Fusion Loop Modified and Mature Dengue Virus Elicits Protective Serum with Minimal Antibody Dependent Enhancement

Reviewer #1 (Public review):

Summary:

Dalben et al. grafted the fusion loop mature (FLM) modification, based on a previously reported D2-FLM, to another serotype DENV4, and adapted them to replicate in Vero cells for live attenuated vaccine (LAV) manufacturing while retaining favorable antigenic profiles, generating two new strains: D2-vFLM and D4-vFLM. Deep sequencing revealed adapted mutations at the junction of envelope domains I and II (EDI and EDII), and both D2-vFLM and D4-vFLM showed no evidence of ADE in the presence of FL-targeting Abs. Sera from D2-vFLM immunized mice displayed strong homotypic and reduced heterotypic neutralization compared to wild-type viruses, with minimal to no ADE potential in vitro. Moreover, D2-vFLM immunization completely protected AG129 mice from lethal challenge with mouse-adapted D220. They demonstrate that the FLM modification platform is transferable across serotypes and yields strains with favorable immunogenicity and reduced ADE risk. The FLM approach provides a promising path toward the development of a safer tetravalent DENV LAV.

Strengths:

The authors carried out a series of experiments to generate and characterize two new strains (D2-vFLM and D4-vFLM) of FLM-modified viruses, and showed their antigenic and immunogenic profiles. The observation that the FLM modification platform is transferable across serotypes and yields strains with favorable immunogenicity and reduced ADE risk is interesting.

Weaknesses:

However, one concern is the total number of mutations (including originally introduced and compensatory mutations) in this FLM vaccine platform, and it is not clear regarding the future directions for the proof-of-concept vaccine in this study.

Reviewer #2 (Public review):

Summary:

In this manuscript, YR Dalben et al describe the generation of DENV2 and DENV4 strains with mutations in the fusion loop (FL) of the E protein and pre-membrane (prM) protein to limit potential antibody-dependent enhancement (ADE) resulting from vaccination with live-attenuated vaccines and adapted these strains for growth in Vero cells. They show that the DENV2 version D2-vFLM is immunogenic and generates neutralizing serum against DENV2 and DENV4 after 2 boosts and is protective against lethal challenge. Serum from D2-vFLM also showed no ADE against DENV4.

Strengths:

Overall, the paper is well written and presented, and the data presented support most of the conclusions made. Grafting D2-FLM mutations to DENV4 and adapting both to growth in Vero cells is a good step to show that this method could be used to generate production-level LAV. The growth and stability data are clear and well-conducted.

Weaknesses:

However, there are several weaknesses, mostly in regard to the immunogenicity data, that limit the overall impact. The FLM mutations were only grafted to DENV4 but not to the other Dengue serotypes. The authors acknowledge that this is a proof-of-concept, but generating mutants of the other serotypes would strengthen the idea that this could be used to develop a tetravalent LAV. Immunizations in mice were only performed for D2-vFLM but not D4-vFLM. Immunogenicity data for D4-vFLM would strengthen this work if it shows that it can be immunogenic, protective, and limit ADE, as is shown for D2-vFLM. ADE from D2-vFLM was only tested against DENV4; does it also limit ADE from the other serotypes? This would better show that these mutations do limit ADE across serotypes and not just a single one.

Additionally, some of the immunization data likely need to be repeated:

The authors should describe why they pooled the sera from the mice and whether they purified total IgG or not (Figure 5). They should also probably repeat the challenge experiment since it was 4 mice (D2) against 5 (D2-vFLM), and it is unclear if there is a statistical difference between the results obtained. It is not even mentioned in the Results section (D2 result vs D2-FLM), and thus unclear if using D2-FLM is an improvement in the way the data is currently presented.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

Dalben et al. grafted the fusion loop mature (FLM) modification, based on a previously reported D2-FLM, to another serotype DENV4, and adapted them to replicate in Vero cells for live attenuated vaccine (LAV) manufacturing while retaining favorable antigenic profiles, generating two new strains: D2-vFLM and D4-vFLM. Deep sequencing revealed adapted mutations at the junction of envelope domains I and II (EDI and EDII), and both D2-vFLM and D4-vFLM showed no evidence of ADE in the presence of FL-targeting Abs. Sera from D2-vFLM immunized mice displayed strong homotypic and reduced heterotypic neutralization compared to wild-type viruses, with minimal to no ADE potential in vitro. Moreover, D2-vFLM immunization completely protected AG129 mice from lethal challenge with mouse-adapted D220. They demonstrate that the FLM modification platform is transferable across serotypes and yields strains with favorable immunogenicity and reduced ADE risk. The FLM approach provides a promising path toward the development of a safer tetravalent DENV LAV.

