Skip Navigation

rDEN4Δ30, a Live Attenuated Dengue Virus Type 4 Vaccine Candidate, Is Safe, Immunogenic, and Highly Infectious in Healthy Adult Volunteers

  1. Anna P. Durbin1,
  2. Stephen S. Whitehead2,
  3. Julie McArthur1,
  4. John R. Perreault1,
  5. Joseph E. Blaney Jr.2,
  6. Bhavin Thumar1,
  7. Brian R. Murphy2 and
  8. Ruth A. Karron1
  1. 1Center for Immunization Research, Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, and
  2. 2Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
  1. Reprints or correspondence: Dr. Anna P. Durbin, Center for Immunization Research, Dept. of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205 (adurbin{at}jhsph.edu)
 
Next Section

Abstract

BackgroundThe live attenuated dengue virus type 4 (DEN-4) vaccine candidate virus rDEN4Δ30 was previously found to be safe and immunogenic at a dose of 105 plaque-forming units (pfu)

MethodsIn a follow-up placebo-controlled phase 2 clinical trial, rDEN4Δ30 was administered as a single inoculation to 3 separate dose cohorts (103 pfu, 102 pfu, or 101 pfu), for further evaluation. Each dose cohort consisted of 20 vaccinees and 4 placebo recipients. Volunteers were monitored closely for adverse events, and serum was collected on study days 28 and 42 for determination of neutralizing antibody titer

ResultsThe vaccine was well tolerated at all doses studied. The most common adverse events observed were a transient asymptomatic rash in >50% of vaccinees and a mild neutropenia in ∼20% of vaccinees. No vaccinee developed a dengue-like illness. The vaccine was highly infectious and immunogenic, with 95%–100% of vaccinees in each dose cohort developing a ⩾4-fold increase in titers of serum neutralizing antibodies against DEN-4

ConclusionsThe rDEN4Δ30 vaccine is safe and induced an antibody response that was broadly neutralizing against genotypically diverse DEN-4 viruses. It is a promising vaccine candidate for inclusion in a tetravalent dengue vaccine formulation

Dengue viruses are positive-sense, single-stranded RNA viruses belonging to the Flavivirus genus of the Flaviviridae family [1]. There are 4 serotypes of dengue virus (DEN-1, DEN-2, DEN-3, and DEN-4), all of which are capable of causing the full spectrum of dengue illness, which ranges from a mild, self-limited febrile illness to life-threatening disease [2]. Dengue is now endemic in >100 countries in tropical and subtropical regions of the world and, according to the World Health Organization, has become the most common mosquito-borne viral disease worldwide. Approximately 2.5 billion persons are at risk from dengue, with an estimated 50 million dengue infections occurring annually worldwide [3]. Infection with dengue virus is among the leading causes of hospitalization and death in children in at least 8 tropical Asian countries [4]. Aside from the human toll of this disease, the economic burden on affected nations is great. A 1995 report estimated the annual cost of dengue hemorrhagic fever/shock syndrome (DHF/DSS) in Thailand to be US $19–51 million [4]

Immunity to dengue is mediated primarily by neutralizing antibodies directed against the structural envelope (E) glycoprotein. Infection with 1 dengue serotype provides lifelong homotypic immunity but only short-lived heterotypic immunity [5]. Epidemiologic studies have demonstrated that the greatest risk factor for development of DHF/DSS is secondary infection with a dengue serotype different from that which caused the primary infection [6]. For this reason, DHF/DSS occurs primarily in regions where dengue is hyperendemic and multiple dengue serotypes circulate simultaneously or sequentially. The goal of immunization is to induce a long-lived neutralizing antibody response against all 4 dengue serotypes. A live attenuated virus vaccine is most likely to achieve this goal economically, as has been demonstrated with the live attenuated 17D vaccine for yellow fever [2]. Despite the urgent need to control dengue disease, a dengue vaccine has not been licensed

We recently reported the results of a phase 1 trial of the recombinant live attenuated DEN-4 vaccine candidate rDEN4Δ30 (previously referred to as “rDEN-2AΔ30”) [7, 8]. This vaccine was found to be safe and immunogenic when administered to 20 healthy adult volunteers at a dose of 105 pfu. The most common adverse events noted in that trial were a mild transient maculopapular rash in 50% of volunteers, a transient increase in alanine aminotransferase (ALT) level in 25% of volunteers, and a transient neutropenia in 20% of volunteers. All 20 volunteers experienced seroconversion to DEN-4, as measured by a plaque-reduction neutralization assay. In the present study, we further evaluated rDEN4Δ30, at progressive 10-fold lower doses, to determine whether the mild reactogenicity observed in the 105 pfu dose cohort could be reduced and to determine the 50% human infectious dose (HID50) of the vaccine. In the present article, we report that rDEN4Δ30 is highly infectious, immunogenic, and safe in healthy adult volunteers after a single inoculation

