Incorporation of IgG Depletion in a Neutralization Assay Facilitates Differential Diagnosis of Zika and Dengue in Secondary Flavivirus Infection Cases.

Zika virus (ZIKV) has emerged as a major global public health concern due to its link as a causative agent of human birth defects. Laboratory diagnosis of suspected ZIKV infections by serological testing of specimens collected a week or more after symptom onset primarily relies on detection of anti-ZIKV-specific IgM antibodies by enzyme-linked immunosorbent assay coupled with detection of ZIKV-specific neutralizing antibody by neutralization tests. A definitive diagnosis based on serological assays is possible during primary ZIKV infections; however, due to the cross-reactivity of antibodies elicited during flaviviral infections, a definitive diagnosis is not always possible, especially among individuals who have previously been exposed to closely related flaviviruses, such as dengue virus (DENV). Here, we investigated the neutralizing IgM antibody profiles of 33 diagnostic specimens collected from individuals with suspected primary and secondary flaviviral infections acquired when visiting areas experiencing active ZIKV transmission in 2015 and 2016. Specimens collected between 1 day and 3 months postexposure were tested for ZIKV and dengue virus type 1 (DENV1) and type 2 (DENV2) by the plaque reduction neutralization test (PRNT) before and after IgG depletion. We found that IgG depletion prior to neutralization testing had little effect in differentiating samples from individuals with secondary infections taken less than 3 weeks postexposure; however, IgG depletion significantly reduced the cross-reactive neutralizing antibody titers and increased the percentage of cases discernible by PRNT from 15.4% (95% confidence interval [CI], 4.3 to 42.2%) to 76.9% (95% CI, 49.7 to 91.8%) for samples collected between roughly 3 and 12 weeks postexposure. These results highlight the potential of IgG depletion to improve the specificity of PRNT for better confirmation and differential diagnosis of flavivirus infections.

Safety and Immunogenicity of a Live Attenuated Tetravalent Dengue Vaccine Candidate in Flavivirus-Naive Adults: A Randomized, Double-Blinded Phase 1 Clinical Trial.

BACKGROUND: Dengue viruses (DENVs) infect >300 million people annually, causing 96 million cases of dengue disease and 22 000 deaths [1]. A safe vaccine that protects against DENV disease is a global health priority [2]. METHODS: We enrolled 72 flavivirus-naive healthy adults in a phase 1 double-blinded, randomized, placebo-controlled dose-escalation trial (low and high dose) of a live attenuated recombinant tetravalent dengue vaccine candidate (TDV) given in 2 doses 90 days apart. Volunteers were followed for safety, vaccine component viremia, and development of neutralizing antibodies to the 4 DENV serotypes. RESULTS: The majority of adverse events were mild, with no vaccine-related serious adverse events. Vaccinees reported injection site pain (52% vs 17%) and erythema (73% vs 25%) more frequently than placebo recipients. Low levels of TDV-serotype 2 (TDV-2), TDV-3, and TDV-4 viremia were observed after the first but not second administration of vaccine. Overall seroconversion rates and geometric mean neutralization titers after 2 doses were 84.2% and 54.1, respectively, for DENV serotype 1 (DENV-1); 92.1% and 292.8, respectively, for DENV-2; 86.8% and 32.3, respectively, for DENV-3; and 71.1% and 15.0, respectively, for DENV-4. More than 90.0% of high-dose recipients had trivalent or broader responses. CONCLUSIONS: TDV was generally well tolerated, induced trivalent or broader neutralizing antibodies to DENV in most flavivirus-naive vaccinees, and is undergoing further development. CLINICAL TRIALS REGISTRATION: NCT01110551.

Safety and immunogenicity of a recombinant live attenuated tetravalent dengue vaccine (DENVax) in flavivirus-naive healthy adults in Colombia: a randomised, placebo-controlled, phase 1 study.

