Blocking NS3-NS4B interaction inhibits dengue virus in non-human primates.

Dengue is a major health threat and the number of symptomatic infections caused by the four dengue serotypes is estimated to be 96 million1 with annually around 10,000 deaths2. However, no antiviral drugs are available for the treatment or prophylaxis of dengue. We recently described the interaction between non-structural proteins NS3 and NS4B as a promising target for the development of pan-serotype dengue virus (DENV) inhibitors3. Here we present JNJ-1802-a highly potent DENV inhibitor that blocks the NS3-NS4B interaction within the viral replication complex. JNJ-1802 exerts picomolar to low nanomolar in vitro antiviral activity, a high barrier to resistance and potent in vivo efficacy in mice against infection with any of the four DENV serotypes. Finally, we demonstrate that the small-molecule inhibitor JNJ-1802 is highly effective against viral infection with DENV-1 or DENV-2 in non-human primates. JNJ-1802 has successfully completed a phase I first-in-human clinical study in healthy volunteers and was found to be safe and well tolerated4. These findings support the further clinical development of JNJ-1802, a first-in-class antiviral agent against dengue, which is now progressing in clinical studies for the prevention and treatment of dengue.

Impact of prior flavivirus immunity on Zika virus infection in rhesus macaques.

Studies have demonstrated cross-reactivity of anti-dengue virus (DENV) antibodies in human sera against Zika virus (ZIKV), promoting increased ZIKV infection in vitro. However, the correlation between in vitro and in vivo findings is not well characterized. Thus, we evaluated the impact of heterotypic flavivirus immunity on ZIKV titers in biofluids of rhesus macaques. Animals previously infected (≥420 days) with DENV2, DENV4, or yellow fever virus were compared to flavivirus-naïve animals following infection with a Brazilian ZIKV strain. Sera from DENV-immune macaques demonstrated cross-reactivity with ZIKV by antibody-binding and neutralization assays prior to ZIKV infection, and promoted increased ZIKV infection in cell culture assays. Despite these findings, no significant differences between flavivirus-naïve and immune animals were observed in viral titers, neutralizing antibody levels, or immune cell kinetics following ZIKV infection. These results indicate that prior infection with heterologous flaviviruses neither conferred protection nor increased observed ZIKV titers in this non-human primate ZIKV infection model.

Experimental dengue virus challenge of human subjects previously vaccinated with live attenuated tetravalent dengue vaccines.

BACKGROUND: Protection against dengue requires immunity against all 4 serotypes of dengue virus (DENV). Experimental challenge may be useful in evaluating vaccine-induced immunity. METHODS: Ten subjects previously vaccinated with a live attenuated tetravalent dengue vaccine (TDV) and 4 DENV-naive control subjects were challenged by subcutaneous inoculation of either 10(3) plaque-forming units (PFU) of DENV-1 or 10(5) PFU of DENV-3. Two additional subjects who did not develop DENV-3 neutralizing antibody (NAb) from TDV were revaccinated with 10(4) PFU of live attenuated DENV-3 vaccine to evaluate memory response. RESULTS: All 5 TDV recipients were protected against DENV-1 challenge. Of the 5 TDV recipients challenged with DENV-3, 2 were protected. All DENV-3-challenge subjects who developed viremia also developed elevated liver enzyme levels, and 2 had values that were >10 times greater than normal. Of the 2 subjects revaccinated with DENV-3 vaccine, 1 showed a secondary response to DENV-2, while neither showed such response to DENV-3. All 4 control subjects developed dengue fever from challenge. Protection was associated with presence of NAb, although 1 subject was protected despite a lack of measurable NAb at the time of DENV-1 challenge. CONCLUSIONS: Vaccination with TDV induced variable protection against subcutaneous challenge. DENV-3 experimental challenge was associated with transient but marked elevations of transaminases.

Cell-Mediated Immunity Generated in Response to a Purified Inactivated Vaccine for Dengue Virus Type 1.

