Evaluation of dengue virus strains for human challenge studies.

Discordance between the measured levels of dengue virus neutralizing antibody and clinical outcomes in the first-ever efficacy study of a dengue tetravalent vaccine (Lancet, Nov 2012) suggests a need to re-evaluate the process of pre-screening dengue vaccine candidates to better predict clinical benefit prior to large-scale vaccine trials. In the absence of a reliable animal model and established correlates of protection for dengue, a human dengue virus challenge model may provide an approach to down-select vaccine candidates based on their ability to reduce risk of illness following dengue virus challenge. We report here the challenge of flavivirus-naïve adults with cell culture-passaged dengue viruses (DENV) in a controlled setting that resulted in uncomplicated dengue fever (DF). This sets the stage for proof-of-concept efficacy studies that allow the evaluation of dengue vaccine candidates in healthy adult volunteers using qualified DENV challenge strains well before they reach field efficacy trials involving children. Fifteen flavivirus-naïve adult volunteers received 1 of 7 DENV challenge strains (n=12) or placebo (n=3). Of the twelve volunteers who received challenge strains, five (two DENV-1 45AZ5 and three DENV-3 CH53489 cl24/28 recipients) developed DF, prospectively defined as ≥2 typical symptoms, ≥48h of sustained fever (>100.4°F) and concurrent viremia. Based on our study and historical data, we conclude that the DENV-1 and DENV-3 strains can be advanced as human challenge strains. Both of the DENV-2 strains and one DENV-4 strain failed to meet the protocol case definition of DF. The other two DENV-4 strains require additional testing as the illness approximated but did not satisfy the case definition of DF. Three volunteers exhibited effusions (1 pleural/ascites, 2 pericardial) and 1 volunteer exhibited features of dengue (rash, lymphadenopathy, neutropenia and thrombocytopenia), though in the absence of fever and symptoms. The occurrence of effusions in milder DENV infections counters the long-held belief that plasma leakage syndromes are restricted to dengue hemorrhagic fever/dengue shock syndromes (DHF/DSS). Hence, the human dengue challenge model may be useful not only for predicting the efficacy of vaccine and therapeutic candidates in small adult cohorts, but also for contributing to our further understanding of the mechanisms behind protection and virulence.

A human challenge model for dengue infection reveals a possible protective role for sustained interferon gamma levels during the acute phase of illness.

Dengue has recently been defined by the World Health Organization as a major international public health concern. Although several vaccine candidates are in various stages of development, there is no licensed vaccine available to assist in controlling the further spread of this mosquito borne disease. The need for a reliable animal model for dengue disease increases the risk to vaccine developers as they move their vaccine candidates into large-scale phase III testing. In this paper we describe the cellular immune responses observed in a human challenge model for dengue infection; a model that has the potential to provide efficacy data for potential vaccine candidates in a controlled setting. Serum levels of sIL-2Rα and sTNF-RII were increased in volunteers who developed illness. Supernatants from in vitro stimulated PBMC were tested for cytokines associated with a T(H)1 or T(H)2 T-cell response (IL-2, TNF-α, IFN-γ, IL-4, IL-10, IL-5) and only IFN-γ was associated with protection against fever and/or viremia. Interestingly, IFN-γ levels drop to 0 pg/mL for volunteers who develop illness after challenge suggesting that some mechanism of immunosuppression may play a role in dengue illness. The human challenge model provides an opportunity to test potential vaccine candidates for efficacy prior to large-scale phase III testing, and hints at a possible mechanism for immune suppression by dengue.

Detection of concurrent infection with multiple dengue virus serotypes in Thai children by ELISA and nested RT-PCR assay.

The prevailing global spread of four dengue virus (DENV) serotypes and the resultant co-circulation of multiple serotypes in the same region have invariably led to conditions supporting the periodic occurrence of simultaneous infection of individuals with more than one DENV serotype. This raises the issue of how best to detect concurrent multiple infections. We report here the use of a nested reverse transcription-polymerase chain reaction (RT-PCR) assay, which detected concurrent infection with three DENV serotypes (DENV-1/DENV-2/DENV-3) and two serotypes (DENV-1/DENV-2 and DENV-2/DENV-4), respectively, in three serum specimens from Thai children hospitalized during the dengue epidemic of 2000-2001. In contrast, an enzyme-linked immunosorbent assay used previously for virus serotype identification failed to detect multiple DENV serotypes in these specimens. Serotype identification by RT-PCR was confirmed by sequence analysis of each amplified PCR product. Phylogenetic analyses performed on PCR-amplified DNA fragments further supported the occurrence of concurrent infections with multiple DENV serotypes in these children. Although the sample set was small, our data suggest that nested RT-PCR is an effective method for the detection of concurrent DENV infections.