BACKGROUND: Dengue virus is the most serious mosquito-borne viral threat to public health and no vaccines or antiviral therapies are approved for dengue fever. The tetravalent DENVax vaccine contains a molecularly characterised live attenuated dengue serotype-2 virus (DENVax-2) and three recombinant vaccine viruses expressing the prM and E structural genes for serotypes 1, 3, and 4 in the DENVax-2 genetic backbone. We aimed to assess the safety and immunogenicity of tetravalent DENVax formulations. METHODS: We undertook a randomised, double-blind, phase 1, dose-escalation trial between Oct 11, 2011, and Nov 9, 2011, in the Rionegro, Antioquia, Colombia. The first cohort of participants (aged 18-45 years) were randomly assigned centrally, via block randomisation, to receive a low-dose formulation of DENvax, or placebo, by either subcutaneous or intradermal administration. After a safety assessment, participants were randomly assigned to receive a high-dose DENVax formulation, or placebo, by subcutaneous or intradermal administration. Group assignment was not masked from study pharmacists, but allocation was concealed from participants, nurses, and investigators. Primary endpoints were frequency and severity of injection-site and systemic reactions within 28 days of each vaccination. Secondary endpoints were the immunogenicity of DENVax against all four dengue virus serotypes, and the viraemia due to each of the four vaccine components after immunisation. Analysis was by intention to treat for safety and per protocol for immunogenicity. Because of the small sample size, no detailed comparison of adverse event rates were warranted. The trial is registered with ClinicalTrials.gov, number NCT01224639. FINDINGS: We randomly assigned 96 patients to one of the four study groups: 40 participants (42%) received low-dose vaccine and eight participants (8%) received placebo in the low-dose groups; 39 participants (41%) received high-dose vaccine, with nine (9%) participants assigned to receive placebo. Both formulations were well tolerated with mostly mild and transient local or systemic reactions. No clinically meaningful differences were recorded in the overall incidence of local and systemic adverse events between patients in the vaccine and placebo groups; 68 (86%) of 79 participants in the vaccine groups had solicited systemic adverse events compared with 13 (76%) of 17 of those in the placebo groups. By contrast, 67 participants (85%) in the vaccine group had local solicited reactions compared with five (29%) participants in the placebo group. Immunisation with either high-dose or low-dose DENVax formulations induced neutralising antibody responses to all four dengue virus serotypes; 30 days after the second dose, 47 (62%) of 76 participants given vaccine seroconverted to all four serotypes and 73 (96%) participants seroconverted to three or more dengue viruses. Infectious DENVax viruses were detected in only ten (25%) of 40 participants in the low-dose group and 13 (33%) of 39 participants in the high-dose group. INTERPRETATION: Our findings emphasise the acceptable tolerability and immunogenicity of the tetravalent DENVax formulations in healthy, flavivirus-naive adults. Further clinical testing of DENVax in different age groups and in dengue-endemic areas is warranted. FUNDING: Takeda Vaccines.

Genetic and phenotypic characterization of manufacturing seeds for a tetravalent dengue vaccine (DENVax).

BACKGROUND: We have developed a manufacturing strategy that can improve the safety and genetic stability of recombinant live-attenuated chimeric dengue vaccine (DENVax) viruses. These viruses, containing the pre-membrane (prM) and envelope (E) genes of dengue serotypes 1-4 in the replicative background of the attenuated dengue-2 PDK-53 vaccine virus candidate, were manufactured under cGMP. METHODOLOGY/PRINCIPAL FINDINGS: After deriving vaccine viruses from RNA-transfected Vero cells, six plaque-purified viruses for each serotype were produced. The plaque-purified strains were then analyzed to select one stock for generation of the master seed. Full genetic and phenotypic characterizations of the master virus seeds were conducted to ensure these viruses retained the previously identified attenuating determinants and phenotypes of the vaccine viruses. We also assessed vector competence of the vaccine viruses in sympatric (Thai) Aedes aegypti mosquito vectors. CONCLUSION/SIGNIFICANCE: All four serotypes of master vaccine seeds retained the previously defined safety features, including all three major genetic loci of attenuation, small plaques, temperature sensitivity in mammalian cells, reduced replication in mosquito cell cultures, and reduced neurovirulence in new-born mice. In addition, the candidate vaccine viruses demonstrated greatly reduced infection and dissemination in Aedes aegypti mosquitoes, and are not likely to be transmissible by these mosquitoes. This manufacturing strategy has successfully been used to produce the candidate tetravalent vaccine, which is currently being tested in human clinical trials in the United States, Central and South America, and Asia.

Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion.

Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion.

Amino acid changes within the E protein hinge region that affect dengue virus type 2 infectivity and fusion.

Fifteen mutant dengue viruses were engineered and used to identify AAs in the molecular hinge of the envelope protein that are critical to viral infection. Substitutions at Q52, A54, or E133 reduced infectivity in mammalian cells and altered the pH threshold of fusion. Mutations at F193, G266, I270, or G281 affected viral replication in mammalian and mosquito cells, but only I270W had reduced fusion activity. T280Y affected the pH threshold for fusion and reduced replication in C6/36 cells. Three different mutations at L135 were lethal in mammalian cells. Among them, L135G abrogated fusion and reduced replication in C6/36 cells, but only slightly reduced the mosquito infection rate. Conversely, L135W replicated well in C6/36 cells, but had the lowest mosquito infection rate. Possible interactions between hinge residues 52 and 277, or among 53, 135, 170, 186, 265, and 276 required for hinge function were discovered by sequence analysis to identify compensatory mutations.

Domain-III FG loop of the dengue virus type 2 envelope protein is important for infection of mammalian cells and Aedes aegypti mosquitoes.

The FG extended loop in domain III of the dengue virus type 2 (DENV2) envelope protein is postulated to be a molecular determinant for host cell infectivity. To determine the contribution of the FG loop to virus infectivity, an infectious cDNA clone of DENV2 was manipulated by deleting amino acids in the loop (VEPGΔ) to mimic tick-borne flaviviruses or by substituting these AAs with RGD or RGDK/S to mimic motifs present in other mosquito-borne flaviviruses. We found the FG loop to be dispensable for infection of C6/36 cells but critical for infection of Aedes aegypti mosquito midguts and mammalian cells. All the FG loop mutants were able to bind to and enter mammalian cells but replication of VEPGΔ in Vero cells at 37 °C was delayed until acquisition of secondary mutations. Reduced binding of DENV2 type-specific monoclonal antibody 3H5 to mutant viruses confirmed the FG loop motif as its target epitope.