Sculpting humoral immunity through dengue vaccination to enhance protective immunity.

Dengue viruses (DENV) are the most important mosquito transmitted viral pathogens infecting humans. DENV infection produces a spectrum of disease, most commonly causing a self-limiting flu-like illness known as dengue fever; yet with increased frequency, manifesting as life-threatening dengue hemorrhagic fever (DHF). Waning cross-protective immunity from any of the four dengue serotypes may enhance subsequent infection with another heterologous serotype to increase the probability of DHF. Decades of effort to develop dengue vaccines are reaching the finishing line with multiple candidates in clinical trials. Nevertheless, concerns remain that imbalanced immunity, due to the prolonged prime-boost schedules currently used in clinical trials, could leave some vaccinees temporarily unprotected or with increased susceptibility to enhanced disease. Here we develop a DENV serotype 1 (DENV-1) DNA vaccine with the immunodominant cross-reactive B cell epitopes associated with immune enhancement removed. We compare wild-type (WT) with this cross-reactivity reduced (CRR) vaccine and demonstrate that both vaccines are equally protective against lethal homologous DENV-1 challenge. Under conditions mimicking natural exposure prior to acquiring protective immunity, WT vaccinated mice enhanced a normally sub-lethal heterologous DENV-2 infection resulting in DHF-like disease and 95% mortality in AG129 mice. However, CRR vaccinated mice exhibited redirected serotype-specific and protective immunity, and significantly reduced morbidity and mortality not differing from naїve mice. Thus, we demonstrate in an in vivo DENV disease model, that non-protective vaccine-induced immunity can prime vaccinees for enhanced DHF-like disease and that CRR DNA immunization significantly reduces this potential vaccine safety concern. The sculpting of immune memory by the modified vaccine and resulting redirection of humoral immunity provide insight into DENV vaccine-induced immune responses.

A detailed mutagenesis study of flavivirus cross-reactive epitopes using West Nile virus-like particles.

Human flavivirus infections elicit virus species-specific and cross-reactive immune responses. The flavivirus envelope (E) glycoprotein is the primary antigen inducing protective immunity; however, the presence of cross-reactive antibodies in human sera creates problems for serodiagnosis. Using a West Nile virus-like particle system, we performed mutagenesis across all three E protein functional domains to identify epitope determinants for a panel of monoclonal antibodies (mAbs) raised against different flaviviruses and exhibiting diverse patterns of cross-reactivity. Residues within the highly conserved fusion peptide were the only epitope determinants identified and were important not only for broadly cross-reactive mAbs recognizing all of the medically important flavivirus serocomplexes, but also for less-broad, complex-reactive mAbs. Moreover, different substitutions at specific fusion peptide residues produced highly variable effects on antibody reactivity and virus-like particle secretion. These results support and extend the conclusion that the fusion peptide region constitutes an immunodominant epitope stimulating antibodies with diverse patterns of cross-reactivity.

Mutation analysis of the fusion domain region of St. Louis encephalitis virus envelope protein.

The immune response to flavivirus infections produces both species-specific and flavivirus cross-reactive antibodies. The presence of cross-reactive antibodies complicates serodiagnosis of flavivirus infections, especially secondary infections caused by a heterologous virus. A successful public health response to the growing global threat posed by flaviviruses necessitates the development of virus-specific diagnostic antigens. The flavivirus envelope (E) glycoprotein is the principle antigen stimulating protective immunity during infection. Using recombinant St. Louis encephalitis virus-like particles (VLPs), we have identified amino acid residues involved in flavivirus cross-reactive epitope determinants. Most significant among the residues studied are three highly conserved amino acids in the fusion peptide: Gly104, Gly106, and Leu107. Substitutions of these residues dramatically influenced VLP secretion and cross-reactive monoclonal antibody reactivity. These results provide critical insight into the antigenic structure of the flaviviral E protein and toward development of species-specific diagnostic antigens that should improve both flavivirus diagnosis and estimates of disease burden.