Previous SectionNext Section

Subjects, Materials, and Methods

Study population This phase 2 trial was conducted at the Center for Immunization Research at the Johns Hopkins Bloomberg School of Public Health (BSPH), located in Baltimore, Maryland. Seventy-two healthy adult volunteers were recruited from the metropolitan Baltimore area. The clinical protocol was reviewed and approved by the Committee on Human Research of the BSPH, and informed consent was obtained from each volunteer, in accordance with the Code of Federal Regulations (Title 21, Part 50—Protection of Human Subjects). Healthy adult male and nonpregnant female volunteers between the ages of 18 and 50 years were enrolled if they met eligibility criteria, as described elsewhere [7]

Study design This study was conducted as a double-blind placebo-controlled trial. In an attempt to determine the HID50 of the vaccine, 3 successively lower doses of vaccine were studied, each in a separate dose cohort (103 pfu, 102 pfu, or 101 pfu). Members of each cohort were vaccinated and followed to completion of data collection prior to vaccination of the next cohort. Twenty-four volunteers were enrolled in each cohort; 20 volunteers received vaccine (rDEN4Δ30), and 4 volunteers received placebo (vaccine diluent). Volunteers were randomly assigned to the vaccine or placebo group. Each volunteer was administered 0.5 mL of vaccine or placebo as a subcutaneous injection. Volunteers were given a digital thermometer and diary card to record their oral temperature 3 times daily for 16 days after vaccination. They returned to the clinic every other day for 16 days after vaccination and then again on study days 21, 28, and 42. At each visit, a physician or nurse practitioner examined the volunteers, and blood was obtained for safety monitoring and virologic or immunologic analysis. Clinical signs and symptoms such as headache, rash, lymphadenopathy, petechiae, abdominal tenderness, anorexia, myalgia, arthralgia, eye pain, and photophobia were assessed at each visit

Vaccine virus rDEN4Δ30 is a live recombinant DEN-4 virus derived from the 814669 (Dominica/81) strain and contains a 30-nt deletion in the 3′ untranslated region (UTR) of the genome (3′ nt 172–143). A vaccine lot of rDEN4Δ30 (DEN4-9) was produced under Good Manufacturing Practice conditions at Novavax (Rockville, MD), as described elsewhere [7]. Vaccine virus was diluted to the appropriate titer just prior to vaccine administration, by use of sterile L-15 suitable for human injection

Virus quantitation and serologic assessment Virus titer was determined by plaque assay after inoculation of serial 10-fold dilutions of serum onto Vero cell monolayer cultures, as described elsewhere [7]. Serum hemagglutination-inhibition and plaque-reduction neutralization titers (PRNTs) were determined as described elsewhere [7, 9]. To determine the ability of serum from vaccinees to neutralize geographically and genetically diverse DEN-4 viruses, the PRNT against genotype 1 and genotype 2 DEN-4 viruses was determined in a subset of 5 randomly selected volunteers from each cohort

Sequencing of virus isolates Virus isolates were prepared from each volunteer with viremia by inoculating Vero cell monolayers with serum collected on the last day of detectable viremia. Genomic RNA was isolated and reverse transcribed, and a polymerase chain reaction (PCR) fragment corresponding to the 3′ UTR was generated using methods described elsewhere [7]. A 100-nt region (nt 10397–10497) of the resulting PCR fragment encompassing the Δ30 mutation was sequenced on both strands by use of dye-terminator reactions and DEN-4–specific primers. The derived sequence was compared to that reported previously for the vaccine virus (GenBank accession no. AF326826)

Data analysis The present study is mostly descriptive. Comparisons of mean peak virus titer, onset and duration of viremia, and onset and duration of rash between groups were performed using the Tukey honestly significant difference test. Comparisons of the ages of the vaccinees and placebo recipients, vaccine reactogenicity, monocyte counts, and absolute neutrophil counts (ANCs) were performed using Student’s t test. Statistical analysis was performed using JMP software (version 5.0.1.2; SAS Institute)

Previous SectionNext Section

Results

Volunteers Seventy-two volunteers were recruited and enrolled into the 3 different dose cohorts and were followed for the duration of the trial. Volunteers ranged in age from 18 years to 50 years. There was no significant difference in mean age between volunteers who received vaccine (32.6 years) and those who received placebo (35 years). There was also no significant difference in mean age of volunteers between dose cohorts (mean age in 103 pfu cohort, 32.3 years; mean age in 102 pfu cohort, 30 years; mean age in 101 pfu cohort, 35.4 years). Of the 60 vaccinees, 34 (57.0%) were female; of the 12 placebo recipients, 7 (58.0%) were female. Of the 72 volunteers, 36 (50.0%) were white, 31 (43.0%) were black, 4 (5.5%) were Hispanic, and 1 (1.4%) was Asian

Reactogenicity Local reactogenicity was minimal in all volunteers and occurred within 3 days of vaccination. Of the 60 vaccinees, 13 (22.0%) had mild (⩽2 cm) injection-site erythema on examination, compared with 2 (17.0%) of 12 placebo recipients. Two vaccinees (3.0%) had mild induration of the injection site (⩽2 cm), and 5 (8.3%) had mild tenderness. Injection-site induration or tenderness was not observed in any placebo recipient. None of these differences was statistically significant

