Safety and immunogenicity of a three dose regimen of two tetravalent live-attenuated dengue vaccines in five- to twelve-year-old Thai children.

Objective: The safety and immunogenicity of tetravalent live-attenuated dengue vaccines after a three dose vaccination series were evaluated in Thai children.

Method: One hundred three healthy flavivirus-seronegative schoolchildren ages 5 to 12 years were randomized to receive either dengue vaccine containing 3, 2, 1 and 2 log10 of the 50% cell culture infective dose, respectively, of the live-attenuated dengue vaccine serotypes 1, 2, 3 and 4 per dose (F3212; n = 40) or 3, 3, 1 and 3 log10 of the 50% cell culture infective dose (F3313; n = 42) or purified Vero cell rabies vaccine (control group; n = 21) given in a two dose schedule (3 to 5 months apart). A third dose was administered 8 to 12 months after the second dose to 90 subjects.

Dengue 2 PDK-53 virus as a chimeric carrier for tetravalent dengue vaccine development.

Attenuation markers of the candidate dengue 2 (D2) PDK-53 vaccine virus are encoded by mutations that reside outside of the structural gene region of the genome. We engineered nine dengue virus chimeras containing the premembrane (prM) and envelope (E) genes of wild-type D1 16007, D3 16562, or D4 1036 virus within the genetic backgrounds of wild-type D2 16681 virus and the two genetic variants (PDK53-E and PDK53-V) of the D2 PDK-53 vaccine virus. Expression of the heterologous prM-E genes in the genetic backgrounds of the two D2 PDK-53 variants, but not that of wild-type D2 16681 virus, resulted in chimeric viruses that retained PDK-53 characteristic phenotypic markers of attenuation, including small plaque size and temperature sensitivity in LLC-MK(2) cells, limited replication in C6/36 cells, and lack of neurovirulence in newborn ICR mice.

Chimeric dengue type 2/type 1 viruses induce immune responses in cynomolgus monkeys.

Chimeric dengue type 2/type 1 (DEN2/1) viruses, which contain the structural genes of the dengue-1 (16007) parental virus and the nonstructural genes of the DEN2-PDK53 virus, have been constructed. These DEN2/1 viruses induce high levels of DEN1 virus-specific neutralizing antibodies in mice. In this study, the DEN2/1 viruses induced DEN1 virus-specific neutralizing antibodies without the development of viremia in cynomolgus monkeys.

Safety and immunogenicity of tetravalent live-attenuated dengue vaccines in Thai adult volunteers: role of serotype concentration, ratio, and multiple doses.

Dengue fever, caused by four serotypes of a mosquito-borne virus, is a growing problem in tropical countries. Currently, there is no treatment or vaccine. We evaluated safety and immunogenicity of two doses, given six months apart, of seven formulations of dengue tetravalent live-attenuated vaccine (containing different concentrations of the component viruses) versus placebo in 59 flavivirus-seronegative Thai adults.

Dengue virus specific T cell responses to live attenuated monovalent dengue-2 and tetravalent dengue vaccines.

The proliferative T cell responses to dengue vaccines were studied using the parental strains of dengue vaccines as antigens in 26 dengue immune individuals who resided in Bangkok which is the endemic area of dengue infection. The magnitude of the T cell responses in subjects with flavivirus cross-reactive neutralizing antibody was much higher and the cross-reactivity was broader than in those with dengue serotype-specific neutralizing antibodies, Japanese encephalitis (JE) specific antibodies or dengue cross-reactive antibodies. The T cell response in those with neutralizing antibody against a single serotype or in those who had dengue cross-reactive neutralizing antibody was relatively low, independent of the level or degree of cross-reactivity of the antibody.

Accelerating the development and future availability of HIV-1 vaccines: why, when, where, and how?

An HIV-1 vaccine offers the best long-term hope to control the AIDS pandemic, especially in less-developed countries. To ensure its future availability we need to increase our research efforts today, including clinical trials. Although small-scale clinical trials of HIV-1 vaccines have been underway since 1987, the first phase III efficacy trials started only recently in the USA and Thailand.

Live attenuated tetravalent dengue vaccine.

The development of a live attenuated tetravalent dengue vaccine is currently the best strategy to obtain a vaccine against dengue viruses. The Mahidol University group developed candidate live attenuated vaccines by attenuation through serial passages in certified primary cell cultures. Dengue serotype 1, 2 and 4 viruses were developed in primary dog kidney cells, whereas dengue serotype 3 was serially passaged in primary African green monkey kidney cells.

Chimeric dengue type 2 (vaccine strain PDK-53)/dengue type 1 virus as a potential candidate dengue type 1 virus vaccine.

