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.

Phase 1 studies of Walter Reed Army Institute of Research candidate attenuated dengue vaccines: selection of safe and immunogenic monovalent vaccines.

We describe the results of initial safety testing of 10 live-attenuated dengue virus (DENV) vaccine candidates modified by serial passage in primary dog kidney (PDK) cells at the Walter Reed Army Institute of Research. The Phase 1 studies, conducted in 65 volunteers, were designed to select an attenuated vaccine candidate for each DENV serotype. No recipient of the DENV candidate vaccines sustained serious injury or required treatment. Three vaccine candidates were associated with transient idiosyncratic reactions in one volunteer each, resulting in their withdrawal from further clinical development. Increasing PDK cell passage of DENV-1, DENV-2, and DENV-3 candidate vaccines increased attenuation for volunteers, yet also decreased infectivity and immunogenicity. This effect was less clear for DENV-4 candidate vaccines following 15 and 20 PDK cell passages. Only one passage level each of the tested DENV-2, -3, and -4 vaccine candidates was judged acceptably reactogenic and suitable for expanded clinical study. Subsequent studies with more recipients will further establish safety and immunogenicity of the four selected vaccine candidates: DENV-1 45AZ5 PDK 20, DENV-2 S16803 PDK 50, DENV-3 CH53489 PDK 20, and DENV-4 341750 PDK 20.

HLA-A and -B allele associations with secondary dengue virus infections correlate with disease severity and the infecting viral serotype in ethnic Thais.

Little is known of the role of classical HLA-A and -B class I alleles in determining resistance, susceptibility, or the severity of acute viral infections. Appropriate paradigms for immunogenetic studies of acute viral infections are dengue fever (DF) and dengue hemorrhagic fever (DHF). Both primary and secondary infections with dengue virus (DEN) serotypes 1, 2, 3 or 4, can result in either clinically less severe DF or the more severe DHF. In secondary exposures, a memory response is induced in immunologically primed individuals, which can both clear the infecting dengue virus and contribute to its pathology. In a case-control study of 263 ethnic Thai patients infected with either DEN-1, -2, -3 or -4, we detected HLA class I associations with secondary infections, but not in immunologically naive patients with primary infections. HLA-A*0203 was associated with the less severe DF, regardless of the secondary infecting virus serotype. By contrast, HLA-A*0207 was associated with susceptibility to the more severe DHF in patients with secondary DEN-1 and DEN-2 infections only. Conversely, HLA-B*51 was associated with the development of DHF in patients with secondary infections, and HLA-B*52 was associated with DF in patients with secondary DEN-1 and DEN-2 infections. Moreover, HLA-B44, B62, B76 and B77 also appeared to be protective against developing clinical disease after secondary dengue virus infection. These results confirm that classical HLA class I alleles are associated with the clinical outcome of exposure to dengue virus, in previously exposed and immunologically primed individuals.

Extinction and rapid emergence of strains of dengue 3 virus during an interepidemic period.

Strains of dengue 3 (DEN-3) virus circulating in Thailand prior to 1992 appear to have disappeared from that location and to have been replaced by two new lineages which have evolved locally, rather than being introduced. Similar DEN-3 virus extinctions may have occurred previously in Thailand in 1962 and 1973. Although no causal relationship could be shown, this strain replacement event was accompanied by DEN-3 replacing DEN-2 as the serotype recovered most frequently from patients in Thailand. Although this implies a change in selection pressure, we found no evidence for positive natural selection at the level of either the E protein or the E protein gene. Further, the extinction of the pre-1992 strains and the appearance of the new lineages occurred during an interepidemic period, suggesting that a genetic bottleneck, rather than selection, might have been important in the emergence of these two new strains of virus. The pre-1992 DEN-3 virus lineage could still be found in 1998, to the west, in Myanmar. The ratio of nonsynonymous-to-synonymous nucleotide changes within a DEN-3 virus population from a single patient was less than the ratio among the consensus sequences of DEN-3 viruses from different patients, suggesting that many of the nonsynonymous nucleotide changes which occurred naturally in the E protein were deleterious and removed by purifying selection.

Use of recombinant envelope proteins for serological diagnosis of Dengue virus infection in an immunochromatographic assay.