Molecular genotyping of dengue viruses by phylogenetic analysis of the sequences of individual genes.

The prevalence of four serotypes of dengue virus (DENV) has risen dramatically in recent years accompanied by an increase in viral genetic diversity. The evolution of DENV has had a major impact on their virulence for humans and on the epidemiology of dengue disease around the world. In order to perform disease surveillance and understand DENV evolution and its effects on virus transmission and disease, an efficient and accurate method for genotype identification is required. Phylogenetic analysis of viral gene sequences is the method used most commonly, with envelope (E) gene the most frequently selected target. To determine which gene might be suitable targets for genotyping DENV, phylogenetic analysis was performed on 10 individual coding genes plus the 3'-non-translated region (3'NTR) for 56 geographically divergent DENV strains representing all identified genotypes. These were reflected in eleven maximum likelihood phylogenetic trees. Based on the bootstrap values (over 90%) supporting the major nodes, the best target genes were identified for each serotype: for DENV-1, the sequences of all coding genes except non-structural gene 4A (NS4A), for DENV-2, PrM/M, E, NS1, NS3, NS4A and NS5, for DENV-3, all coding genes and the 3'NTR, and for DENV-4, C, PrM/M, E, NS1, NS2A, NS2B, NS4A and NS5.

Nucleotide sequence of envelope protein of Japanese encephalitis virus SA14-14-2 adapted to vero cells.

Live attenuated Japanese encephalitis (JE) virus SA(14)-14-2 produced in primary dog kidney cells (PDK) was adapted to Vero cells. In an effort to gain insight into the molecular basis of the biological characteristics of the SA14-14-2(Vero) strain, the 1500 nucleotide sequence encoding the envelope (E) gene which possesses major neutralizing epitopes was determined and compared with the sequences of two other attenuated JE virus strains, SA14-14-2(PHK) and SA14-14-2(PDK). The amino acid sequence of the C-terminal region (a.a. 280-500) was found to be identical for all three strains, while the N-terminal region (a.a. 1-279) shows sequence variation. The distribution of mutations in the N-terminal region was nearly the same among the three attenuated strains, suggesting that the N-terminal sequences might be related with virus-host cell specificity. However, it was found that Lys and Val (a.a. 138 and 176, respectively), known to be responsible for attenuation, are still conserved in SA(14)-14-2(Vero). Animal testing showed that SA(14)-14-2(Vero) has an attenuation phenotype similar to that of the parent SA(14)-14-2(PDK) strain in mice.

Development of a purified, inactivated, dengue-2 virus vaccine prototype in Vero cells: immunogenicity and protection in mice and rhesus monkeys.

The feasibility of a purified, inactivated dengue (DEN) vaccine made in Vero cells was explored. A DEN-2 virus candidate was chosen for production of a monotypic, purified, inactivated vaccine (PIV). Virus was harvested from roller bottle culture supernatants, concentrated, and purified on sucrose gradients. The purified virus was inactivated with 0.05% formalin at 22 degrees C. After inactivation, the virus retained its antigenicity and was immunogenic in mice and rhesus monkeys, in which it elicited high titers of DEN-2 virus-neutralizing antibody. Mice were completely protected against challenge with live, virulent virus after receiving two 0.15-microg doses of PIV. Monkeys vaccinated with three doses ranging as low as 0.25 microg demonstrated complete absence or a significant reduction in the number of days of viremia after challenge with homologous virus. These results warrant further testing and development of PIVs for other DEN virus serotypes.

Immunogenic and protective response in mice immunized with a purified, inactivated, Dengue-2 virus vaccine prototype made in fetal rhesus lung cells.