We constructed chimeric dengue type 2/type 1 (DEN-2/DEN-1) viruses containing the nonstructural genes of DEN-2 16681 virus or its vaccine derivative, strain PDK-53, and the structural genes (encoding capsid protein, premembrane protein, and envelope glycoprotein) of DEN-1 16007 virus or its vaccine derivative, strain PDK-13. We previously reported that attenuation markers of DEN-2 PDK-53 virus were encoded by genetic loci located outside the structural gene region of the PDK-53 virus genome. Chimeric viruses containing the nonstructural genes of DEN-2 PDK-53 virus and the structural genes of the parental DEN-1 16007 virus retained the attenuation markers of small plaque size and temperature sensitivity in LLC-MK(2) cells, less efficient replication in C6/36 cells, and attenuation for mice.

Attenuation markers of a candidate dengue type 2 vaccine virus, strain 16681 (PDK-53), are defined by mutations in the 5' noncoding region and nonstructural proteins 1 and 3.

The genome of a candidate dengue type 2 (DEN-2) vaccine virus, strain PDK-53, differs from its DEN-2 16681 parent by nine nucleotides. Using infectious cDNA clones, we constructed 18 recombinant 16681/PDK-53 viruses to analyze four 16681-to-PDK-53 mutations, including 5' noncoding region (5'NC)-57 C-to-T, premembrane (prM)-29 Asp-to-Val (the only mutation that occurs in the structural proteins), nonstructural protein 1 (NS1)-53 Gly-to-Asp, and NS3-250 Glu-to-Val. The viruses were studied for plaque size, growth rate, and temperature sensitivity in LLC-MK(2) cells, growth rate in C6/36 cells, and neurovirulence in newborn mice.

Recombinant bacillus Calmette-Guérin as a potential vector for preventive HIV type 1 vaccines.

In August 1997, the World Health Organization (WHO) and the Joint United Nations Programme on HIV/AIDS (UNAIDS) convened an expert working group to discuss current strategies for the development of HIV type 1 vaccines. Based on the recent findings of investigators from Japan's National Institute of Infectious Diseases (NIID) in Tokyo using recombinant bacillus Calmette-Guérin (rBCG) as a potential vectored vaccine for HIV, a recommendation was made that further work in this area is a priority. As a result, the working group reconvened in September 1998 to discuss the progress to date with this vaccine approach, as well as areas of related research to assess the feasibility of a BCG-vectored HIV vaccine.

Dynamics of susceptibility and transmissibility of the live, attenuated, candidate vaccines dengue-1 PDK13, dengue-3 PGMK30F3, and dengue-4 PDK48 after oral infection in Aedes aegypti.

Dengue-1 virus PDK13 and isolates from vaccinees (dengue-1 Ib1 and dengue-1 Ib 10), dengue-3 PGMK30F3, and dengue-4 PDK48 were studied for their abilities to infect, disseminate, and replicate in Aedes aegypti mosquitoes by the oral route. In general, infection and dissemination rates were poorer for the vaccine compared with the parent viruses. The transmissibility was also lower for dengue-1 PDK13 than the parent virus, whereas it was not detected for isolates from vaccinees and dengue-3 PGMK30F3.

The use of Toxorhynchites splendens for identification and quantitation of serotypes contained in the tetravalent live attenuated dengue vaccine.

Assurance of identity and quantity is an indispensable part of quality control in the manufacture of vaccines. Dengue-1 PDK13, dengue-2 PDK53, dengue-3 PGMK30F3 and dengue-4 PDK48 in the live attenuated tetravalent dengue vaccine were assayed by identification and quantitation in a mosquito system (Toxorhynchites splendens). Each serotype of dengue virus was identified by dengue specific monoclonal antibodies in the indirect fluorescent antibody test.

The micro-focus reduction neutralization test for determining dengue and Japanese encephalitis neutralizing antibodies in volunteers vaccinated against dengue.

A micro-focus reduction neutralization test (mFRNT) was evaluated as an alternative test to the ordinary plaque reduction neutralization test (PRNT) for the determination of dengue virus and Japanese encephalitis virus neutralizing antibody responses in persons receiving dengue vaccine. The 2 tests were similar in terms of titres and ability to detect seroconversion. Although the neutralizing antibody titres obtained by mFRNT were slightly lower than those given by PRNT, the differences were less than two-fold, indicating than mFRNT was reliable.

Construction of infectious cDNA clones for dengue 2 virus: strain 16681 and its attenuated vaccine derivative, strain PDK-53.

