A tetravalent dengue vaccine based on a complex adenovirus vector provides significant protection in rhesus monkeys against all four serotypes of dengue virus.

Nearly a third of the human population is at risk of infection with the four serotypes of dengue viruses, and it is estimated that more than 100 million infections occur each year. A licensed vaccine for dengue viruses has become a global health priority. A major challenge to developing a dengue vaccine is the necessity to produce fairly uniform protective immune responses to all four dengue virus serotypes. We have developed two bivalent dengue virus vaccines, using a complex adenovirus vector, by incorporating the genes expressing premembrane (prM) and envelope (E) proteins of dengue virus types 1 and 2 (dengue-1 and -2, respectively) (CAdVax-Den12) or dengue-3 and -4 (CAdVax-Den34). Rhesus macaques were vaccinated by intramuscular inoculation of a tetravalent dengue vaccine formulated by combining the two bivalent vaccine constructs. Vaccinated animals produced high-titer antibodies that neutralized all four serotypes of dengue viruses in vitro. The ability of the vaccine to induce rapid, as well as sustained, protective immune responses was examined with two separate live-virus challenges administered at 4 and 24 weeks after the final vaccination. For both of these virus challenge studies, significant protection from viremia was demonstrated for all four dengue virus serotypes in vaccinated animals. Viremia from dengue-1 and dengue-3 challenges was completely blocked, whereas viremia from dengue-2 and dengue-4 was significantly reduced, as well as delayed, compared to that of control-vaccinated animals. These results demonstrate that the tetravalent dengue vaccine formulation provides significant protection in rhesus macaques against challenge with all four dengue virus serotypes.

Induction of bivalent immune responses by expression of dengue virus type 1 and type 2 antigens from a single complex adenoviral vector.

There are approximately 100 million new cases of dengue (DEN) virus infection each year. Infection can result in illness ranging from a mild fever to hemorrhaging, shock, or even death. There are four serotypes of dengue virus (DEN1-4), and immunity to one serotype does not cross protect from infection with other serotypes. Currently there are no approved vaccines for dengue fever. In this report, we describe the construction of a bivalent dengue virus vaccine using a complex recombinant adenovirus approach to express multiple genes of DEN1 and DEN2 serotypes. In vaccinated mice, this vector induced humoral immune responses against all four dengue serotypes as measured by enzyme-linked immunosorbent assay. However, the neutralizing antibody responses were specific for DEN1 and DEN2 serotypes. Expansion of this vaccine development platform towards the DEN3 and DEN4 serotypes can lead towards the development of an adenovirus-based tetravalent dengue vaccine.

A complex adenovirus vaccine against chikungunya virus provides complete protection against viraemia and arthritis.

Chikungunya virus, a mosquito-borne alphavirus, recently caused the largest epidemic ever seen for this virus. Chikungunya disease primarily manifests as a painful and debilitating arthralgia/arthritis, and no effective drug or vaccine is currently available. Here we describe a recombinant chikungunya virus vaccine comprising a non-replicating complex adenovirus vector encoding the structural polyprotein cassette of chikungunya virus. A single immunisation with this vaccine consistently induced high titres of anti-chikungunya virus antibodies that neutralised both an old Asian isolate and a Réunion Island isolate from the recent epidemic. The vaccine also completely protected mice against viraemia and arthritic disease caused by both virus isolates.

Protection of nonhuman primates against two species of Ebola virus infection with a single complex adenovirus vector.

Ebola viruses are highly pathogenic viruses that cause outbreaks of hemorrhagic fever in humans and other primates. To meet the need for a vaccine against the several types of Ebola viruses that cause human diseases, we developed a multivalent vaccine candidate (EBO7) that expresses the glycoproteins of Zaire ebolavirus (ZEBOV) and Sudan ebolavirus (SEBOV) in a single complex adenovirus-based vector (CAdVax). We evaluated our vaccine in nonhuman primates against the parenteral and aerosol routes of lethal challenge. EBO7 vaccine provided protection against both Ebola viruses by either route of infection. Significantly, protection against SEBOV given as an aerosol challenge, which has not previously been shown, could be achieved with a boosting vaccination. These results demonstrate the feasibility of creating a robust, multivalent Ebola virus vaccine that would be effective in the event of a natural virus outbreak or biological threat.

A complex adenovirus-vectored vaccine against Rift Valley fever virus protects mice against lethal infection in the presence of preexisting vector immunity.

Rift Valley fever virus (RVFV) has been cited as a potential biological-weapon threat due to the serious and fatal disease it causes in humans and animals and the fact that this mosquito-borne virus can be lethal in an aerosolized form. Current human and veterinary vaccines against RVFV, however, are outdated, inefficient, and unsafe. We have incorporated the RVFV glycoprotein genes into a nonreplicating complex adenovirus (CAdVax) vector platform to develop a novel RVFV vaccine. Mice vaccinated with the CAdVax-based vaccine produced potent humoral immune responses and were protected against lethal RVFV infection. Additionally, protection was elicited in mice despite preexisting immunity to the adenovirus vector.

Vaccine to confer to nonhuman primates complete protection against multistrain Ebola and Marburg virus infections.

Filoviruses (Ebola and Marburg viruses) are among the deadliest viruses known to mankind, with mortality rates nearing 90%. These pathogens are highly infectious through contact with infected body fluids and can be easily aerosolized. Additionally, there are currently no licensed vaccines available to prevent filovirus outbreaks. Their high mortality rates and infectious capabilities when aerosolized and the lack of licensed vaccines available to prevent such infectious make Ebola and Marburg viruses serious bioterrorism threats, placing them both on the category A list of bioterrorism agents. Here we describe a panfilovirus vaccine based on a complex adenovirus (CAdVax) technology that expresses multiple antigens from five different filoviruses de novo. Vaccination of nonhuman primates demonstrated 100% protection against infection by two species of Ebola virus and three Marburg virus subtypes, each administered at 1,000 times the lethal dose. This study indicates the feasibility of vaccination against all current filovirus threats in the event of natural hemorrhagic fever outbreak or biological attack.

