A heterologous prime-boost strategy for immunization against Dengue virus combining the Tetra DIIIC subunit vaccine candidate with the TV005 live-attenuated tetravalent vaccine.

The development of live-attenuated vaccines against Dengue virus (DENV) has been problematic. Dengvaxia, licensed in several countries where DENV is endemic, has shown low efficacy profiles and there are safety concerns prohibiting its administration to children younger than 9 years old, and the live-attenuated tetravalent vaccine (LATV) developed by NIAID has proven too reactogenic during clinical trialing. In this work we examined whether the combination of TV005, a LATV-derived formulation, with Tetra DIIIC, a subunit vaccine candidate based on fusion proteins derived from structural proteins from all four DENV serotypes, can overcome the respective limitations of these two vaccine approaches.

A Tetravalent Formulation Based on Recombinant Nucleocapsid-like Particles from Dengue Viruses Induces a Functional Immune Response in Mice and Monkeys.

Despite the considerable effort that has been invested in elucidating the mechanisms of protection and immunopathogenesis associated with dengue virus infections, a reliable correlate of protection against the disease remains to be found. Neutralizing Abs, long considered the prime component of a protective response, can exacerbate disease severity when present at subprotective levels, and a growing body of data is challenging the notion that their titers are positively correlated with disease protection. Consequently, the protective role of cell-mediated immunity in the control of dengue infections has begun to be studied.

Dengue infection.

Dengue is widespread throughout the tropics and local spatial variation in dengue virus transmission is strongly influenced by rainfall, temperature, urbanization and distribution of the principal mosquito vector Aedes aegypti. Currently, endemic dengue virus transmission is reported in the Eastern Mediterranean, American, South-East Asian, Western Pacific and African regions, whereas sporadic local transmission has been reported in Europe and the United States as the result of virus introduction to areas where Ae. aegypti and Aedes albopictus, a secondary vector, occur.

Dengue encephalitis-associated immunopathology in the mouse model: Implications for vaccine developers and antigens inducer of cellular immune response.

Despite the many efforts made by the scientific community in the development of vaccine candidates against dengue virus (DENV), no vaccine has been licensed up to date. Although the immunopathogenesis associated to the disease is a key factor to take into account by vaccine developers, the lack of animal models that reproduce the clinical signs of the disease has hampered the vaccine progress. Non-human primates support viral replication, but they are very expensive and do not show signs of disease.

A novel tetravalent formulation combining the four aggregated domain III-capsid proteins from dengue viruses induces a functional immune response in mice and monkeys.

Our group developed a subunit vaccine candidate against dengue virus based on two different viral regions: the domain III of the envelope protein and the capsid protein. The novel chimeric protein from dengue-2 virus [domain III-capsid (DIIIC-2)], when presented as aggregated incorporating oligodeoxynucleotides, induced anti-viral and neutralizing antibodies, a cellular immune response and conferred significant protection to mice and monkeys. The remaining constructs were already obtained and properly characterized.

The protein DIIIC-2, aggregated with a specific oligodeoxynucleotide and adjuvanted in alum, protects mice and monkeys against DENV-2.

Previously, we reported the ability of the chimeric protein DIIIC-2 (domain III of the dengue envelope protein fused to the capsid protein of dengue-2 virus), to induce immunity and protection in mice, when it is highly aggregated with a non-defined oligodeoxynucleotide (ODN) and adjuvanted in alum. In this work, three different defined ODNs were studied as aggregating agents. Our results suggest that the nature of the ODN influences the capacity of protein DIIIC-2 to activate cell-mediated immunity in mice.

Capsid protein: evidences about the partial protective role of neutralizing antibody-independent immunity against dengue in monkeys.

The role of cellular immune response in dengue virus infection is not yet fully understood. Only few studies in murine models propose that CD8(+) T-cells are associated with protection from infection and disease. At the light of recent reports about the protective role of CD8(+) T-cells in humans and the no correlation between neutralizing antibodies and protection observed in several studies, a vaccine based on cell-mediated immunity constitute an attractive approach.

Recombinant dengue 2 virus NS3 protein conserves structural antigenic and immunological properties relevant for dengue vaccine design.

The NS3 protein is a multifunctional non-structural protein of flaviviruses implicated in the polyprotein processing. The predominance of cytotoxic T cell lymphocytes epitopes on the NS3 protein suggests a protective role of this protein in limiting virus replication. In this work, we studied the antigenicity and immunogenicity of a recombinant NS3 protein of the Dengue virus 2.

A tetravalent dengue vaccine containing a mix of domain III-P64k and domain III-capsid proteins induces a protective response in mice.