The feasibility of a purified, inactivated vaccine (PIV) against dengue type 2 (DEN-2) virus was explored. Dengue-2 virus strain 16681 was used for producing a monotypic PIV. Virus adapted to fetal rhesus lung (FRhL-2) cells was harvested from roller bottle culture supernatant fluids, concentrated, and purified on sucrose gradients. Analysis of purified virus preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting showed primarily envelope (E) and premembrane (prM) antigens. These preparations had a purity, estimated from silver-stained gels, of approximately 70%, and a yield, based on recovery of virus and viral antigen, of 10-20%. The purified virus was inactivated with 0.05% formalin at 22 degrees C, or alternatively, with 7 mRads from a 60Co source. Vaccinated mice developed high titers of anti-DEN-2 virus neutralizing antibody and were partially protected from virus challenge. These results warrant further testing and development of PIVs for the other DEN virus serotypes.

Dengue-2 virus infection of human bone marrow: characterization of dengue-2 antigen-positive stromal cells.

Dengue is often associated with neutropenia and thrombocytopenia, suggesting that cells of the bone marrow may be targets of dengue viral infections. In this study we infected long-term marrow cultures with dengue type-2 (DEN-2) virus and characterized the viral antigen-positive cells. Using immunofluorescence microscopy and immunohistochemical staining, we demonstrated two types of stromal cells that were positive for DEN-2 virus antigens. The first was a population of relatively small (approximately 25 microns) CD11b/CD18 (MAC-1)-positive cells. When stained with anti-DEN-2 polyclonal antibody, these cells showed viral antigen-positive inclusions and, when stained with anti-tubulin or anti-vimentin antibodies, they showed a diffuse pattern of fluorescence, consistent with mobile dendritic cells with phagocytic functions. The second population of DEN-2 antigen-positive cells comprised a smaller proportion of the total cells. It was made up of larger cells (> 100 microns) that had a well-formed cytoskeletal system as demonstrated by intense staining with anti-actin, anti-tubulin, and anti-vimentin antibodies. When stained with anti-DEN-2 antibody, these cells showed a more diffuse pattern of fluorescence in the perinuclear and Golgi regions, consistent with ongoing virus replication. These large, strongly adherent cells were positive for nerve growth factor receptor, consistent with their identification as adventitial reticular cells. The molecule that mediates the virus interaction with susceptible cells has not previously been identified. Using plasma membrane proteins isolated from K562 cells, virus-binding studies suggest that an approximately 100-kD membrane protein may be involved in the initial virus-cell interaction.

Dengue type-2 virus envelope protein made using recombinant baculovirus protects mice against virus challenge.

The gene coding for the envelope (E) glycoprotein of dengue-2 virus was cloned into baculovirus (Autographa californica nuclear polyhedrosis virus). The recombinant virus contained the entire E protein gene, preceded by 38 nucleotides from the end of the prematrix glycoprotein gene and followed by the first 83 nucleotides of nonstructural protein 1. When expressed in Spodoptera frugiperda (Sf9) cells, approximately 1 mg of recombinant E antigen was made per 10(9) cells. This antigen reacted with polyclonal, anti-dengue type-2 antibody and a dengue type-2-specific, neutralizing monoclonal antibody. BALB/c mice immunized with the recombinant antigen produced only non-neutralizing antibody against dengue-2 virus, but were partially protected against morbidity and mortality after intracranial challenge with virulent dengue-2 virus.

Purification of native dengue-2 viral proteins and the ability of purified proteins to protect mice.

Both the envelope structural protein and the non-structural NS1 protein have been purified from the flavivirus dengue-2 by high-pressure liquid chromatography. These purified proteins maintain their reactivity with monoclonal antibodies. When tested in mice, the envelope protein elicited neutralizing antibodies and partially protected the animals against a lethal viral challenge. The mice responded to the non-structural protein by producing antibodies; however, these antibodies were not neutralizing and the mice were not protected.

Mapping of a region of dengue virus type-2 glycoprotein required for binding by a neutralizing monoclonal antibody.