Dengue is the most prevalent arboviral disease afflicting humans, and a vaccine appears to be the most rational means of control. Dengue vaccine development is in a critical phase, with the first vaccine licensed in some countries where dengue is endemic but demonstrating insufficient efficacy in immunologically naive populations. Since virus-neutralizing antibodies do not invariably correlate with vaccine efficacy, other markers that may predict protection, including cell-mediated immunity, are urgently needed. Previously, the Walter Reed Army Institute of Research developed a monovalent purified inactivated virus (PIV) vaccine candidate against dengue virus serotype 1 (DENV-1) adjuvanted with alum. The PIV vaccine was safe and immunogenic in a phase I dose escalation trial in healthy, flavivirus-naive adults in the United States. From that trial, peripheral blood mononuclear cells obtained at various time points pre- and postvaccination were used to measure DENV-1-specific T cell responses. After vaccination, a predominant CD4+ T cell-mediated response to peptide pools covering the DENV-1 structural proteins was observed. Over half (13/20) of the subjects produced interleukin-2 (IL-2) in response to DENV peptides, and the majority (17/20) demonstrated peptide-specific CD4+ T cell proliferation. In addition, analysis of postvaccination cell culture supernatants demonstrated an increased rate of production of cytokines, including gamma interferon (IFN-γ), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Overall, the vaccine was found to have elicited DENV-specific CD4+ T cell responses as measured by enzyme-linked immunosorbent spot (ELISpot), intracellular cytokine staining (ICS), lymphocyte proliferation, and cytokine production assays. Thus, together with antibody readouts, the use of a multifaceted measurement of cell-mediated immune responses after vaccination is a useful strategy for more comprehensively characterizing immunity generated by dengue vaccines.IMPORTANCE Dengue is a tropical disease transmitted by mosquitoes, and nearly half of the world's population lives in areas where individuals are at risk of infection. Several vaccines for dengue are in development, including one which was recently licensed in several countries, although its utility is limited to people who have already been infected with one of the four dengue viruses. One major hurdle to understanding whether a dengue vaccine will work for everyone-before exposure-is the necessity of knowing which marker can be measured in the blood to signal that the individual has protective immunity. This report describes an approach measuring multiple different parts of immunity in order to characterize which signals one candidate vaccine imparted to a small number of human volunteers. This approach was designed to be able to be applied to any dengue vaccine study so that the data can be compared and used to inform future vaccine design and/or optimization strategies.

Route of inoculation and mosquito vector exposure modulate dengue virus replication kinetics and immune responses in rhesus macaques.

Dengue virus (DENV) is transmitted by infectious mosquitoes during blood-feeding via saliva containing biologically-active proteins. Here, we examined the effect of varying DENV infection modality in rhesus macaques in order to improve the DENV nonhuman primate (NHP) challenge model. NHPs were exposed to DENV-1 via subcutaneous or intradermal inoculation of virus only, intradermal inoculation of virus and salivary gland extract, or infectious mosquito feeding. The infectious mosquito feeding group exhibited delayed onset of viremia, greater viral loads, and altered clinical and immune responses compared to other groups. After 15 months, NHPs in the subcutaneous and infectious mosquito feeding groups were re-exposed to either DENV-1 or DENV-2. Viral replication and neutralizing antibody following homologous challenge were suggestive of sterilizing immunity, whereas heterologous challenge resulted in productive, yet reduced, DENV-2 replication and boosted neutralizing antibody. These results show that a more transmission-relevant exposure modality resulted in viral replication closer to that observed in humans.

Phase 2 clinical trial of three formulations of tetravalent live-attenuated dengue vaccine in flavivirus-naïve adults.