An immunochromatographic test that incorporates recombinant antigens (Dengue Duo Rapid Strip Test; PanBio, Brisbane, Australia) has recently become commercially available. This assay is performed in 15 min and detects both immunoglobulin M (IgM) and IgG in a capture format. The four recombinant proteins used represent the N-terminal 80% of the viral envelope glycoproteins of dengue viruses 1, 2, 3, and 4, respectively. The sensitivity and specificity of the recombinant-antigen-based assay were 90 and 86%, respectively. The similar diagnostic performance of these antigens to that of enzyme-linked immunosorbent assays using whole dengue virus suggests that they mimic whole dengue viruses in primary structure and epitope conformation. These results suggest that recombinant proteins can be used in diagnostic assays for dengue to overcome safety issues associated with the use of whole virus.

Attenuation and immunogenicity in humans of a live dengue virus type-4 vaccine candidate with a 30 nucleotide deletion in its 3'-untranslated region.

The recombinant dengue virus type-4 vaccine candidate 2AA30 was attenuated in rhesus monkeys due to an engineered 30-nucleotide deletion in the 3'-untranslated region of the viral genome. A clinical trial to evaluate the safety and immunogenicity of a single dose of 2Adelta30 was conducted with 20 adult human volunteers. The vaccine candidate was well tolerated and did not cause systemic illness in any of the 20 volunteers. Viremia was detectable in 14 volunteers at a mean level of 1.6 log10 plaque-forming units/ml of serum, although all 20 volunteers seroconverted with a seven-fold or greater increase in serum neutralizing antibody titer on day 28 post-vaccination (mean titer = 1:580). A mild, asymptomatic, macular rash developed in 10 volunteers, and a transient elevation in the serum level of alanine aminotransferase was noted in five volunteers. The low level of reactogenicity and high degree of immunogenicity of this vaccine candidate warrant its further evaluation and its use to create chimeric vaccine viruses expressing the structural genes of dengue virus types 1, 2, and 3.

A purified inactivated Japanese encephalitis virus vaccine made in Vero cells.

A second generation, purified, inactivated vaccine (PIV) against Japanese encephalitis (JE) virus was produced and tested in mice where it was found to be highly immunogenic and protective. The JE-PIV was made from an attenuated strain of JE virus propagated in certified Vero cells, purified, and inactivated with formalin. Its manufacture followed current GMP guidelines for the production of biologicals. The manufacturing process was efficient in generating a high yield of virus, essentially free of contaminating host cell proteins and nucleic acids. The PIV was formulated with aluminum hydroxide and administered to mice by subcutaneous inoculation. Vaccinated animals developed high-titered JE virus neutralizing antibodies in a dose dependent fashion after two injections. The vaccine protected mice against morbidity and mortality after challenge with live, virulent, JE virus. Compared with the existing licensed mouse brain-derived vaccine, JE-Vax, the Vero cell-derived JE-PIV was more immunogenic and as effective as preventing encephalitis in mice. The JE-PIV is currently being tested for safety and immunogenicity in volunteers.

T cell receptor Vbeta gene usage in Thai children with dengue virus infection.

T lymphocyte activation during dengue is thought to contribute to the pathogenesis of dengue hemorrhagic fever (DHF). We examined the T cell receptor Vbeta gene usage by a reverse transcriptase-polymerase chain reaction assay during infection and after recovery in 13 children with DHF and 13 children with dengue fever (DF). There was no deletion of specific Vbeta gene families. We detected significant expansions in usage of single Vbeta families in six subjects with DHF and three subjects with DF over the course of infection, but these did not show an association with clinical diagnosis, viral serotype, or HLA alleles. Differences in Vbeta gene usage between subjects with DHF and subjects with DF were of borderline significance. These data suggest that the differences in T cell activation in DHF and DF are quantitative rather than qualitative and that T cells are activated by conventional antigen(s) and not a viral superantigen.

Development of a fluorogenic RT-PCR system for quantitative identification of dengue virus serotypes 1-4 using conserved and serotype-specific 3' noncoding sequences.

A fluorogenic reverse transcriptase-polymerase chain reaction (RT-PCR) system was developed for use as a rapid diagnostic test for determining dengue viremia. The dengue virus 3'-noncoding sequence was utilized to formulate serotype-specific RT-PCR assays for quantitative identification of the four different dengue virus serotypes. A generic RT primer set containing two dengue specific anti-sense primers (DV-L1 and DV-L2) could be used to transcribe extracted viral RNA of all four dengue virus types to complimentary DNA (cDNA). The resultant dengue viral cDNA could be quantitatively identified at the serotype level by the 5'-3' exonuclease assay using four serotype-specific sense primers. The fluorogenic dengue type-specific RT-PCR can detect each of the four dengue types at similar low detection limits, i.e. 20-50 plaque forming units per milliliter of serum. Two panels with four dengue reference serotypes and 134 clinical samples were used to validate detection sensitivity and specificity of the dengue serotype RT-PCR assay, using virus isolation in cell culture as the criterion standard. By analyzing sera samples from Puerto Rico that were collected from 1999 through 2000, the assay demonstrated high level detection sensitivity and specificity of 92.8 and 92.4%, respectively, for all four dengue virus serotypes.