The vaccine was well tolerated by the volunteers. No volunteer’s condition met the definition of systemic illness (table 1). There was no significant difference in the occurrence of any solicited symptom (headache, eye pain, photophobia, myalgia, arthralgia, or nausea) between vaccinees and placebo recipients. One vaccinee in the 102 pfu dose cohort recorded a single temperature elevation, to 100.5°F, on study day 3. She had no physical complaints at the time of the temperature elevation and had no other elevations of her temperature throughout the study. One vaccinee in the 103 pfu cohort had a minor increase in ALT level (1.5 times the upper limit of normal)

Table 1
View larger version:
Table 1

Clinical and virological response of volunteers to rDEN4Δ30, a live attenuated dengue virus type 4 vaccine

A mild maculopapular rash was noted consistently in vaccinees in all 3 cohorts (table 2). The rash started on the trunk and extended to the proximal upper extremities, rarely involving the neck or face. The rash was completely asymptomatic in all but 4 affected volunteers. Two volunteers complained of mild pruritus that lasted 1 day and resolved without treatment, and 2 other volunteers complained of pruritus that resolved after 1 dose of oral diphenhydramine

Table 2
View larger version:
Table 2

Incidence, onset, and duration of rash

A transient neutropenia was noted in 20%–25% of vaccinees in each cohort (14/60 vaccinees overall). In all but 2 of the vaccinees, the ANC remained ⩾1000 cells/mm3. Only 1 volunteer had an ANC <500 cells/mm3 (490 cells/mm3). This volunteer received 102 pfu of vaccine, and the ANC nadir occurred on day 14 after vaccination. The mean±SE ANC of those volunteers who subsequently became neutropenic was significantly lower (P<.001) on study day 0, prior to vaccination (2014±350 cells/mm3), than those of both placebo recipients (3500±364 cells/mm3) and vaccinees who did not become neutropenic (3724±166 cells/mm3). The neutropenia experienced by the volunteers resolved uneventfully in all cases

Monocyte counts (absolute and percentage) were recorded as part of the white-blood-cell count differential. There was no significant difference between the prevaccination monocyte counts of vaccinees (472; 8.2%) and placebo recipients (447; 7.7%). The difference between peak postvaccination and prevaccination monocyte count was calculated. Compared with placebo recipients, vaccinees were noted to have a significant increase in both absolute monocyte count (184 for vaccinees vs. 83 for placebo recipients; P<.0036) and monocyte percentage (4.6% for vaccinees vs. 1.0% for placebo recipients; P<.0001)

Viremia Vaccine virus was recovered from the serum of 31 vaccinees (52.0%) (table 3). The mean peak titer of virus among viremic volunteers did not differ significantly between cohorts and was very low in each cohort (0.5–0.7 log10 pfu/mL). The mean day of onset of viremia and mean number of days of viremia in each dose cohort also did not differ significantly

Table 3
View larger version:
Table 3

Incidence, magnitude, onset, and duration of viremia

Sequence analysis The nucleotide sequence surrounding the Δ30 mutation was determined for virus isolated from 30 volunteers on their last day of detectable viremia (study days 8–16). Sequence analysis revealed that the Δ30 mutation occurring after nt 10476 remained unchanged in each isolate. Only the following point mutations in the surrounding region were noted: isolate 2, an insertion of G at nt 10467 and a substitution at nt 10473 (C→A); isolate 5, a substitution at nt 10452 (C→U); isolate 22, a substitution at nt 10487 (U→C); isolate 23, an insertion of G at nt 10467; and isolate 29, an insertion of A at nt 10463

Serologic response to rDEN4Δ30 rDEN4Δ30 was highly immunogenic in vaccinees at all doses tested. Overall, 97% of vaccinees experienced seroconversion to DEN-4, as defined by a ⩾4-fold increase in serum neutralizing antibody titer (table 4). All volunteers in the 101 pfu dose cohort experienced seroconversion to DEN-4 and had mean serum neutralizing antibody titers on study days 28 and 42 that were comparable to those of volunteers who received 105 pfu of vaccine in the phase 1 trial. Of the 20 volunteers in each of the 102 and 103 dose cohorts, 19 experienced seroconversion to DEN-4 by study day 42. Serum neutralizing antibodies induced by the rDEN4Δ30 vaccine were broadly cross-protective against all 5 DEN-4 wild-type viruses tested (table 5). The different wild-type viruses were chosen because they are representative of the diversity among DEN-4 viruses. Only 1 of the volunteers tested did not experience seroconversion to all 5 wild-type viruses. This volunteer did not develop neutralizing antibodies against the DEN-4 Thailand/85 virus

Table 4
View larger version:
Table 4

Immunologic responses induced by dengue virus type 4 vaccine candidate rDEN4Δ30 at different doses