Sixteen dose formulations of our live-attenuated tetravalent dengue virus vaccines (TDV) were previously evaluated for safety and immunogenicity. Two of the sixteen candidate TDV formulations (Formulations 13 and 14) were selected for further evaluation. A new TDV formulation, Formulation 17, using a higher primary dog kidney (PDK) cell passage Dengue-1 virus (DENV-1) and a lower PDK cell passage DENV-4, was developed to optimize the neutralizing antibody response. All three formulations consist of combinations of 10exp3-5 pfu/dose of the four dengue vaccine virus serotypes. This double-blind, randomized trial in 71 healthy adult subjects evaluated vaccine safety, reactogenicity and immunogenicity. TDV's were given subcutaneously in the deltoid on Day 0 and 180 (6 months). Subjects were seen in clinic on Study Days 0, 10, 28, 180, 190 and 208 and filled out daily symptom diaries for 21 days after each vaccination. Formulation 13 was the most reactogenic, while both Formulations 14 and 17 were similar in reported reactions. Seventy-five percent, 31% and 31% of subjects were viremic on Day 10 after primary vaccination with Formulations 13, 14 and 17 respectively. Viremia was not detected in any subject following the second dose of vaccine. The immunogenicity endpoint was neutralizing antibody titer one month after the second vaccination. Thirty-six percent, 40% and 63% of vaccinated subjects developed tetravalent neutralizing antibodies after two doses of Formulations 13, 14 and 17, respectively. Formulation 17 was selected for further clinical evaluation based on this study.

Safety and immunogenicity of a tetravalent live-attenuated dengue vaccine in flavivirus naive children.

A live-attenuated tetravalent dengue virus (DENV) vaccine candidate has been well tolerated and immunogenic in healthy, US flavivirus naive adult volunteers. We conducted a pilot, safety, and immunogenicity trial of the vaccine candidate in healthy Thai children (6-7 years of age) to prepare for its eventual evaluation in Thai infants. In an uncontrolled, open clinical trial, the investigational vaccine was administered on study Days 0 and 180 to seven volunteers residing in Bangkok without neutralizing antibodies to DENV1-4 or to Japanese encephalitis virus (JEV). Clinical and laboratory safety assessments were completed during the 30 days after each vaccine dose, and immunogenicity was determined at Day 30. In this study, the vaccine was well tolerated with no serious adverse events or alert laboratory values. One volunteer experienced fever (38.2 degrees C, < 2 days) and associated DENV4 vaccine viremia 7 days after Dose 2. One month after Dose 2, six volunteers in the per-protocol analysis exhibited a tetravalent neutralizing antibody response with DENV1-4 geometric mean titers of 55, 475, 350, and 171, respectively. Ten weeks (~75 days) after Dose 2, five of the six volunteers continued to exhibit a tetravalent neutralizing antibody profile; one volunteer's DENV4 PRNT50 titer fell below the assay cut-off (29 --> < 10); (clinicaltrials.gov NCT00384670).

Safety and Immunogenicity of a Dengue Virus Serotype-1 Purified-Inactivated Vaccine: Results of a Phase 1 Clinical Trial.

We describe the results from a human clinical trial of a dengue virus serotype-1, purified-inactivated vaccine (DENV-1 PIV) adjuvanted with aluminum hydroxide. This first-in-man, Phase 1, open-label clinical trial consisted of two groups of flavivirus-naïve healthy adult volunteers that received two intramuscular vaccine doses of either 2.5 µg or 5 µg of DENV-1 PIV administered on days 0 and 28. Following vaccination, both vaccine doses exhibited an acceptable safety profile with minimal injection site and systemic reactions. By study day 42, 2 weeks following the second vaccine dose, all volunteers in both vaccine groups developed serum-neutralizing antibodies against DENV-1. Additional testing using an enzyme-linked immunosorbent assay demonstrated induction of a humoral immune response following both vaccine doses. The DENV-1 PIV was safe and immunogenic in a small number of volunteers supporting development and further testing of a tetravalent DENV PIV formulation.