Safety and immunogenicity of attenuated dengue virus vaccines (Aventis Pasteur) in human volunteers.

A randomized, controlled, double-blinded study was conducted to determine safety and immunogenicity of five live attenuated dengue vaccines produced by Aventis Pasteur (AvP). The study was completed with 40 flavivirus non-immune volunteers: five recipients of each monovalent (dengue-1, dengue-2, dengue-3, or dengue-4) vaccine, ten recipients of tetravalent (dengue-1, dengue-2, dengue-3, and dengue-4) vaccine, and ten recipients of vaccine vehicle alone. All vaccines were administered in a single subcutaneous dose (range, 3.6-4.4 log(10) plaque forming units). No serious adverse reactions occurred in volunteers followed for 6 months after vaccination. Five vaccine recipients developed fever (T > or = 38.0 degrees C), including four tetravalent vaccinees between days 8 and 10 after vaccination. Dengue-1, dengue-2, dengue-3, or dengue-4 vaccine recipients reported similar frequency of mild symptoms of headache, malaise, and eye pain. Tetravalent vaccinees noted more moderate symptoms with onset from study days 8-11 and developed maculopapular rashes distributed over trunk and extremities. Transient neutropenia (white blood cells < 4000/mm3) was noted after vaccination but not thrombocytopenia (platelets < 100,000/mm3). All dengue-3, dengue-4, and tetravalent vaccine recipients were viremic between days 7 and 12 but viremia was rarely detected in dengue-1 or dengue-2 vaccinees. All dengue-2, dengue-3, and dengue-4, and 60% of dengue-1 vaccine recipients developed neutralizing and/or immunoglobulin M antibodies. All tetravalent vaccine recipients were viremic with dengue-3 virus and developed neutralizing antibodies to dengue-3 virus. Seven volunteers also had multivalent antibody responses, yet the highest antibody titers were against dengue-3 virus. The AvP live attenuated dengue virus vaccines are safe and tolerable in humans. The live attenuated tetravalent dengue vaccine was most reactogenic, and preferential replication of dengue-3 virus may have affected its infectivity and immunogenicity.

Hepatitis E virus DNA vaccine elicits immunologic memory in mice.

Injection of an expression vector pJHEV containing hepatitis E virus (HEV) structural protein open reading frame 2 gene generates a strong antibody response in BALB/c mice that can bind to and agglutinate HEV. In this study, we tested for immunologic memory in immunized mice whose current levels of IgG to HEV were low or undetectable despite 3 doses of HEV DNA vaccine 18 months earlier. Mice previously vaccinated with vector alone were controls. All mice were administered a dose of HEV DNA vaccine to simulate an infectious challenge with HEV. The endpoint was IgG to HEV determined by ELISA. Ten days after the vaccine dose, 5 of 9 mice previously immunized with HEV DNA vaccine had a slight increase in IgG to HEV. By 40 days after the vaccine dose, the level of IgG to HEV had increased dramatically in all 9 mice (108-fold increase in geometric mean titer). In contrast, no control mice became seropositive. These results indicate that mice vaccinated with 3 doses of HEV DNA vaccine retain immunologic memory. In response to a small antigenic challenge delivered as DNA, possibly less than delivered by a human infective dose of virus, mice with memory were able to generate high levels of antibody in less time than the usual incubation period of hepatitis E. We speculate that this type of response could protect a human from overt disease.

Analysis of plasma viral RNA levels during acute dengue virus infection using quantitative competitor reverse transcription-polymerase chain reaction.