Envelope glycoprotein E of flaviviruses is exposed at the surface of the virion, and is responsible for eliciting a neutralizing antibody (Ab) response, as well as protective immunity in the host. In this report, we describe a method for the fine mapping of a linear sequence of the E protein of dengue virus type-2 (DEN-2), recognized by a type-specific and neutralizing monoclonal Ab (mAb), 3H5. First, an Escherichia coli expression vector containing a heat-inducible lambda pL promoter was used to synthesize several truncated, and near-full length E polypeptides. Reactivities of these polypeptides with polyclonal mouse hyperimmune sera, as well as the 3H5 mAb revealed the location of the 3H5-binding site to be within a region of 166 amino acids (aa) between aa 255 and 422. For fine mapping, a series of targeted deletions were made inframe within this region using the polymerase chain reaction (PCR). The hydrophilicity pattern of this region was used as a guide to systematically delete the regions encoding the various groups of surface aa residues within the context of a near-full-length E polypeptide by using PCR. The 3H5-binding site was thus precisely mapped to a region encoding 12 aa (between aa 386 and 397). A synthetic peptide containing this sequence was able to bind to the 3H5 mAb specifically, as shown by enzyme-linked immunosorbent assay. In addition, we show that rabbit Abs raised against the synthetic peptide of 12 aa were able to bind to the authentic E protein, and to neutralize DEN-2 virus in a plaque reduction assay.

Dengue-1 virus envelope glycoprotein gene expressed in recombinant baculovirus elicits virus-neutralizing antibody in mice and protects them from virus challenge.

In order to test the feasibility of baculovirus (Autographa californica nuclear polyhedrosis virus, AcNPV) expression vectors for making immunogens against dengue-1 (DEN-1) virus, a portion of the envelope (E) glycoprotein gene of DEN-1 virus was cloned and expressed. The recombinant baculovirus contains 107 nucleotides from the 3' terminus of the DEN-1 matrix (M) gene, which encodes a hydrophobic signal peptide and extends through the first 1, 245 nucleotides of E, terminating 243 nucleotides before the 3' terminus of E. When the recombinant virus was grown in Spodoptera frugiperda cells, about 1 mg of E antigen was made per 10(9) cells. Recombinant E antigen reacted with E protein-specific monoclonal antibodies and stimulated production of DEN-1 virus neutralizing antibody in BALB/c mice. Mice immunized with recombinant E antigen or with heat-inactivated DEN-1 virus were protected significantly against lethal DEN-1 virus challenge. A dose/response effect was observed, with increasing amounts of recombinant antigen leading to increased survival. These results demonstrate the utility of baculovirus for producing immunogens against DEN-1 virus.

Protection of mice against lethal Japanese encephalitis with a recombinant baculovirus vaccine.

Genes coding for the E and NS1 glycoproteins of Japanese encephalitis virus (JEV) were cloned into baculovirus expression vectors. The recombinant baculoviruses obtained were used to infect Spodoptera frugiperda cells. The infected cells were used to immunize C57/B mice, which were then challenged with live JEV. Survival was increased from about 30% in unimmunized mice to 70% in E and polyprotein recipients (P less than 0.005), but was not increased in NS1 recipients despite the development of antibody against NS1 by these mice. Virus neutralizing antibody was demonstrated in 18/20 E glycoprotein recipients and 15/20 polyprotein recipients. The baculovirus expressed E glycoprotein stimulated antibody which was protective and neutralizing in this system.

Sequence analysis of cloned dengue virus type 2 genome (New Guinea-C strain).

Sequences totalling 5472 nucleotides (nt) from four complementary DNA (cDNA) clones of the dengue virus type 2 (DEN-2) RNA (New Guinea strain, NGS-C) have been reported previously [Yaegashi et al., Gene 46 (1986) 257-267; Putnak et al., Virology 163 (1988) 93-103]. This report describes the complete nucleotide sequence, with the exception of about 7 nt at the 5'-noncoding region, of this RNA genome derived from several cDNA clones. It is 10,723 nt in length and contains a single long open reading frame of 10,173 nt, encoding a polyprotein of 3391 amino acids. The genomic organization is similar to that of other flaviviruses that have recently been reported. Among the three DEN-2 strains - the Jamaica genotype (DEN-2JAM), the DEN-2NGS-C, and the S1 candidate vaccine strain derived from Puerto Rico (PR)-159 isolate (DEN-2S1) - which have been sequenced to date, the amino acid sequences of the polyproteins bear 94%-99% similarity. When the amino acid sequences of DEN-2NGS-C are compared with those of the other two strains, the variations are greater in the DEN-2S1 than in the DEN-2JAM. When DEN-2 and DEN-4 are compared, the overall amino acid identities range from 30% to 80% in both the structural and nonstructural proteins; whereas between DEN-2 and DEN-1, they range from 68% to 79% in the region encoding the structural proteins and the nonstructural protein NS1.