The Quest for a Dengue Vaccine

Dengue fever is a viral disease and its prevalence has grown dramatically and globally in recent decades. Dengue fever is caused by the dengue virus (DENV), an arthropod-borne pathogen transmitted to vertebrates by infected mosquitos, which has evolved as four closely related viruses, DENV 1 to 4. Immunity from one serotype does not grant immunity against the others. In fact, the immunity arising from the initial infection might influence the disease outcome once infection is established by a second, distinct serotype, through a process referred to as a antibody-mediated disease enhancement.

The existence of four DENV serotypes has complicated vaccine design: an ideal dengue vaccine would confer balanced immune defense against all four serotypes, and incomplete protection against just one of these serotypes might increase the risk of severe dengue.

That is exactly what happened with a recent vaccine, Dengvaxia, which acted as a silent primary infection in seronegative individuals, creating an increased risk of severe dengue. With no specific drugs effective against DENV, prevention from insect bites remains the major defence, and an anti-DENV vaccine would represent a major advance in the control of the disease.

In their recent work, Shan et al. investigated the concept of using a virion-assembly defective DENV for the development of a new type of vaccine that, according to the authors, would be safe, effective, and stable.

Let’s dig into their work to find out.

The building of DENV

DENV is an enveloped, positive-sense RNA virus. Its ∼11 kb genome encodes a large polyprotein precursor of about 3400 amino acids, which is processed by viral and cellular proteases to give rise to three structural (C, prM, and E) and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. The structural proteins constitute the viral particle. The non-structural proteins carry out specific functions in viral assembly, viral RNA replication, and modulation of the host cell responses, all highly regulated and coordinated processes that are often shared among the members of the Flaviviridae family.

NS2A is a small trans-membrane protein, which is part of the replication complex and has been shown to be involved in virus assembly. In a previous study, this group had identified a series of mutations in the NS2A of DENV serotype 2 (DENV-2) producing distinct defects during the viral infection cycle. One set of NS2A mutations (D125A and G200A) selectively abolished viral RNA synthesis (through blocking the N-terminal cleavage of the NS2A protein, leading to an unprocessed NS1-NS2A protein). The other set of NS2A mutations (E100A, Q187A, and K188A) specifically impaired virion assembly and/or release, without significantly affecting viral RNA synthesis.

The modification of DENV

To minimize the risk of their engineered DENV reverting back to a more dangerous form, in this work the authors deleted those amino acids that they had previously identified as critical for virion assembly, thus generating mutants E100-del, Q187-del, and K188-del. By this approach, it was possible to generate a non-infectious virus, or so-called pseudoinfectious virus (PIV_NS2A).

Because of these mutations, PIV_NS2A virions should be unable to complete viral assembly, yet remain competent in viral RNA replication.

Indeed, when viral RNA synthesis was measured using a transient luciferase replicon, both wild-type and NS2A-deletion replicons generated equivalent luciferase signals at 48 h post transfection, demonstrating that the three deletions do not compromise viral RNA replication. Also, once the deletion-carrying RNA genomes were transfected into a human cell line (BHK-21 cells), the number of cells positive for the viral envelope (E-positive cells) failed to increase during the 4 days post-transfection, showing that the deletion-carrying viruses are unable to spread.

These data show that, as hypothesised, the authors’ engineered PIV- NS2A virions can initiate only a single-round infection in parental cells.

Such a virion holds much potential for vaccine development because would be safe (non-infectious beyond a single-round infection) yet present the whole virus (rather than just one or two antigens) to the target’s immune system. Below we look how Shan et al. test the potential of their K188-del PIV_NS2A as an anti-dengue vaccine.

Selection for adaptive mutations to improve K188-del PIV_NS2A yield

Achieving sufficient yield is an obstacle to producing an assembly-deficient virion as a vaccine. If your engineered virion is good for only a single-round infection, how to mass-produce it? Shan et al. overcome this obstacle by trans-complementation with exogenous wild-type NS2A protein: when the three deleted RNAs dengue genomes were individually transfected into BHK-21 cells that constitutively expressed wild-type NS2A protein, infectious PIV_NS2As were produced, as evidenced by an increasing number of viral E positive cells at day 4 post transfection.

Continuous culturing of K188-del PIV_NS2A in NS2A-expressing BHK-21 cells led to the accumulation of additional adaptive mutations in both structural and non-structural genes, leading to higher PIV_NS2A titers over 30 passages. The role of these adaptive mutations was tested in the context of the NS2A-K1888del RNA, to understand the advantage that they may confer to DENV. The mutations tested were in the prM, E, NS2A, NS2B, and NS3 genes and contributed to increased PIV production in NS2A-expressing cells; however, they could not rescue the assembly defects in parental BHK-21 cells.

So, having addressed the yield problem, can PIV_NS2A work as a vaccine? The authors tested this by immunizing mice by subcutaneous injection of PIV_NS2A. Mice immunized with PIV_NS2A produced high levels of neutralizing antibodies and were protected from virulent wild-type virus challenge.

Have Shan el al. developed an effective, safe, and stable vaccine approach?

Effective

The concept of engineering replication deficient viruses as vaccines has previously been explored for a number of flaviviruses. Defective PIVs have been made by removing a functional copy of the capsid gene from their genomes. Since these engineered viruses can still produce extracellular prM-E proteins in the form of secreted subviral particles, they can constitute effective immunogens. Compared with these defective PIVs, PIV_NS2A contains all intact structural and non-structural proteins and should resemble authentic wild-type viruses. The authors demonstrated the feasibility of generating high titers of PIV_NS2A. Thus, these defective flaviviruses could be produced in large scale under low biocontainment conditions.

Safe

In vitro and in vivo results demonstrate the safety of the PIV_NS2A vaccine: i) it is safer than the live attenuated vaccine because the infection is limited to a single round; ii) no infectious virus was recovered after continuous culturing of PIV_NS2A on NS2A expressing cells for over 4 months or after inoculation in mice.

Stable

Reversion of the K188-NS2A deletion was carefully monitored after passaging in cells and inoculation in mice and such a deletion was retained without secondary alterations. Rather, adaptive mutations accumulated in the k188del genomic RNA, leading to an improved PIV_NS2A titer without restoring assembly.

This formulation has the advantage of presenting to the immune system also DENV non-structural proteins, which have been shown to elicit a protective T cell response, although such a response was not tested in this study. Whether this would convey better protection then the current Dengvaxia formulation (where the NS proteins belong to yellow fever vaccine strain) needs to be clarified; however the issue of raising comparable levels of immunity against different serotypes to avoid antibody-dependent enhancement -currently the main problem of Dengvaxia- still needs to be addressed, even for attenuated viruses.

Although the authors demonstrated the safety and stability of their PIV_NS2A with in vitro and in vivo experiments, it remains sensible to proceed with extra caution when developing such close-to-wild-type viruses as vaccines, especially when replication is preserved, as RNA viruses are notorious for their ability to escape, recombine, and out-manoeuvre us.