Table 5
View larger version:
Table 5

Immunogenicity of vaccine candidate rDEN4Δ30 against both dengue virus type 4 (DEN-4) genotypes

Previous SectionNext Section

Discussion

To date, the results of phase 1 and phase 2 clinical trials evaluating several live attenuated monovalent and tetravalent dengue vaccines have been published [10 –17]. Although they appeared promising in preclinical studies, most of these vaccine candidates were found to be either under- or overattenuated when administered to healthy human volunteers. When combined into a tetravalent formulation, there appeared to be some interference between the serotypes, which induced a variable antibody response to individual serotypes in the formulation [18, 19]. These vaccine candidate viruses were derived by serial passage in various cell lines. Mutations that accumulate during serial passage in foreign host tissue can occur throughout the genome, and their effects can be unpredictable. The availability of recombinant DNA technology now permits the introduction of specific mutations into the dengue virus genome and provides the means for targeted dengue vaccine development

rDEN4Δ30 was the first recombinant dengue vaccine candidate to be evaluated in clinical trials [7]. Unlike previous dengue virus vaccine candidates, whose attenuation phenotypes are conferred by point mutations introduced by passage in foreign host cells, rDEN4Δ30 is attenuated by a 30-nt deletion in the 3′ UTR of the genome [20]. An important principle in the design of a recombinant dengue virus vaccine is to specifically avoid introducing mutations into the E protein. This is done (1) to preserve the infectivity of the vaccine, since the E protein mediates both attachment to cells and fusion of viral and cell membranes, and (2) to preserve the immunogenicity of the vaccine, since E is the major protective antigen [2, 21]. Mutations present in the E protein of another dengue virus vaccine candidate have been associated with a decrease in its infectivity for humans. Specifically, the HID50 for the DEN-1 16007 PDK-13 vaccine candidate, a virus containing 5 mutations in the E protein, was 104.0 pfu, whereas the HID50 for the highly infectious DEN-2 PDK-53 vaccine candidate, a virus lacking mutations in the E protein, was 100.7 pfu [22 –24]. The rDEN4Δ30 vaccine virus that was evaluated in the present study, like DEN-2 PDK-53, possesses an authentic wild-type E glycoprotein and is highly infectious in human volunteers, with an HID50 of <101.0 pfu. This high level of infectivity, if preserved in a tetravalent formulation, should make this vaccine inexpensive to manufacture, which is an important consideration for vaccines destined for use in developing countries. Although a decrease in the infectivity of a vaccine can be influenced by factors other than the sequence of the E protein, the rDEN4Δ30 vaccine virus with an authentic E protein did indeed maintain a high level of infectivity, despite being highly restricted (peak titer of <0.7 log10) in its replication in humans

The authenticity of the sequence of the E protein of the rDEN4Δ30 vaccine virus was also associated with a preservation of its immunogenicity against the parent wild-type virus and against other divergent DEN-4 viruses. Dengue viruses are geographically and genetically diverse, even among strains of the same serotype [25, 26]. DEN-4 viruses have been classified into genotypes 1 and 2 [26, 27] and, although they are highly related antigenically, they exhibit sequence divergence in 4% of the amino acid residues of the E protein [25, 27]. The introduction of mutations into the E protein of a dengue vaccine candidate may be advantageous in decreasing viscerotropism of the virus [28], but it could also have the undesired property of reducing its immunogenicity to naturally occurring DEN viruses. This unintended consequence has been documented with another flavivirus vaccine candidate, ChimeriVax-JE, a live attenuated chimeric virus for Japanese encephalitis virus (JEV) in which the genes encoding the JEV attenuated SA14-14-2 vaccine-strain prM and E proteins (with 10 amino acid substitutions in the E protein) were substituted for the corresponding genes of the attenuated 17D yellow fever vaccine virus [28]. The antibody response of ChimeriVax-JE recipients was more frequent and of greater magnitude against the vaccine virus than against either the parent JE wild-type virus or other strains of wild-type JEV [28, 29]. The rDEN4Δ30 virus, however, contains the authentic prM and E glycoproteins of the wild-type parent Dominica/81 virus and induced broadly neutralizing antibodies against members of DEN-4 genotypes 1 and 2, representative of the genetic diversity of the circulating DEN-4 viruses. This finding suggests that the rDEN4Δ30 should be highly protective against genetically distinct DEN-4 viruses. This high level of immunogenicity was seen at all doses tested, indicating that it is ultimately the level of replication of the vaccine virus in the host, and not the absolute dose administered, that is primarily responsible for the induction of antibodies. Thus, the rDEN4Δ30 vaccine candidate was both highly infectious and broadly immunogenic in human volunteers, and the authenticity of the rDEN4Δ30 E protein was likely an important determinant of these features

