Mapping the neutralizing antibody response to natural dengue virus infection

Dengue virus (DENV) is the most common mosquito-borne human viral disease worldwide, and infection with any of the four serotypes (DENV1-4) can result in a range of disease outcomes, from inapparent infection to classical dengue fever (DF) to the most severe forms, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The four DENV serotypes can cause repeat infections in a single individual, each time with a new serotype. While primary infection generates what is thought to be life-long neutralizing antibodies to the infecting serotype, secondary DENV infections elicit cross-neutralizing antibodies to two or more serotypes, as well as type-specific antibodies to the infecting serotype. While natural infection studies have provided valuable insights into the immune response to DENV infection, the complex antibody response needs further elucidation, especially in the light of recent vaccine trial results. In this dissertation, the functional properties and epitope specificity of the antibody response in primary and secondary natural DENV infections are explored./nRecent advances in the characterization of human antibody responses to primary DENV infection indicate that highly neutralizing type-specific antibodies target quaternary epitopes on the DENV envelope protein. Among such type-specific antibodies is 5J7, a human monoclonal antibody (hmAb) isolated from a traveler infected with DENV3. Using two recombinant chimeric DENV4 viruses containing differently sized transplants of the 5J7 epitope, we measured the levels and kinetics of neutralizing antibodies targeting this epitope in post-primary DENV3 infection plasma in a large number of individuals in Nicaragua. We found that DENV3 neutralizing antibodies in post-primary DENV3 infections track with the 5J7 epitope to varying degrees and can be detected even years after infection. Some individuals, however, presented little to no recognition of this epitope even in the expanded transplant version, which suggested the existence of additional epitopes within the DENV3 repertoire that required further investigation. In addition to measuring the prevalence of antibodies directed to the 5J7 epitope in a population in a dengue-endemic area, this study validated and helped define the footprint of a quaternary highly neutralizing epitope. Having demonstrated that chimeric viruses are a powerful tool for mapping type-specific neutralizing antibody responses, we carried out a similar approach to measure type-specific neutralizing antibody responses to the epitope bound by 1F4, a DENV1 type-specific hmAb. This time, we analyzed post-primary DENV1 infection antibody response in individuals from two endemic regions -- Asia and the Americas -- where different genotypes of DENV1 are circulating. The 1F4 epitope was prevalent in both areas, although a higher proportion was found in the Asian population, supporting the hypothesis that intra-serotype diversity can affect neutralization potential. Our second finding was that both populations presented a bimodal pattern of recognition of the 1F4 epitope, suggesting that additional epitopes within the DENV1 repertoire may contribute to type-specific neutralization of the DENV1 serotype./nAfter a secondary DENV infection, the neutralizing antibody response gains breadth, and potent broadly neutralizing antibodies may be elicited. In an attempt to investigate the molecular pathways that generate broadly neutralizing antibodies, together with collaborators at Vanderbilt University, we isolated and sequenced hmAbs from EBV-transformed PBMCs collected after secondary DENV infection. Subsequently, we performed deep sequencing of the immunoglobulin (Ig) genes of B cells in matched PBMCs after the first DENV infection of the same individual at time-points prior to the second infection. Although the limited sample size from our pediatric subjects did not allow the necessary depth for tracing the clonal lineages of broadly neutralizing antibodies, we gained substantial knowledge about the molecular and functional properties of hmAbs isolated from memory B cells (MBC) after secondary DENV infection. In our panel of hmAbs, we observed a predominance of serotype cross-neutralizing and somatically hypermutated clones. In fact, we observed a higher degree of somatic hypermutation (SHM) in cross-reactive clones compared to type-specific clones. Notably, we identified a number of potent DENV3 type-specific neutralizing hmAbs isolated after secondary DENV infection. In accordance with our previous study that predicted additional epitopes within the DENV3 repertoire, we mapped three additional novel antigenic sites for DENV3 neutralization, as well as one epitope that overlapped with the already characterized 5J7 site. Strikingly, the sequence of infecting serotypes may modulate the epitope and potency of DENV3 type-specific hmAbs. Moreover, the newly identified DENV3 hmAbs are protective against DENV3 challenge in a mouse model of DENV infection./nAs an increasing number of natural infection and immunization studies underscore the importance of the quality as well as the quantity of the antibody response, we investigated the contribution of type-specific versus cross-reactive antibodies to polyclonal neutralization after a first, second and third DENV infection in the same individual. Through depletion of antibody subpopulations in collaboration with collaborators at the University of North Carolina at Chapel Hill, we concluded that after the first infection, type-specific antibody populations drive a substantial proportion of the polyclonal neutralization. Following second and third infections, the cross-reactive populations dominate, although type-specific populations to each of the infecting serotypes can still be detected. Strikingly, in one case, we observed an increase in the proportion of the type-specific antibody population to the first infecting serotype and no evidence of a type-specific response to another serotype in a subsequent inapparent infection, suggesting homotypic reinfection. Depletion of antibody subpopulations enables detection of non-typical DENV infections and assessment of the frequency of heterotypic infections, homotypic reinfections and heterologous boosts, providing critical information regarding the epidemiology and long-term immunity to DENV./nTaken together, the work included in this dissertation has made significant contributions to the field’s efforts to decipher the specificity and functional properties of the neutralizing antibody response following primary and secondary natural DENV infections.

Advisor: Harris, Eva