We identified nine nucleotide differences between the genomes of dengue-2 (DEN-2) 16681 virus and its vaccine derivative, strain PDK-53. These included a C-to-T (16681-to-PDK-53) mutation at nucleotide position 57 of the 5'-untranslated region, three silent mutations, and substitutions prM-29 Asp to Val, NS1-53 Gly to Asp, NS2A-181 Leu to Phe, NS3-250 Glu to Val, and NS4A-75 Gly to Ala. Unpassaged PDK-53 vaccine contained two genetic variants as a result of partial mutation at NS3-250.

Possible evidence of endothelial cell activation and disturbance in thalassemia: an in vitro study.

Activation of vascular endothelium is considered as an important facet of inflammation, thrombosis, and vasculitis. Activated endothelial cells express a number of immunologically relevant surface markers which are not detected in dormant condition. These surface markers on endothelial cell may involve in adhesion reaction and migration of blood cell components.

Dengue viruses induce cell proliferation and morphological changes of endothelial cells.

Replication of dengue viruses (type 1, 2, 3 and 4) in vitro in endothelial cells from human umbilical cord vein was demonstrated by virus titers and immunofluorescent antibody studies. Both showed highest peak at Day 6 after inoculation and declined to origin at Day 14. Some of the cultured endothelial cells detached from the culture well.

In vivo and in vitro studies on the morphological change in the monkey epidermal Langerhans cells following exposure to dengue 2 (16681) virus.

A direct comparison of skin Langerhans cell (LC) morphologic change following in vivo and in vitro exposure to dengue-2 (DEN-2) virus (16681) was performed in the monkey to investigate any differences in functional activity profiles. Time-lapse study of skin biopsy at the intradermal (id) virus injection sites, and thin skin sheets removed from the monkey with exposure to virus in culture medium, revealed a highly active migration of epidermal LCs in both sets of experimental specimens. The migration led to a relatively higher number of dendritic cells (DC) which appeared in active migrational profiles, in the superficial dermis.

Immunohistochemical characterization of a new monoclonal antibody reactive with dengue virus-infected cells in frozen tissue using immunoperoxidase technique.

This paper presents a novel monoclonal antibody shown to react with cytoplasmic antigens in various dengue infected human frozen organs from autopsy and necropsy specimens. Strong reactivity was found in hematopoietic cells, including immunoblasts, lymphocytes, plasma cells and macrophages of spleen, lymph node, lung, kidney and stomach. Strikingly, strong positivity was demonstrated in cerebral cortex neurones, Purkinje cells, choroid plexus and blood vessels in addition to astrocytes and microglia.

Langerhans cell density and serological changes following intradermal immunisation of mice with dengue 2 virus.

After the introduction of the dengue-2 (16681) virus by intradermal (i.d.) injection into the footpads of mice, Langerhans cells (LCs) increased in numbers within 24 h at the site of injection and neutralising antibody developed.

Testing of a dengue 2 live-attenuated vaccine (strain 16681 PDK 53) in ten American volunteers.

A live-attenuated dengue 2 vaccine (strain 16681 PDK 53) developed at Mahidol University, Thailand was evaluated for safety and immunogenicity by administering 10(4) p.f.u.

Alterations in vascular endothelial cell-related plasma proteins in thalassaemic patients and their correlation with clinical symptoms.

An increased level of plasma thrombomodulin (TM) in alpha- and beta-thalassaemia was demonstrated using an enzyme-linked immunosorbent assay (ELISA). Nonsplenectomized patients with beta-thalassaemia/haemoglobin E (BE) had higher levels of TM than splenectomized cases (BE-S). Patients with leg ulcers (BE-LU) were found to have the highest increase in TM level.

Diagnosing dengue virus infection in archived autopsy tissues by means of the in situ PCR method: a case report.

Background: The pathogenesis of the severe form of dengue virus infection, dengue hemorrhagic fever, is still obscure. A major research objective has been to determine which body organs are being damaged by dengue virus in this form of dengue. Research has been difficult because dengue hemorrhagic fever is sporadic and tends to occur in parts of the world where modern facilities are scarce and fresh or frozen patient materials are not available.

Infection, dissemination, transmission, and biological attributes of dengue-2 PDK53 candidate vaccine virus after oral infection in Aedes aegypti.

The capacity for oral infection, dissemination, and transmission of the dengue-2 candidate vaccine virus DEN-2 PDK53 and an isolate from a vaccinate individual, DEN-2 Ia8, were compared with the parent strain DEN-2 16681. Capacity for oral infection and dissemination to the brain and salivary gland tissues were significantly lower in the first two than in the parent strain (P < 0.001).