The reactogenicity and immunogenicity profiles of rDEN4Δ30 suggest that this virus is a suitable vaccine candidate for the prevention of DEN-4 disease. The vaccine was well tolerated by volunteers at all doses studied. As noted in the previous phase 1 trial [7], the most common adverse events experienced by volunteers in the present trial were a mild transient maculopapular rash and a transient neutropenia. In the present study, the frequency of occurrence of rash (⩾50% of vaccinees) and neutropenia was similar at each dose tested. Although the rash was similar in location to the rash described in wild-type dengue infection, it lacked the intensely pruritic or petechial quality described by Sabin [30, 31]. Monocyte counts peaked on mean±SE study day 10.6±0.4, which was after the mean onset of viremia (study day 9.5±0.4) and before the mean onset of rash (study day 11.7±0.2). Monocytes, tissue macrophages, and myeloid dendritic cells are postulated to be the predominant target cells for dengue virus infection [32 –35]. It is unclear whether the rash observed in some vaccinees was related to the increase in monocyte count; however, several investigators have demonstrated the production of vasoactive factors by dengue-infected monocytes in vitro [36 –38]. Further analysis of the role that monocytes may play in the clinical and immunologic response to live attenuated dengue vaccines is warranted

There did appear to be a relationship between the quantity of virus administered and the development of elevated serum ALT levels, which is in contrast to the lack of such a relationship for the development of rash and neutropenia. Of 20 volunteers who received 105 pfu of rDEN4Δ30, 5 experienced an elevation in serum ALT level, yet only 1 of 60 volunteers who received 101, 102, or 103 pfu in the present trial experienced an elevation in serum ALT level. This volunteer received 103 pfu of vaccine and was noted to have a maximum ALT level of 91 U/L on study day 14. However, because the volunteer’s ALT level was elevated on study day 0 (78 U/L), just prior to vaccination, it is unclear how significantly the rDEN4Δ30 vaccine contributed to this mild elevation. None of the volunteers experienced any combination of symptoms that would be classified as dengue fever or dengue-like illness. Thus, of the 3 clinical findings associated with the rDEN4Δ30 vaccine—that is, rash, neutropenia, and elevated ALT level—only the third finding appeared to be decreased by dose. In general, it has been the experience of others that it is difficult to abrogate clinical illness or laboratory abnormalities in recipients of a dengue virus vaccine by decreasing the dose of virus administered [39, 40, 41]. Rather, it appears that, once infection is initiated, the level of replication and tissue distribution specified by the genetic program of the virus determines the outcome

There were no significant differences in mean peak viremia, mean duration of viremia, or mean onset of viremia among the dose cohorts in this study. Importantly, the level of viremia (mean peak titers of 100.5–100.7 pfu/mL) was much lower than the level in patients with clinically significant dengue fever or DHF/DSS (105–108 pfu/mL) [42]. This finding of reduced viremia compared with naturally occurring dengue virus infection is consistent with the high level of attenuation and lack of clinical symptoms among seronegative vaccinees in the present study. Since rDEN4Δ30 is attenuated by a deletion mutation rather than by a point mutation, reversion to the wild-type phenotype is highly unlikely. The Δ30 mutation was stable, as expected, after multiple rounds of replication in human volunteers

In summary, the live attenuated rDEN4Δ30 vaccine candidate was safe, highly infectious, and broadly immunogenic when administered to healthy adult volunteers in this phase 2 study. This vaccine appears to be a promising candidate for inclusion in a tetravalent dengue vaccine formulation, although future trials need to be continued beyond study day 42, to assess the durability of the antibody response. The Δ30 mutation appears to be useful for the attenuation of other dengue viruses [43], and its presence contributes to the overall attenuation of antigenic chimeric viruses DEN2/4Δ30 [44], DEN3/4Δ30 [45], and WN/DEN4Δ30 [46]

Previous SectionNext Section

Acknowledgments

We thank Sabrina Weaver, Felipe Troncoso, Kim Wanionek, Zainab Adetoro, Aileen Velez-Cabessa, Janece Lovchik, and Christopher T. Hanson for their assistance in the recruitment of volunteers and expert technical assistance. We also thank all of the volunteers who participated in the trial and whose efforts have contributed to the development of a dengue vaccine

Previous SectionNext Section

Footnotes

  • ↵(See the editorial commentary by Edelman, on pages 647–9.)

  • ↵Presented in part: American Society of Tropical Medicine and Hygiene Annual Meeting, Philadelphia, 3–6 December 2003 (abstract 379)

    Potential conflicts of interest: The rDEN4Δ30 vaccine candidate has been patented by the National Institute of Allergy and Infectious Diseases (NIAID), the institute with which B.R.M., S.S.W., and J.E.B. are affiliated. Through the execution of licensing agreements, the NIAID makes the rDEN4Δ30 vaccine candidate available to parties interested in its further development and commercialization

    Financial support: National Institutes of Health (contract N01A115444)

