The increased activity of Dengue virus in the tropical regions of the world and the recent movement of West Nile virus from the eastern to the western hemisphere emphasize the fact that vector-borne flaviviruses are medically important emerging infectious diseases These facts warrant continued efforts to decode all facets of flavivirus immunology This chapter reviews current understanding of the antigenic fine structure of flaviviral structural and nonstructural (NS) proteins and their involvement in B- and T-cell host responses The virion structural glycoprotein E elicits both virus-neutralizing antibodies and antiviral TH-cell responses Consistent with the current hypothesis of the MHC class I pathway of protein processing, immunodominant flaviviral TC-cell epitopes mainly reside on the NS proteins To prepare effective and inexpensive subunit vaccines, we will need to continue to better understand these structure–function relationships of flavivirus proteins

Antigenic structure of flavivirus proteins.

Antibody-mediated neutralization of flaviviruses: a reductionist view.

Anti-dengue 2 virus core protein monoclonal antibodies (MAbs) reacted with antigens in the cytoplasm and in, or on, the nucleus of dengue 2 and dengue 4, but not dengue 1, dengue 3, Kunjin or Murray Valley encephalitis virus-infected cells. These MAbs also reacted with the core protein from dengue 1, 2 and 4 virions in Western blots. The antigens detected by these MAbs could not be detected in uninfected or heat-shocked cells, but were first detected in infected cells approximately 32 h post-infection. PEPSCAN epitope mapping suggested that all the MAbs react with a region of the dengue 2 virus core protein (9RNTPFNMLKRE19) which is adjacent to a putative nuclear localization sequence (6KKAR9) and spans a possible second site for the initiation of synthesis of core protein (12PFN↓MLKR18).

http://vir.sgmjournals.org/content/73/11/2999.full.pdf

Nuclear localization of dengue 2 virus core protein detected with monoclonal antibodies.

In a step towards a tetravalent dengue virus subunit vaccine which is economical to produce, highly immunogenic and stable, a hybrid dengue virus envelope (E) protein molecule has been constructed. It consists of 36 amino acids from the membrane protein, the N-terminal 288 amino acids of the dengue-2 virus E protein plus amino acids 289-424 of the dengue-3 virus E protein. It has been engineered for secretory expression by fusion to a mellitin secretory signal sequence and truncation of the hydrophobic transmembrane segment. Using the baculovirus expression system and serum-free conditions, more than 95% of recombinant dengue-2 virus-dengue-3 virus hybrid E protein (rD2D3E) was secreted into the cell culture supernatant in a stable form with multiple features indicative of preserved conformation. The hybrid molecule reacted with a panel of dengue virus- and flavivirus-specific MAbs which recognize linear or conformational epitopes on dengue virions. Human dengue virus-specific antisera also reacted with the protein. The hybrid rD2D3E protein was able to inhibit the in vitro binding of dengue-2 and dengue-3 viruses to human myelomonocytic cells, suggesting that the receptor-binding epitope(s) was preserved. Adjuvant-free immunization with the hybrid protein induced an antibody response to both dengue-2 and dengue-3 virus in outbred mice, comparable in strength to that of individual rD2E and rD3E proteins. Notably, these antibody responses were primarily of the IgG2a and IgG2b isotype. A strong dengue virus cross-reactive T cell response was also induced in the mice, suggesting that dengue virus hybrid E proteins could form the basis of an efficacious multivalent dengue virus vaccine.

Analysis of a recombinant dengue-2 virus dengue-3 virus hybrid envelope protein expressed in a secretory baculovirus system

The importance of viral structural and non-structural antigens in flavivirus pathogenesis has not been satisfactorily assessed. This review examines the antigenic interrelationships of the flaviviruses, considers some of the implications of antigenic cross reactivity in virus virulence and highlights potential consequences of virus-antibody interactions.

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Antigenicity of flaviviruses

Mice immunized with DEN-1 pre-matrix protein produced antibody which reacted with dengue virus infected cells and intact virions but failed to neutralize virus in vitro or in vivo. Newborn mice passively immunized with anti-DEN-1 pre-matrix antibody also failed to survive intracerebral infection with 100 LD50 of any dengue virus serotype.

Jennifer Fletcher

Papers

Failure of a dengue 1 sub-unit vaccine to protect mice against a lethal dengue virus infection.