There is increasing recognition of the potential importance of viral burden in the pathogenesis of dengue hemorrhagic fever (DHF). There is little data available, however, describing the kinetics of viral replication in humans with natural dengue virus (DV) infection. Standard procedures for measuring titers of infectious virus in clinical specimens are either laborious or insensitive. We developed a method for measurement of DV RNA in plasma samples based on reverse transcription-polymerase chain reaction (RT-PCR) using a mutant RNA target as a competitor. This technique was reproducible and accurate for samples containing any of the four DV serotypes, and could be applied to samples containing as few as 250 copies of RNA per reaction. We examined plasma viral RNA levels in 80 children with acute DV infection; sequential plasma samples were tested in 34 of these children. Plasma viral RNA levels ranged as high as 10(9) RNA copies/ml, and correlated with titers of infectious virus measured in mosquitoes (r= 0.69). Plasma viral RNA levels fell rapidly during the last several days of the febrile period. We did not find a significant difference in maximal plasma viral RNA levels between children with DHF and children with dengue fever, but peak viral RNA levels were identified in only 16 subjects. We conclude that this quantitative RT-PCR method will be valuable for further studies of natural DV infections.

TT virus infection in acute non-A to E hepatitis in northern Thailand.

TT virus is a novel DNA virus widely distributed in the general population. We examined the prevalence of TTV infection in a population with acute non-A to E hepatitis and in comparison groups located in Northern Thailand. The prevalence of TTV in subjects with non-A-E hepatitis was 19% (21/112), 6% (4/72) in healthy volunteers, 17% (12/72) in those with hepatitis A or B, and 17% (8/48) in hospitalized patients with non-hepatitis illnesses. A significant association with TTV infection and non-A-E hepatitis was seen in all groups (OR 3.9, p = 0.02) and in children (OR 25.8, p = 0.001). Among subjects with non-A-E hepatitis, TTV was associated with higher alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (significant for AST, p = 0.02). Our observations suggest that TTV in our study population may be associated with non-A-E hepatitis and that children in particular may be at risk of hepatocellular injury as a result of TTV infection.

Mechanisms of hemorrhage in dengue without circulatory collapse.

To characterize the molecular basis for the hemostatic defects of dengue infections, a study was conducted in Bangkok, Thailand. Febrile children (n = 68) hospitalized with suspected dengue were enrolled before their clinical syndromes were classified as either dengue fever (DF) or dengue hemorrhagic fever (DHF). Hospital course and outcome were recorded; blood was obtained during the febrile illness (S1), after defervescence (S2), and 1 month after onset of disease (S4). Patients were classified as DF (n = 21) and DHF grades 1, 2, and 3; (DHF1, n = 8; DHF2, n = 30; and DHF3, n = 9). All had marked thrombocytopenia. Bleeding scores were assigned on the basis of bleeding site. Although there was no correlation between bleeding scores and pleural effusion index (a measure of vascular leakage) or bleeding scores and platelet counts, there was a correlation between pleural effusion index and platelet counts. Bleeding scores did not correlate with hemostatic data. Activated partial thromboplastin time was prolonged, with trends toward decreased fibrinogen and increased levels of prothrombin fragment F1.2 in the acute-phase samples. However, no factor level was dramatically decreased. We conclude that most patients with DF or DHF, even without overt hemorrhage, have consumptive coagulopathy. Nevertheless, hemorrhage in dengue without circulatory collapse is most likely due to activation of platelets rather than coagulopathy, which is well compensated. Our data suggest that vascular alteration may be the principal factor involved in the association of thrombocytopenia and hemorrhage with disease severity.

Invited commentary: Dengue lessons from Cuba.

An 18-year interval between a dengue virus type 1 outbreak in 1977-1979 and a dengue virus type 2 outbreak in 1997 in Santiago de Cuba, Cuba, provided a unique opportunity to evaluate risk factors for dengue disease. All patients with symptomatic dengue, including 205 cases of dengue hemorrhagic fever and 12 deaths, were adults born before the dengue virus type 1 epidemic, and nearly all (98%) experienced secondary dengue virus infections. In contrast, almost all of those who seroconverted without illness (97%) experienced primary dengue virus infection. This provides epidemiologic support for the immune enhancement theory of dengue pathogenesis. The Cuban experience suggests that immune enhancement can be seen even 20 years after the primary dengue virus infection. It also supports the contention that primary infections with dengue virus type 2 (and dengue virus type 4) are largely subclinical. These observations have implications for dengue vaccine development based on live-attenuated viruses.

Hepatitis B virus infection in Thai children.