  • Received July 27, 2004.
  • Accepted October 8, 2004.
Previous Section
 

References

  1. 1.↵
    1. Knipe DM,
    2. Howley PM
    1. Lindenback B,
    2. Rice C
    . Flaviviridae: the viruses and their replication. In: Knipe DM, Howley PM, editors. Fields virology. 4th ed. Vol. 1. Baltimore: Lippincott Williams & Wilkins; 2001. p. 963-1041.
  2. 2.↵
    1. Knipe DM,
    2. Howley PM
    1. Burke DS,
    2. Monath TP
    . Flaviviruses. In: Knipe DM, Howley PM, editors. Fields virology. 4th ed. Vol. 1. Baltimore: Lippincott Williams and Wilkins; 2001. p. 1043-125.
  3. 3.↵
    1. World Health Organization
    . Dengue prevention and control: report by the Secretariat. Fifty‐Fifth World Health Assembly (provisional agenda item 13.14, document no. A55/19). Geneva: WHO; 2002.
  4. 4.↵
    1. World Health Organization
    . Dengue haemorrhagic fever: diagnosis, treatment, prevention and control. 2nd ed. Geneva: WHO; 1997.
  5. 5.↵
    1. Sabin A
    . Recent advances in our knowledge of dengue and sandfly fever. Am J Trop Med Hyg 1955;4:198-207.
    Abstract/FREE Full Text
  6. 6.↵
    1. Burke DS,
    2. Nisalak A,
    3. Johnson DE,
    4. Scott RM
    . A prospective study of dengue infections in Bangkok. Am J Trop Med Hyg 1988;38:172-80.
    Abstract/FREE Full Text
  7. 7.↵
    1. Durbin AP,
    2. Karron RA,
    3. Sun W,
    4. et al
    . Attenuation and immunogenicity in humans of a live dengue virus type‐4 vaccine candidate with a 30 nucleotide deletion in its 3′‐untranslated region. Am J Trop Med Hyg 2001;65:405-13.
    Abstract
  8. 8.↵
    1. Troyer JM,
    2. Hanley KA,
    3. Whitehead SS,
    4. et al
    . A live attenuated recombinant dengue‐4 virus vaccine candidate with restricted capacity for dissemination in mosquitoes and lack of transmission from vaccinees to mosquitoes. Am J Trop Med Hyg 2001;65:414-9.
    Abstract
  9. 9.↵
    1. Clarke DH,
    2. Casals J
    . Techniques for hemagglutination and hemagglutination‐inhibition with arthropod‐borne viruses. Am J Trop Med Hyg 1958;7:561-73.
    Abstract/FREE Full Text
  10. 10.↵
    1. Wisseman CJ,
    2. Sweet B,
    3. Rosenzweig E,
    4. Eylar O
    . Attenuated living type 1 dengue vaccines. Am J Trop Med Hyg 1963;12:620-3.
    Abstract/FREE Full Text
  11. 11.↵
    1. Bhamarapravati N,
    2. Yoksan S,
    3. Chayaniyayothin T,
    4. Angsubphakorn S,
    5. Bunyaratvej A
    . Immunization with a live attenuated dengue‐2‐virus candidate vaccine (16681‐PDK 53): clinical, immunological and biological responses in adult volunteers. Bull World Health Organ 1987;65:189-95.
    MedlineWeb of Science
  12. 12.↵
    1. Hoke CH Jr,
    2. Malinoski FJ,
    3. Eckels KH,
    4. et al
    . Preparation of an attenuated dengue 4 (341750 Carib) virus vaccine. II. Safety and immunogenicity in humans. Am J Trop Med Hyg 1990;43:219-26.
    Abstract/FREE Full Text
  13. 13.↵
    1. Bancroft WH,
    2. Scott RM,
    3. Eckels KH,
    4. et al
    . Dengue virus type 2 vaccine: reactogenicity and immunogenicity in soldiers. J Infect Dis 1984;149:1005-10.
    Abstract/FREE Full Text
  14. 14.↵
    1. McKee KT Jr,
    2. Bancroft WH,
    3. Eckels KH,
    4. Redfield RR,
    5. Summers PL,
    6. Russell PK
    . Lack of attenuation of a candidate dengue 1 vaccine (45AZ5) in human volunteers. Am J Trop Med Hyg 1987;36:435-42.
    Abstract/FREE Full Text
  15. 15.↵
    1. Kanesa‐Thasan N,
    2. Edelman R,
    3. Tacket CO,
    4. et al
    . Phase 1 studies of Walter Reed Army Institute of Research candidate attenuated dengue vaccines: selection of safe and immunogenic monovalent vaccines. Am J Trop Med Hyg 2003;69:17-23.
    Abstract/FREE Full Text
  16. 16.↵
    1. Sun W,
    2. Edelman R,
    3. Kanesa‐Thasan N,
    4. et al
    . Vaccination of human volunteers with monovalent and tetravalent live‐attenuated dengue vaccine candidates. Am J Trop Med Hyg 2003;69:24-31.
    Abstract/FREE Full Text
  17. 17.↵
    1. Edelman R,
    2. Wasserman SS,
