruses. We tested the neutralizing and enhancing potential of well-characterized broadly neutralizing human anti-DENV monoclonal antibodies (HMAbs) and human DENV immune sera against ZIKV using neutralization and ADE assays. We show that anti-DENV HMAbs, cross-react, do not neutralize, and greatly enhance ZIKV infection in vitro. DENV immune sera had varying degrees of neutralization against ZIKV and similarly enhanced ZIKV infection. Our results suggest that pre-existing DENV immunity may enhance ZIKV infection in vivo and may lead to increased disease severity. Understanding the interplay between ZIKV and DENV will be critical in informing public health responses and will be particularly valuable for ZIKV and DENV vaccine design and implementation strategies.

Protective Capacity of the Human Anamnestic Antibody Response during Acute Dengue Virus Infection

ABSTRACTHalf of the world's population is exposed to the risk of dengue virus infection. Although a vaccine for dengue virus is now available in a few countries, its reported overall efficacy of about 60% is not ideal. Protective immune correlates following natural dengue virus infection remain undefined, which makes it difficult to predict the efficacy of new vaccines. In this study, we address the protective capacity of dengue virus-specific antibodies that are produced by plasmablasts a few days after natural secondary infection. Among a panel of 18 dengue virus-reactive human monoclonal antibodies, four groups of antibodies were identified based on their binding properties. While antibodies targeting the fusion loop of the glycoprotein of dengue virus dominated the antibody response, two smaller groups of antibodies bound to previously undescribed epitopes in domain II of the E protein. The latter, largely serotype-cross-reactive antibodies, demonstrated increased stability of binding at pH 5. These antibodies possessed weak to moderate neutralization capacity in vitro but were the most efficacious in promoting the survival of infected mice. Our data suggest that the cross-reactive anamnestic antibody response has a protective capacity despite moderate neutralization in vitro and a moderate decrease of viremia in vivo.IMPORTANCE Antibodies can protect from symptomatic dengue virus infection. However, it is not easy to assess which classes of antibodies provide protection because in vitro assays are not always predictive of in vivo protection. During a repeat infection, dengue virus-specific immune memory cells are reactivated and large amounts of antibodies are produced. By studying antibodies cloned from patients with heterologous secondary infection, we tested the protective value of the serotype-cross-reactive “recall” or “anamnestic” response. We found that results from in vitro neutralization assays did not always correlate with the ability of the antibodies to reduce viremia in a mouse model. In addition, a decrease of viremia in mice did not necessarily improve survival. The most protective antibodies were stable at pH 5, suggesting that antibody binding in the endosomes, after the antibody-virus complex is internalized, might be important to block virus spread in the organism.

Precisely Molded Nanoparticle Displaying DENV-E Proteins Induces Robust Serotype-Specific Neutralizing Antibody Responses.

Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic fever. The virus is endemic in over 120 countries, causing over 350 million infections per year. Dengue vaccine development is challenging because of the need to induce simultaneous protection against four antigenically distinct DENV serotypes and evidence that, under some conditions, vaccination can enhance disease due to specific immunity to the virus. While several live-attenuated tetravalent dengue virus vaccines display partial efficacy, it has been challenging to induce balanced protective immunity to all 4 serotypes. Instead of using whole-virus formulations, we are exploring the potentials for a particulate subunit vaccine, based on DENV E-protein displayed on nanoparticles that have been precisely molded using Particle Replication in Non-wetting Template (PRINT) technology. Here we describe immunization studies with a DENV2-nanoparticle vaccine candidate. The ectodomain of DENV2-E protein was expressed as a secreted recombinant protein (sRecE), purified and adsorbed to poly (lactic-co-glycolic acid) (PLGA) nanoparticles of different sizes and shape. We show that PRINT nanoparticle adsorbed sRecE without any adjuvant induces higher IgG titers and a more potent DENV2-specific neutralizing antibody response compared to the soluble sRecE protein alone. Antigen trafficking indicate that PRINT nanoparticle display of sRecE prolongs the bio-availability of the antigen in the draining lymph nodes by creating an antigen depot. Our results demonstrate that PRINT nanoparticles are a promising platform for delivering subunit vaccines against flaviviruses such as dengue and Zika.

A Tetravalent Sub-unit Dengue Vaccine Formulated with Ionizable Cationic Lipid Nanoparticle induces Significant Immune Responses in Rodents and Non-Human Primates.