Recombinant fusion proteins containing domain III of the dengue virus envelope protein fused to the P64k protein from Neisseria meningitidis and domain III of dengue virus type 2 (D2) fused to the capsid protein of this serotype were immunogenic and conferred protection in mice against lethal challenge, as reported previously. Combining the domain III-P64k recombinant proteins of dengue virus types 1, 3 and 4 (D1, D3, and D4) with the domain III-capsid protein from D2, we obtained a novel tetravalent formulation containing different antigens. Here, the IgG and neutralizing antibody response, the cellular immune response, and the protective capacity against lethal challenge in mice immunized with this tetravalent formulation were evaluated.

Evaluation in mice of the immunogenicity and protective efficacy of a tetravalent subunit vaccine candidate against dengue virus.

A dengue vaccine must induce protective immunity against the four serotypes of the virus. Our group has developed chimeric proteins consisting of the protein P64k from Neisseria meningitidis and the domain III from the four viral envelope proteins. In this study, the immunogenicity of a tetravalent vaccine formulation using aluminum hydroxide as adjuvant was evaluated in mice.

Purified and highly aggregated chimeric protein DIIIC-2 induces a functional immune response in mice against dengue 2 virus.

It was previously reported that DIIIC-2 (a fusion protein composed of domain III of the envelope protein and the capsid protein from dengue 2 virus), as an aggregate antigen from a partially purified preparation, induced a functional protective immune response against dengue 2 virus in the mouse encephalitis model. In the present work, a purification procedure was developed for DIIIC-2, and soluble and aggregated fractions of the purified protein were characterized and evaluated in mice. The purification process rendered a protein preparation of 91 % purity, and the remaining 9 % consisted of fragments and aggregates of the same recombinant protein.

Serotype specificity of recombinant fusion protein containing domain III and capsid protein of dengue virus 2.

Recombinant fusion protein containing domain III of the dengue envelope protein fused to capsid protein from dengue 2 virus was immunogenic and conferred protection in mice against lethal challenge in previously report. Here, the antigenic specificity of this recombinant protein using anti-dengue antibodies from mice and humans and the cross-reactive humoral and cellular response induced in immunized mice were evaluated. The homologous anti-dengue antibodies showed a higher reactivity to the recombinant protein compared to the wide cross-reactivity observed for viral antigen as determined by ELISA.

Recombinant nucleocapsid-like particles from dengue-2 induce functional serotype-specific cell-mediated immunity in mice.

The interplay of different inflammatory cytokines induced during dengue virus infection plays a role in either protection or increased disease severity. In this sense, vaccine strategies incorporating whole virus are able to elicit both functional and pathological responses. Therefore, an ideal tetravalent vaccine candidate against dengue should be focused on serotype-specific sequences.

Long-term persistence of clinical symptoms in dengue-infected persons and its association with immunological disorders.

Objectives: The acute manifestations of dengue are well known. The clinical symptoms that present during the convalescent phase of infection are less well characterized, but may be autoimmune-based. This study was undertaken to determine the prevalence of persistent clinical symptoms among individuals infected during the 2006 Cuban epidemic and to evaluate the immunological and genetic factors associated with their occurrence.

Asymptomatic dengue infection in a Cuban population confirms the protective role of the RR variant of the FcgammaRIIa polymorphism.

The role of human Fcgamma receptors (FcgammaR) has been recognized considerably over the last years. These receptors vary in their affinity for IgG subclasses and the intracellular signals elicited by them. Allelic variants of FcgammaR genes may influence the biological phagocyte activity, accounting for an inherited pre-disposition to disease.

Serotype-specificity of recombinant fusion proteins containing domain III of dengue virus.

Here, the antigenic specificity of the recombinant fusion proteins containing aa 286-426 of the dengue envelope protein fused to P64k from Neisseria meningitidis and the cross-reactive antibody response induced in immunized mice and monkeys were evaluated. The anti-dengue mice antibodies showed a higher reactivity to the homologous recombinant proteins compared to the wide cross-reactivity observed by dot blot to the viral antigens. The immune response induced by the recombinant proteins in mice and monkeys, was highly serotype specific.

Immunogenicity and protective efficacy of a recombinant fusion protein containing the domain III of the dengue 1 envelope protein in non-human primates.

Recombinant fusion proteins containing the aa 286-426 of the dengue envelope protein fused to P64k protein from Neisseria meningitidis have been previously reported. Particularly, the immunogenicity and protective capacity of the dengue 2 recombinant protein was demonstrated in Macaca fascicularis monkeys. Here we evaluate the recombinant fusion protein containing the domain III of the dengue 1 envelope protein (PD10) in non-human primates (M.

Primary and secondary infections of Macaca fascicularis monkeys with Asian and American genotypes of dengue virus 2.

The goal of this study was to compare the immune response and the protection capacity induced by the dengue virus 2 (DENV-2) American and Asian genotypes in Macaca fascicularis monkeys. Animals were infected with American or Asian DENV-2 strains and challenged 1 year later with a DENV-2 Asian genotype strain. The viremia and monkey antibody levels were similar for the different strains after primary and secondary infection; however, the functionality of the antibody response was different.