An adjuvanted, tetravalent dengue virus purified inactivated vaccine candidate induces long-lasting and protective antibody responses against dengue challenge in rhesus macaques.

The immunogenicity and protective efficacy of a candidate tetravalent dengue virus purified inactivated vaccine (TDENV PIV) formulated with alum or an Adjuvant System (AS01, AS03 tested at three different dose levels, or AS04) was evaluated in a 0, 1-month vaccination schedule in rhesus macaques. One month after dose 2, all adjuvanted formulations elicited robust and persisting neutralizing antibody titers against all four dengue virus serotypes. Most of the formulations tested prevented viremia after challenge, with the dengue serotype 1 and 2 virus strains administered at 40 and 32 weeks post-dose 2, respectively. This study shows that inactivated dengue vaccines, when formulated with alum or an Adjuvant System, are candidates for further development.

Vaccination of human volunteers with monovalent and tetravalent live-attenuated dengue vaccine candidates.

Four serotypes of monovalent live attenuated dengue virus vaccine candidates were tested for reactogenicity and immunogenicity in 49 flavivirus non-immune adult human volunteers. The four monovalent candidates were then combined into a tetravalent formulation and given to another 10 volunteers. Neutralizing antibody seroconversion rates after a single-dose monovalent vaccination ranged from 53% to 100%. Solicited reactogenicity was scored by each volunteer. A composite index, the Reactogenicity Index, was derived by these self-reported scores. Reactogenicity differed among the four serotype candidates with serotype-1 associated with the most vaccine related side effects. A second dose of monovalent vaccines at either 30 days or 90 days was much less reactogenic but did not significantly increase seroconversion rates. Seroconversion rates in the 10 volunteers who received a single dose of tetravalent vaccine ranged from 30% to 70% among the four serotypes. Similar to the monovalent vaccines, a second dose of the tetravalent vaccine at one month was less reactogenic and did not increase seroconversion. A third dose of the tetravalent vaccine at four months resulted in three of four volunteers with trivalent or tetravalent high-titer neutralizing antibody responses.

Safety and immunogenicity of a tetravalent live-attenuated dengue vaccine in flavivirus-naive infants.

A Phase I/II observer-blind, randomized, controlled trial evaluated the safety and immunogenicity of a dengue virus (DENV) vaccine candidate in healthy Thai infants (aged 12-15 months) without measurable pre-vaccination neutralizing antibodies to DENV and Japanese encephalitis virus. Fifty-one subjects received two doses of either DENV (N = 34; four received 1/10th dose) or control vaccine (N = 17; dose 1, live varicella; dose 2, Haemophilus influenzae type b). After each vaccine dose, adverse events (AEs) were solicited for 21 days, and non-serious AEs were solicited for 30 days; serious AEs (SAEs) were recorded throughout the study. Laboratory safety assessments were performed at 10 and 30 days; neutralizing antibodies were measured at 30 days. The DENV vaccine was well-tolerated without any related SAEs. After the second dose, 85.7% of full-dose DENV vaccinees developed at least trivalent and 53.6% developed tetravalent neutralizing antibodies ≥ 1:10 to DENV (control group = 0%). This vaccine candidate, therefore, warrants continued development in this age group (NCT00322049; clinicaltrials.gov).

Development of a recombinant tetravalent dengue virus vaccine: immunogenicity and efficacy studies in mice and monkeys.