Strengths:

The authors carried out a series of experiments to generate and characterize two new strains (D2-vFLM and D4-vFLM) of FLM-modified viruses, and showed their antigenic and immunogenic profiles. The observation that the FLM modification platform is transferable across serotypes and yields strains with favorable immunogenicity and reduced ADE risk is interesting.

We thank reviewer 1 for the encouraging comments for our work.

Weaknesses:

However, one concern is the total number of mutations (including originally introduced and compensatory mutations) in this FLM vaccine platform, and it is not clear regarding the future directions for the proof-of-concept vaccine in this study.

Author response table 1.

We summarize the mutations in the FLM platform below.

The maturation mutations are located at the furin cleavage site, which is buried within the membrane or virion. As a result, only five mutations are surface exposed, two of which are in the fusion loop region targeted for removal. Therefore, for a proof-of-concept study, the total number of mutations remains well within the genetic diversity observed among DENV genotypes.

Compensatory mutations may affect overall DENV antigenicity. Notably, one such mutation, K204R, has been reported to alter antigenicity and could contribute to the improved safety profile of the vaccine. However, we have also shown that multiple adaptive pathways can support Vero cell adaptation, and our data indicate that K204R is not absolutely required for this process.

Reviewer #2 (Public review):

Summary:

In this manuscript, YR Dalben et al describe the generation of DENV2 and DENV4 strains with mutations in the fusion loop (FL) of the E protein and pre-membrane (prM) protein to limit potential antibody-dependent enhancement (ADE) resulting from vaccination with live-attenuated vaccines and adapted these strains for growth in Vero cells. They show that the DENV2 version D2-vFLM is immunogenic and generates neutralizing serum against DENV2 and DENV4 after 2 boosts and is protective against lethal challenge. Serum from D2-vFLM also showed no ADE against DENV4.

Strengths:

Overall, the paper is well written and presented, and the data presented support most of the conclusions made. Grafting D2-FLM mutations to DENV4 and adapting both to growth in Vero cells is a good step to show that this method could be used to generate production-level LAV. The growth and stability data are clear and well-conducted.

We thank reviewer 2 for the encouraging comments for our work.

Weaknesses:

However, there are several weaknesses, mostly in regard to the immunogenicity data, that limit the overall impact. The FLM mutations were only grafted to DENV4 but not to the other Dengue serotypes. The authors acknowledge that this is a proof-of-concept, but generating mutants of the other serotypes would strengthen the idea that this could be used to develop a tetravalent LAV.

We selected DENV2 and DENV4 because they are the most genetically divergent. Currently, our data should support the FLM mutations that can be grafted on both DENV2 and DENV4, likely extend to their corresponding genotypes. We agree that the FLM mutations should be evaluated in additional serotypes. We also have promising preliminary data for FLM mutation grafting in DENV1 and are currently applying the same approach to DENV3. We hope to include these results, whether positive or negative, in the revised manuscript.

Immunizations in mice were only performed for D2-vFLM but not D4-vFLM. Immunogenicity data for D4-vFLM would strengthen this work if it shows that it can be immunogenic, protective, and limit ADE, as is shown for D2-vFLM.

We are currently immunizing AG129 mice with DV4 and D4-vFLM, followed by heterotypic challenge with D220. Because DENV vaccine-related hospitalization in clinical trials typically occurs 3 - 4 years after vaccination, we are cautious about whether this experimental design will fully capture the added safety benefit of the FLM mutations. We are also developing a passive immunization model in AG129 mice using diluted DENV4 serum to better mimic long-term waning antibody titers. We will include the future findings in the revised manuscript.

ADE from D2-vFLM was only tested against DENV4; does it also limit ADE from the other serotypes? This would better show that these mutations do limit ADE across serotypes and not just a single one.

We are trying to keep the scope of the paper within DENV2 and DENV4, however, we will perform ADE and neutralization assays for all four serotypes in the revised manuscript.

Additionally, some of the immunization data likely need to be repeated:

The authors should describe why they pooled the sera from the mice and whether they purified total IgG or not (Figure 5).

We used pooled serum, consisting of equal volumes from each mouse, rather than purified IgG. In Figure 5, our goal was to show the overall increase in serum titer after each immunization using cheek-bleed samples from individual animals. Because the available sample volume was limited, we pooled the sera for this analysis. We also measured end-point serum titers for each individual animal.

They should also probably repeat the challenge experiment since it was 4 mice (D2) against 5 (D2-vFLM), and it is unclear if there is a statistical difference between the results obtained. It is not even mentioned in the Results section (D2 result vs D2-FLM), and thus unclear if using D2-FLM is an improvement in the way the data is currently presented.

This experiment was designed to determine whether D2-vFLM protects AG129 mice against homotypic challenge as effectively as DV2-WT. Although the sample size was small, the results support our conclusion. However, we agree with the reviewer that the study should include more animals, and we will increase the group size to n > 8 to 10 in the revised experiment.