An adenovirus prime/plasmid boost strategy for induction of equipotent immune responses to two dengue virus serotypes.

BACKGROUND: Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. It is a mosquito-borne viral disease, caused by dengue (DEN) viruses, which are members of the Flaviviridae family. There are four closely related serotypes, DEN-1, DEN-2, DEN-3 and DEN-4, each of which is capable of causing disease. As immunity to any one serotype can potentially sensitize an individual to severe disease during exposure to a heterologous serotype, the general consensus is that an effective vaccine should be tetravalent, that is, it must be capable of affording protection against all four serotypes. The current strategy of creating tetravalent vaccine formulations by mixing together four monovalent live attenuated vaccine viruses has revealed the phenomenon of viral interference leading to the manifestation of immune responses biased towards a single serotype. RESULTS: This work stems from the emergence of (i) the DEN virus envelope (E) domain III (EDIII) as the most important region of the molecule from a vaccine perspective and (ii) the adenovirus (Ad) as a promising vaccine vector platform. We describe the construction of a recombinant, replication-defective Ad (rAd) vector encoding a chimeric antigen made of in-frame linked EDIIIs of DEN virus serotypes 2 and 4. Using this rAd vector, in conjunction with a plasmid vector encoding the same chimeric bivalent antigen, in a prime-boost strategy, we show that it is possible to elicit equipotent neutralizing and T cell responses specific to both DEN serotypes 2 and 4. CONCLUSION: Our data support the hypothesis that a DEN vaccine targeting more than one serotype may be based on a single DNA-based vector to circumvent viral interference. This work lays the foundation for developing a single Ad vector encoding EDIIIs of all four DEN serotypes to evoke a balanced immune response against each one of them. Thus, this work has implications for the development of safe and effective tetravalent dengue vaccines.

Induction of neutralizing antibodies specific to dengue virus serotypes 2 and 4 by a bivalent antigen composed of linked envelope domains III of these two serotypes.

There is no vaccine to prevent dengue fever, a mosquito-borne viral disease, caused by four serotypes of dengue viruses. In this study, which has been prompted by the emergence of dengue virus envelope domain III as a promising sub-unit vaccine candidate, we have examined the possibility of developing a chimeric bivalent antigen with the potential to elicit neutralizing antibodies against two serotypes simultaneously. We created a chimeric dengue antigen by splicing envelope domain IIIs of serotypes 2 and 4. It was expressed in Escherichia coli and purified to near homogeneity. This protein retains the antigenic identities of both its precursors. It elicited antibodies that could efficiently block host cell binding of both serotypes 2 and 4 of dengue virus and neutralize their infectivity (neutralizing antibody titers approximately 1:40 and ~1:80 for dengue virus serotypes 2 and 4, respectively). This work could be a forerunner to the development of a single envelope domain III-based tetravalent antigen.

An adenovirus type 5 (AdV5) vector encoding an envelope domain III-based tetravalent antigen elicits immune responses against all four dengue viruses in the presence of prior AdV5 immunity.

Dengue is a mosquito-borne viral disease caused by four antigenically distinct serotypes of dengue viruses (DENVs). This disease, which is prevalent in over a hundred tropical and sub-tropical countries of the world, represents a significant global public health problem. A tetravalent dengue vaccine capable of protecting against all four DENV serotypes has been elusive so far. Current efforts are focused on producing a tetravalent vaccine by mixing four monovalent vaccine components. In this work, we have utilized a discrete carboxy-terminal region of the major DENV envelope (E) protein, known as domain III (EDIII), which mediates virus entry into target cells and contains multiple serotype-specific neutralizing epitopes, to create a chimeric tetravalent antigen. This antigen derived by in-frame fusion of the EDIII-encoding sequences of the four DENV serotypes was expressed using a replication-defective recombinant human adenovirus type 5 (rAdV5) vaccine vector. This rAdV5 vector induced cell-mediated immune responses and virus-neutralizing antibodies specific to each of the four DENVs in mice. Interestingly, anti-AdV5 antibodies did not suppress the induction of DENV-specific neutralizing antibodies. We observed that anti-AdV5 antibodies in the sera of immunized mice could promote uptake of a rAdV5-derived reporter vector into U937 cells, suggesting that pre-existing immunity to AdV5 may in fact facilitate the uptake of rAdV5 vectored vaccines into antigen presenting cells. This work presents an alternative approach to developing a single component tetravalent vaccine that bypasses the complexities inherent in the currently adopted four-in-one physical mixture approach.

An envelope domain III-based chimeric antigen produced in Pichia pastoris elicits neutralizing antibodies against all four dengue virus serotypes.

There is currently no vaccine to prevent dengue (DEN) virus infection, which is caused by any one of four closely related serotypes, DEN-1, DEN-2, DEN-3, or DEN-4. A DEN vaccine must be tetravalent, because immunity to a single serotype does not offer cross-protection against the other serotypes. We have developed a novel tetravalent chimeric protein by fusing the receptor-binding envelope domain III (EDIII) of the four DEN virus serotypes. This protein was expressed in the yeast, Pichia pastoris, and purified to near homogeneity in high yields. Antibodies induced in mice by the tetravalent protein, formulated in different adjuvants, neutralized the infectivity of all four serotypes. This, coupled with the high expression potential of the P. pastoris system and easy one-step purification, makes the EDIII-based recombinant protein a potentially promising candidate for the development of a safe, efficacious, and inexpensive, tetravalent DEN vaccine.

Induction of neutralizing antibodies and T cell responses by dengue virus type 2 envelope domain III encoded by plasmid and adenoviral vectors.

Dengue is a re-emerging public health problem in many developing nations. There is neither a specific antiviral therapy to treat nor a licensed vaccine to prevent dengue infections. In recent years, the carboxy-terminal region of the major dengue virion envelope (E) protein, known as domain III, has emerged as a significant sub-unit vaccine candidate. In this study, we created a recombinant adenovirus capable of expressing the E domain III (EDIII) of dengue virus type 2 (DEN-2) and tested it in combination with a plasmid encoding the same domain to determine its potential as a possible dengue vaccine candidate. We examined the relative efficacies of plasmid prime/Ad boost (P/A) and Ad prime/plasmid boost (A/P) regimens in eliciting DEN-2 virus-specific immune responses. Both regimens resulted in the induction of antibodies that specifically bound to and neutralized the infectivity of DEN-2 virus. Splenocytes from immunized mice, stimulated in vitro, manifested a significant proliferative response accompanied by the production of high levels of interferon-gamma, but moderately elevated levels of interleukin-4, indicative of a predominantly Th1 type response. Based on a comparison of the parameters investigated, the immune response induced by the A/P regimen appeared to be relatively more potent. Our data suggest that EDIII may be valuable in the efforts to develop plasmid- and Ad-vectored dengue vaccines.

A synthetic dengue virus antigen elicits enhanced antibody titers when linked to, but not mixed with, Mycobacterium tuberculosis HSP70 domain II.

Domain II of Mycobacterium tuberculosis (Mtb) heat shock protein 70 (HSP70), spanning amino acid residues 161-370, was covalently linked to a recently described synthetic dengue virus antigen to study the influence of the former on the immunogenicity of the latter. Using an enzyme-linked immunosorbent assay, dengue antigen-specific antibody titers elicited by the fusion protein in Balb/c mice were an order of magnitude higher than those elicited by either the synthetic dengue antigen alone or a physical mixture of the dengue antigen plus Mtb HSP70 domain II protein. Our data demonstrate that (i) Mtb HSP70 domain II is capable of potentiating B-cell response and (ii) it should be covalently linked to the target antigen to do so.