We studied hepatitis B virus (HBV) transmission among 7416 Thai children from 148 schools in Kamphaeng Phet province, a rural part of northern Thailand. Their age ranged from 2 to 16 years (median 9 years). Between May 1991 and June 1992, 61 of 2593 (2.4%) in the cohort of susceptible children acquired anti-HBc immunoglobulin. Forty-seven of the 148 schools had children who acquired anti-HBc. School seroconversion rates to anti-HBc varied from 0% to 23%. There was no correlation between percent of carriers in schools and percent of anti-HBc acquisition. Of the 61 children who acquired anti-HBc, eight (13%) became HBsAg carriers but only two were symptomatic, for a clinical to subclinical infection ration of 1 : 30. One of the two symptomatic children became an HBsAg carrier. Three (38%) of the eight who were persistently antigenemic developed antibody to hepatitis B virus e antigen. Males were 2.5 times (95% CI 1.4-4.3) more likely to acquire anti-HBc than females. Risk factors for acquisition of HBc in Thailand over a 9-month period were examined in a subset of 2412 susceptible children and later in a case-control study of 22 children who acquired anti-HBc and 59 age and sex-matched controls. Risks for acquiring anti-HBc were male gender and a history of bleeding gums. In comparing this study to an earlier pilot study among 9848 children from the same area in Thailand, the yearly antibody acquisition rate to anti-HBc among Thai children dropped from 5.7% in 1989 to 2.4% in 1992. A random sample of children in the pilot study showed that 16% were HBsAg positive and 27% had anti-HBc at the beginning of the study. 34% had markers for either anti-HBc or HBsAg. 12% were repeatedly positive for HBsAg a year later.

Safety and immunogenicity of NYVAC-JEV and ALVAC-JEV attenuated recombinant Japanese encephalitis virus--poxvirus vaccines in vaccinia-nonimmune and vaccinia-immune humans.

A controlled, randomized, double-blind clinical trial evaluated whether two attenuated recombinant poxviruses with identical Japanese encephalitis virus (JEV) gene insertions, NYVAC-JEV and ALVAC-JEV, were safe and immunogenic in volunteers. Groups of 10 volunteers distinguished by vaccinia immune status received two doses of each vaccine. The vaccines appeared to be equally safe and well tolerated in volunteers, but more reactogenic than licensed formalin-inactivated JE and placebo vaccines given as controls. NYVAC-JEV and ALVAC-JEV vaccine recipients had frequent occurrence of local warmth, erythema, tenderness, and/or arm pain after vaccination. There was no apparent effect of vaccinia immune status on frequency or magnitude of local and systemic reactions. NYVAC-JEV elicited antibody responses to JEV antigens in recipients but ALVAC-JEV vaccine poorly induced antibody responses. However, NYVAC-JEV vaccine induced neutralizing antibody responses only in vaccinia-nonimmune recipients while vaccinia-immune volunteers failed to develop protective antibodies (5/5 vs. 0/5 seroconversion, p<0.01). These data suggest that preexisting immunity to poxvirus vector may suppress antibody responses to recombinant gene products.

Etiology of interepidemic periods of mosquito-borne disease.

Dengue viruses and malaria protozoa are of increasing global concern in public health. The diseases caused by these pathogens often show regular seasonal patterns in incidence because of the sensitivity of their mosquito vectors to climate. Between years in endemic areas, however, there can be further significant variation in case numbers for which public health systems are generally unprepared. There is an acute need for reliable predictions of within-year and between-year epidemic events. The prerequisite for developing any system of early warning is a detailed understanding of the factors involved in epidemic genesis. In this report we discuss the potential causes of the interepidemic periods in dengue hemorrhagic fever in Bangkok and of Plasmodium falciparum malaria in a highland area of western Kenya. The alternative causes are distinguished by a retrospective analysis of two unique and contemporaneous 33-year time series of epidemiological and associated meteorological data recorded at these two sites. We conclude that intrinsic population dynamics offer the most parsimonious explanation for the observed interepidemic periods of disease in these locations.

Phylogenetic analysis suggests only one serotype of Japanese encephalitis virus.

Phylogenetic analysis was performed for different genome regions of Japanese encephalitis virus (JEV). Similar genetic groupings were identified for all analyzed genome regions including complete genomes. More extensive analysis was performed for 92 isolates (complete envelope sequences) available in the GenBank. Results of phylogenetic analysis were compared with those performed for human positive strand RNA viruses with well characterized serotypes - poliovirus (PV) and dengue virus (DEN). The observed level of the JEV inter-genotype diversity was much less than that observed across PV and DEN serotypes and was consistent with the genetic diversity observed within PV or DEN serotypes. This genetic analysis supports the contention that all known JEV isolates comprise a single serotype.