Truncated recombinant dengue virus envelope protein subunits (80E) are efficiently expressed using the Drosophila Schneider-2 (S2) cell expression system. Binding of conformationally sensitive antibodies as well as X-ray crystal structural studies indicate that the recombinant 80E subunits are properly folded native-like proteins. Combining the 80E subunits from each of the four dengue serotypes with ISCOMATRIX adjuvant, an adjuvant selected from a set of adjuvants tested for maximal and long lasting immune responses, results in high titer virus neutralizing antibody responses. Immunization of mice with a mixture of all four 80E subunits and ISCOMATRIX adjuvant resulted in potent virus neutralizing antibody responses to each of the four serotypes. The responses to the components of the tetravalent mixture were equivalent to the responses to each of the subunits administered individually. In an effort to evaluate the potential protective efficacy of the Drosophila expressed 80E, the dengue serotype 2 (DEN2-80E) subunit was tested in both the mouse and monkey challenge models. In both models protection against viral challenge was achieved with low doses of antigen in the vaccine formulation. In non-human primates, low doses of the tetravalent formulation induced good virus neutralizing antibody titers to all four serotypes and protection against challenge with the two dengue virus serotypes tested. In contrast to previous reports, where subunit vaccine candidates have generally failed to induce potent, protective responses, native-like soluble 80E proteins expressed in the Drosophila S2 cells and administered with appropriate adjuvants are highly immunogenic and capable of eliciting protective responses in both mice and monkeys. These results support the development of a dengue virus tetravalent vaccine based on the four 80E subunits produced in the Drosophila S2 cell expression system.

Comparative evaluation of three assays for measurement of dengue virus neutralizing antibodies.

Plaque reduction neutralization tests (PRNTs) are commonly used for measuring levels of dengue virus (DENV) neutralizing antibodies. However, these assays lack a standardized format, generally have a low sample throughput, and are labor-intensive. The objective of the present study was to evaluate two alternative DENV neutralizing antibody assays: an enzyme-linked immunosorbent assay-based microneutralization (MN) assay, and a fluorescent antibody cell sorter-based, DC-SIGN expresser dendritic cell (DC) assay. False-positive rates, serotype specificity, reproducibility, sensitivity, and agreement among the assay methods were assessed using well-characterized but limited numbers of coded test sera. Results showed that all three assays had false-positive rates of less than 10% with titers near the cut-off and generally below the estimated limits of detection. All three methods demonstrated a high degree of specificity and good agreement when used to assay sera and serum mixtures from monovalent vaccinees and sera from patients after primary natural infection, with the only notable exception being moderate-to-high neutralizing antibody titers against DENV 2 measured by PRNT in a mixture containing only DENV 3 and DENV 4 sera. The MN and DC assays demonstrated good reproducibility. All three assays were comparable in their sensitivity, except that the PRNT was less sensitive for measuring DENV 4 antibody, and the MN and DC assays were less sensitive for measuring DENV 2 antibody. However, when used to test sera from persons after tetravalent DENV vaccination or secondary DENV infection, there was poor specificity and poor agreement among the different assays.

Protection of Rhesus monkeys against dengue virus challenge after tetravalent live attenuated dengue virus vaccination.

Rhesus monkeys develop viremia after dengue virus (DENV) inoculation and have been used as an animal model to study DENV infection and DENV vaccine candidates. We evaluated, in this model, the protective efficacy of a live attenuated tetravalent DENV vaccine (TDV) candidate against parenteral challenge with parental near-wild-type DENV strains. Twenty monkeys were vaccinated with TDV at 0 and 1 month, and 20 unvaccinated monkeys served as controls. Vaccinated animals and their controls were inoculated with 10(3)-10(4) pfu of challenge virus 4.5 months after the second vaccination. Primary vaccination resulted in 95%, 100%, 70%, and 15% seroconversion to DENV serotypes 1, 2, 3, and 4 (DENV-1, -2, -3, and -4), respectively. After the second vaccination, the seropositivity rates were 100%, 100%, 90%, and 70%, respectively. Vaccination with TDV resulted in complete protection against viremia from DENV-2 challenge and in 80%, 80%, and 50% protection against challenge with DENV-1, -3, and -4, respectively. Our results suggest that the TDV can elicit protective immunity against all 4 DENV serotypes. Interference among the 4 vaccine viruses may have resulted in decreased antibody responses to DENV-3 and -4, which would require reformulation or dose optimization to minimize this interference during testing of the vaccine in humans.