Dengue virus has emerged as an important arboviral infection worldwide. As a complex pathogen, with four distinct serotypes, the development of a successful Dengue virus vaccine has proven to be challenging. Here, we describe a novel Dengue vaccine candidate that contains truncated, recombinant, Dengue virus envelope protein from all four Dengue virus serotypes (DEN-80E) formulated with ionizable cationic lipid nanoparticles (LNPs). Immunization studies in mice, Guinea pigs, and in Rhesus macaques, revealed that LNPs induced high titers of Dengue virus neutralizing antibodies, with or without co-administration or encapsulation of a Toll-Like Receptor 9 agonist. Importantly, LNPs were also able to boost DEN-80E specific CD4+ and CD8+ T cell responses. Cytokine and chemokine profiling revealed that LNPs induced strong chemokine responses without significant induction of inflammatory cytokines. In addition to being highly efficacious, the vaccine formulation proved to be well-tolerated, demonstrating no elevation in any of the safety parameters evaluated. Notably, reduction in cationic lipid content of the nanoparticle dramatically reduced the LNP’s ability to boost DEN-80E specific immune responses, highlighting the crucial role for the charge of the LNP. Overall, our novel studies, across multiple species, reveal a promising tetravalent Dengue virus sub-unit vaccine candidate.

N-Desmethylclozapine, Fluoxetine, and Salmeterol Inhibit Postentry Stages of the Dengue Virus Life Cycle.

Around 10,000 people die each year due to severe dengue disease, and two-thirds of the world population lives in a region where dengue disease is endemic. There has been remarkable progress in dengue virus vaccine development; however, there are no licensed antivirals for dengue disease, and none appear to be in clinical trials. We took the approach of repositioning approved drugs for anti-dengue virus activity by screening a library of pharmacologically active compounds. We identified N-desmethylclozapine, fluoxetine hydrochloride, and salmeterol xinafoate as dengue virus inhibitors based on reductions in the numbers of infected cells and viral titers. Dengue virus RNA levels were diminished in inhibitor-treated cells, and this effect was specific to dengue virus, as other flaviviruses, such as Japanese encephalitis virus and West Nile virus, or other RNA viruses, such as respiratory syncytial virus and rotavirus, were not affected by these inhibitors. All three inhibitors specifically inhibited dengue virus replication with 50% inhibitory concentrations (IC50s) in the high-nanomolar range. Estimation of negative-strand RNA intermediates and time-of-addition experiments indicated that inhibition was occurring at a postentry stage, most probably at the initiation of viral RNA replication. Finally, we show that inhibition is most likely due to the modulation of the endolysosomal pathway and induction of autophagy.

Dengue Virus Envelope Dimer Epitope Monoclonal Antibodies Isolated from Dengue Patients Are Protective against Zika Virus

ABSTRACTZika virus (ZIKV) is a mosquito-borne flavivirus responsible for thousands of cases of severe fetal malformations and neurological disease since its introduction to Brazil in 2013. Antibodies to flaviviruses can be protective, resulting in lifelong immunity to reinfection by homologous virus. However, cross-reactive antibodies can complicate flavivirus diagnostics and promote more severe disease, as noted after serial dengue virus (DENV) infections. The endemic circulation of DENV in South America and elsewhere raises concerns that preexisting flavivirus immunity may modulate ZIKV disease and transmission potential. Here, we report on the ability of human monoclonal antibodies and immune sera derived from dengue patients to neutralize contemporary epidemic ZIKV strains. We demonstrate that a class of human monoclonal antibodies isolated from DENV patients neutralizes ZIKV in cell culture and is protective in a lethal murine model. We also tested a large panel of convalescent-phase immune sera from humans exposed to primary and repeat DENV infection. Although ZIKV is most closely related to DENV compared to other human-pathogenic flaviviruses, most DENV immune sera (73%) failed to neutralize ZIKV, while others had low (50% effective concentration [EC50], <1:100 serum dilution; 18%) or moderate to high (EC50, >1:100 serum dilution; 9%) levels of cross-neutralizing antibodies. Our results establish that ZIKV and DENV share epitopes that are targeted by neutralizing, protective human antibodies. The availability of potently neutralizing human monoclonal antibodies provides an immunotherapeutic approach to control life-threatening ZIKV infection and also points to the possibility of repurposing DENV vaccines to induce cross-protective immunity to ZIKV.