Dengue virus: are we forgetting the genotypes?

With almost 400 million infections every year, the impact of dengue virus (DENV) on global health is of substantial concern. The presence of four co-circulating serotypes, and the development of non-neutralizing enhancing immunity following heterotypic infections is the main cause of exacerbated disease and also of vaccine inefficacy in naïve populations. However, the paradigm is that the immune response after the first encounter with DENV confers life-long protection against the same serotype.

Is this entirely correct?

The genotypes

DENV is an RNA virus, and as such new mutations occur during replication giving rise to genetically distinct genotypes within each serotype. Between different serotypes, the envelope (E) proteins (against which most of the antibody response is mounted) differ of about 70%, with some very conserved (e.g. the fusion loop) and some highly variable regions. Different genotypes are far less diverse than this, and are all supposed to be controlled by the same immune response; however few studies so far have investigated if this is really the case. Gallichotte and colleagues try to fill this gap by looking at the genetic diversity within serotype 4.

The genotypes within serotype 4

There are five genotypes within DENV-4, numbered I to V, with genotype II divided in IIa and IIb. Genotypes I and II are the most common. How does an antibody response against their slightly different E proteins compare?

To answer this question without mudding the water with unrelated differences in other viral proteins, Gallichotte and colleagues exploit the power of reverse genetics by engineering E proteins from different DENV-4 genotypes within the same DENV-4 backbone. Sequence analyses show that these proteins differ primarily in the EDIII region, as well as in the hinge region between EDI and EDII.

Any difference?

Growth

Differences between these viruses could already be observed in growth kinetics and foci morphology in the mammalian Vero cells, while more uniform replication was seen in the mosquito cell line C6/36. Genotype V stuck out, as it was the most attenuated in Vero cells, but the only one causing syncytia in C6/36, possibly suggesting adaptation for higher growth in mosquito.

Maturation

While no changes were observed in thermostability between different genotypes, the authors observed differences in the virus maturation state, with genotypes I and III being the least mature, and IV and V the most mature. During maturation, the viral precursor protein prM is cleaved by furin in the Golgi and the pr region is dissociated in the neutral pH of the extracellular environment after exocytosis. The efficiency of this process varies and strongly influences infectivity as well as enhancement This is because antibodies against prM are suggested to be poorly neutralizing and responsible for ADE. While infection with all viruses was enhances by anti-prM antibodies, no clear correlation with the different maturation states was observed. As the furin cleavage site was the same across the virus panel, and the aminoacids known to interact with pr were conserved, this suggests that E itself may affect virus maturation through residues that do not directly interact with pr, but still contribute stabilizing it.

Glycosylation

Interestingly, a difference in glycosylation was also observed for genotype V, as changes in Asn-153 (in EDI) disrupted the glycosylation motif both in the strain examined and in >87% of the genotype V viruses examined by phylogenetic analysis. This is a very interesting observation, as glycosylation is important for receptor binding and for antibody neutralization.

Binding and neutralization

Besides differences in infectivity, are these viruses recognized and neutralized by the same antibodies? This is an important question, as it may impact vaccine design.

Binding

DENV-4 serotype specific antibodies D4-126 and D4-131, which recognize partially overlapping epitopes in the EDI/EDII hinge, and D4-141, which recognizes an EDIII epitope, bound to all the genotypes tested in an ELISA.

Conversely, the non-human primate monoclonal 5H2, which binds to an epitope on EDI, displayed a higher binding variability, and did not bind genotype III, likely due to a polymorphism in residue 174.

The cross-reactive human monoclonal C10 and B7, which recognize envelope dimer epitopes (EDE) spanning across neighboring monomers, bound all genotype, with the exception of genotype V for B7, which requires a glycan in position 153.

Neutralization

As often observed, particularly for DENV, binding and neutralization did not necessarily follow the same pattern, and in general a difference of 1-2 logs in neutralization titres was observed across the panel. D4-126, for instance, which strongly bound genotype III, failed to neutralize the virus in a focus reduction neutralization test. Equally 5H2, which only weakly bound serotype V, effectively neutralized the same virus.

Do human sera neutralize different genotypes?

Natural infection

Sera from convalescent patients also showed variable neutralization titres, although all genotypes were neutralized. It is worth remembering that these are convalescent sera, so even higher variability may be observed several years after infection.

Monovalent vaccine

A larger spread in neutralization titres was observed in individuals vaccinated with a monovalent vaccine based on genotype II of DENV-4. As expected, genotype II was the genotype neutralized more strongly, but some samples failed to neutralize different genotypes. The difference in neutralization titres was not due to cross-reactive antibodies, as minimal changes were observed after their depletion.

Tetravalent vaccine

Similar results were seen from subjects immunized with the tetravalent Dengvaxia, which is known to induce a strong antibody response against the immunizing genotype II of DENV-4. Vaccine-matched genotype II was neutralized between 3 and 20 folds more effectively than other genotypes, and some samples failed to neutralize circulating serotypes I and III.

Together these observations suggest differences in the immune response generated against different genotypes, with stronger response against the immunizing genotype and variable (or even zero) protection against the other.

Outlook

What are the implications of this study? Presumably that the differences between genotypes should not be totally ignored in vaccine design, or when testing the neutralizing titres of antibodies. Infection after re-exposure to the same serotype or after vaccination is a fairly rare occurrence, and presumably when talking of DENV many other more pressing issues come to mind first. However, so far the immunological implications of exposure to different genotypes have been very poorly explored, and it is not at all clear what level of intra-serotype divergence could escape protection or compromised vaccine efficacy/safety.

This study calls for further studies into this genetic variability and its impact in a population context, but also for the inclusion of larger and more representative samples of viruses in neutralization panels, as already recommended by the WHO. In the race to make a DENV vaccine more effective and safer, it seems that the more complexity we are ready to embrace, the more chances of success we may have.