    3. Bodison SA,
    4. et al
    . Phase I trial of 16 formulations of a tetravalent live‐attenuated dengue vaccine. Am J Trop Med Hyg 2003;69:48-60.
    Abstract/FREE Full Text
  18. 18.↵
    1. Bhamarapravati N,
    2. Sutee Y
    . Live attenuated tetravalent dengue vaccine. Vaccine 2000;18:44-7.
    CrossRefMedlineWeb of Science
  19. 19.↵
    1. Kanesa‐Thasan N,
    2. Sun W,
    3. Kim‐Ahn G,
    4. et al
    . Safety and immunogenicity of attenuated dengue virus vaccines (Aventis Pasteur) in human volunteers. Vaccine 2001;19:3179-88.
    CrossRefMedlineWeb of Science
  20. 20.↵
    1. Men R,
    2. Bray M,
    3. Clark D,
    4. Chanock RM,
    5. Lai CJ
    . Dengue type 4 virus mutants containing deletions in the 3′ noncoding region of the RNA genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in rhesus monkeys. J Virol 1996;70:3930-7.
    Abstract/FREE Full Text
  21. 21.↵
    1. Anderson R,
    2. King AD,
    3. Innis BL
    . Correlation of E protein binding with cell susceptibility to dengue 4 virus infection. J Gen Virol 1992;73:2155-9.
    Abstract/FREE Full Text
  22. 22.↵
    1. Gubler D,
    2. Kuno G
    . Dengue and dengue hemorrhagic fever. New York: CAB International; 1997.
  23. 23.↵
    1. Huang CY,
    2. Butrapet S,
    3. Pierro DJ,
    4. et al
    . Chimeric dengue type 2 (vaccine strain PDK‐53)/dengue type 1 virus as a potential candidate dengue type 1 virus vaccine. J Virol 2000;74:3020-8.
    Abstract/FREE Full Text
  24. 24.↵
    1. Kinney RM,
    2. Butrapet S,
    3. Chang GJ,
    4. et al
    . Construction of infectious cDNA clones for dengue 2 virus: strain 16681 and its attenuated vaccine derivative, strain PDK‐53. Virology 1997;230:300-8.
    CrossRefMedlineWeb of Science
  25. 25.↵
    1. Wang E,
    2. Ni H,
    3. Xu R,
    4. et al
    . Evolutionary relationships of endemic/epidemic and sylvatic dengue viruses. J Virol 2000;74:3227-34.
    Abstract/FREE Full Text
  26. 26.↵
    1. Henchal EA,
    2. Repik PM,
    3. McCown JM,
    4. Brandt WE
    . Identification of an antigenic and genetic variant of dengue‐4 virus from the Caribbean. Am J Trop Med Hyg 1986;35:393-400.
    Abstract/FREE Full Text
  27. 27.↵
    1. Lanciotti RS,
    2. Gubler DJ,
    3. Trent DW
    . Molecular evolution and phylogeny of dengue‐4 viruses. J Gen Virol 1997;78:2279-84.
    Abstract/FREE Full Text
  28. 28.↵
    1. Guirakhoo F,
    2. Zhang Z,
    3. Myers G,
    4. et al
    . A single amino acid substitution in the envelope protein of chimeric yellow fever‐dengue 1 vaccine virus reduces neurovirulence for suckling mice and viremia/viscerotropism for monkeys. J Virol 2004;78:9998-10008.
    Abstract/FREE Full Text
  29. 29.↵
    1. Monath TP,
    2. McCarthy K,
    3. Bedford P,
    4. et al
    . Clinical proof of principle for ChimeriVax: recombinant live, attenuated vaccines against flavivirus infections. Vaccine 2002;20:1004-18.
    CrossRefMedlineWeb of Science
  30. 30.↵
    1. Monath TP,
    2. Guirakhoo F,
    3. Nichols R,
    4. et al
    . Chimeric live, attenuated vaccine against Japanese encephalitis (ChimeriVax‐JE): phase 2 clinical trials for safety and immunogenicity, effect of vaccine dose and schedule, and memory response to challenge with inactivated Japanese encephalitis antigen. J Infect Dis 2003;188:1213-30.
    Abstract/FREE Full Text
  31. 31.↵
    1. Sabin AB,
    2. Schlesinger RW
    . Production of immunity to dengue with virus modified by propagation in mice. Science 1945;101:640-2.
    Abstract/FREE Full Text
  32. 32.↵
    1. Rivers T,
    2. Horsfall F
    1. Sabin A
    . Dengue. In: Rivers T, Horsfall F, editors. Viral and rickettsial infections of man. 3rd ed. Philadelphia: JB Lippincott Company; 1959. p. 361-73.
  33. 33.↵
    1. Halstead SB,
    2. O’Rourke EJ,
    3. Allison AC
    . Dengue viruses and mononuclear phagocytes. II. Identity of blood and tissue leukocytes supporting in vitro infection. J Exp Med 1977;146:218-29.
    Abstract/FREE Full Text
  34. 34.↵
    1. Scott RM,
    2. Nisalak A,
    3. Cheamudon U,
    4. Seridhoranakul S,
    5. Nimmannitya S
    . Isolation of dengue viruses from peripheral blood leukocytes of patients with hemorrhagic fever. J Infect Dis 1980;141:1-6.
    Abstract/FREE Full Text
  35. 35.↵
    1. Wu SJ,
    2. Grouard‐Vogel G,
    3. Sun W,
    4. et al
    . Human skin Langerhans cells are targets of dengue virus infection. Nat Med 2000;6:816-20.
    CrossRefMedlineWeb of Science
  36. 36.↵
    1. Libraty DH,
    2. Pichyangkul S,
    3. Ajariyakhajorn C,
    4. Endy TP,
    5. Ennis FA
    . Human dendritic cells are activated by dengue virus infection: enhancement by gamma interferon and implications for disease pathogenesis. J Virol 2001;75:3501-8.
    Abstract/FREE Full Text
  37. 37.↵
    1. Hober D,
    2. Poli L,
    3. Roblin B,
    4. et al
    . Serum levels of tumor necrosis factor‐alpha (TNF‐alpha), interleukin‐6 (IL‐6), and interleukin‐1 beta (IL‐1 beta) in dengue‐infected patients. Am J Trop Med Hyg 1993;48:324-31.
    Abstract/FREE Full Text
  38. 38.↵
    1. Shaio MF,
    2. Cheng SN,
    3. Yuh YS,
    4. Yang KD
    . Cytotoxic factors released by dengue virus‐infected human blood monocytes. J Med Virol 1995;46:216-23.
    MedlineWeb of Science
  39. 39.↵
    1. Anderson R,
    2. Wang S,
    3. Osiowy C,
    4. Issekutz AC
    . Activation of endothelial cells via antibody‐enhanced dengue virus infection of peripheral blood monocytes. J Virol 1997;71:4226-32.
    Abstract/FREE Full Text
  40. 40.↵
    1. Sabchareon A,
    2. Lang J,
    3. Chanthavanich P,
    4. et al
    . Safety and immunogenicity of a three dose regimen of two tetravalent live‐attenuated dengue vaccines in five‐ to twelve‐year‐old Thai children. Pediatr Infect Dis J 2004;23:99-109.
    CrossRefMedlineWeb of Science
  41. 41.↵
    1. Sabchareon A,
    2. Lang J,
    3. Chanthavanich P,
    4. et al
    . Safety and immunogenicity of tetravalent live‐attenuated dengue vaccines in Thai adult volunteers: role of serotype concentration, ratio, and multiple doses. Am J Trop Med Hyg 2002;66:264-72.
    Abstract
  42. 42.↵
    1. Vaughn DW,
    2. Green S,
    3. Kalayanarooj S,
    4. et al
    . Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect Dis 2000;181:2-9.
    Abstract/FREE Full Text
  43. 43.↵
    1. Whitehead SS,
    2. Falgout B,
    3. Hanley KA,
    4. Blaney JE Jr,
    5. Markoff L,
    6. Murphy BR
    . A live, attenuated dengue virus type 1 vaccine candidate with a 30‐nucleotide deletion in the 3′ untranslated region is highly attenuated and immunogenic in monkeys. J Virol 2003;77:1653-7.
    Abstract/FREE Full Text
  44. 44.↵
    1. Whitehead SS,
    2. Hanley KA,
    3. Blaney JE Jr,
    4. Gilmore LE,
    5. Elkins WR,
    6. Murphy BR
    . Substitution of the structural genes of dengue virus type 4 with those of type 2 results in chimeric vaccine candidates which are attenuated for mosquitoes, mice, and rhesus monkeys. Vaccine 2003;21:4307-16.
    CrossRefMedlineWeb of Science
  45. 45.↵
    1. Blaney JE Jr,
    2. Hanson CT,
    3. Firestone CY,
    4. Hanley KA,
    5. Murphy BR,
    6. Whitehead SS
    . Genetically modified, live attenuated dengue virus type 3 vaccine candidates. Am J Trop Med Hyg 2004;71:811-21.
    Abstract/FREE Full Text
  46. 46.↵
    1. Pletnev AG,
    2. Claire MS,
    3. Elkins R,
    4. Speicher J,
    5. Murphy BR,
    6. Chanock RM
    . Molecularly engineered live‐attenuated chimeric West Nile/dengue virus vaccines protect rhesus monkeys from West Nile virus. Virology 2003;314:190-5.
    CrossRefMedlineWeb of Science
| Table of Contents

This Article

  1. J Infect Dis. (2005) 191 (5): 710-718. doi: 10.1086/427780
  1. AbstractFree
  2. » Full Text (HTML)Free
  3. Full Text (PDF)Free

Classifications

Share

  1. Email this article

Navigate This Article

Current Issue

  1. March 1, 2012 205 (5)
  1. Journal of Infectious Diseases
  1. Alert me to new issues

Most