Multi-antigen vaccines based on complex adenovirus vectors induce protective immune responses against H5N1 avian influenza viruses.

There are legitimate concerns that the highly pathogenic H5N1 avian influenza virus could adapt for human-to-human transmission and cause a pandemic similar to the 1918 "Spanish flu" that killed 50 million people worldwide. We have developed pandemic influenza vaccines by incorporating multiple antigens from both avian and Spanish influenza viruses into complex recombinant adenovirus vectors. In vaccinated mice, these vaccines induced strong humoral and cellular immune responses against pandemic influenza virus antigens, and protected vaccinated mice against lethal H5N1 virus challenge. These results indicate that this multi-antigen, broadly protective vaccine may serve as a safer and more effective approach than traditional methods for development of a pandemic influenza vaccine.

Two complex, adenovirus-based vaccines that together induce immune responses to all four dengue virus serotypes.

Dengue virus infections can cause hemorrhagic fever, shock, encephalitis, and even death. Worldwide, approximately 2.5 billion people live in dengue-infested regions with about 100 million new cases each year, although many of these infections are believed to be silent. There are four antigenically distinct serotypes of dengue virus; thus, immunity from one serotype will not cross-protect from infection with the other three. The difficulties that hamper vaccine development include requirements of the natural conformation of the envelope glycoprotein to induce neutralizing immune responses and the necessity of presenting antigens of all four serotypes. Currently, the only way to meet these requirements is to use a mixture of four serotypes of live attenuated dengue viruses, but safety remains a major problem. In this study, we have developed the basis for a tetravalent dengue vaccine using a novel complex adenovirus platform that is capable of expressing multiple antigens de novo. This dengue vaccine is constructed as a pair of vectors that each expresses the premembrane and envelope genes of two different dengue virus serotypes. Upon vaccination, the vaccine expressed high levels of the dengue virus antigens in cells to mimic a natural infection and induced both humoral and cellular immune responses against multiple serotypes of dengue virus in an animal model. Further analyses show the humoral responses were indeed neutralizing against all four serotypes. Our studies demonstrate the concept of mimicking infections to induce immune responses by synthesizing dengue virus membrane antigens de novo and the feasibility of developing an effective tetravalent dengue vaccine by vector-mediated expression of glycoproteins of the four serotypes.

Complex adenovirus-mediated expression of West Nile virus C, PreM, E, and NS1 proteins induces both humoral and cellular immune responses.

West Nile Virus (WNV), a member of the family Flaviviridae, was first identified in Africa in 1937. In recent years, it has spread into Europe and North America. The clinical manifestations of WNV infection range from mild febrile symptoms to fatal encephalitis. Two genetic lineages (lineages I and II) are recognized; lineage II is associated with mild disease, while lineage I has been associated with severe disease, including encephalitis. WNV has now spread across North America, significantly affecting both public and veterinary health. In the efforts to develop an effective vaccine against all genetic variants of WNV, we have studied the feasibility of inducing both neutralizing and cellular immune responses by de novo synthesis of WNV antigens using a complex adenoviral vaccine (CAdVax) vector. By expressing multiple WNV proteins from a single vaccine vector, we were able to induce both humoral and cellular immune responses in vaccinated mice. Neutralization assays demonstrated that the antibodies were broadly neutralizing against both lineages of WNV, with a significant preference for the homologous lineage II virus. The results from this study show that multiple antigens synthesized de novo from a CAdVax vector are capable of inducing both humoral and cellular immune responses against WNV and that a multiantigen approach may provide broad protection against multiple genetic variants of WNV.

Complex adenovirus-vectored vaccine protects guinea pigs from three strains of Marburg virus challenges.

The Marburg virus (MARV), an African filovirus closely related to the Ebola virus, causes a deadly hemorrhagic fever in humans, with up to 90% mortality. Currently, treatment of disease is only supportive, and no vaccines are available to prevent spread of MARV infections. In order to address this need, we have developed and characterized a novel recombinant vaccine that utilizes a single complex adenovirus-vectored vaccine (cAdVax) to overexpress a MARV glycoprotein (GP) fusion protein derived from the Musoke and Ci67 strains of MARV. Vaccination with the cAdVaxM(fus) vaccine led to efficient production of MARV-specific antibodies in both mice and guinea pigs. Significantly, guinea pigs vaccinated with at least 5 x 10(7) pfu of cAdVaxM(fus) vaccine were 100% protected against lethal challenges by the Musoke, Ci67 and Ravn strains of MARV, making it a vaccine with trivalent protective efficacy. Therefore, the cAdVaxM(fus) vaccine serves as a promising vaccine candidate to prevent and contain multi-strain infections by MARV.

Development of a cAdVax-based bivalent ebola virus vaccine that induces immune responses against both the Sudan and Zaire species of Ebola virus.

Ebola virus (EBOV) causes a severe hemorrhagic fever for which there are currently no vaccines or effective treatments. While lethal human outbreaks have so far been restricted to sub-Saharan Africa, the potential exploitation of EBOV as a biological weapon cannot be ignored. Two species of EBOV, Sudan ebolavirus (SEBOV) and Zaire ebolavirus (ZEBOV), have been responsible for all of the deadly human outbreaks resulting from this virus. Therefore, it is important to develop a vaccine that can prevent infection by both lethal species. Here, we describe the bivalent cAdVaxE(GPs/z) vaccine, which includes the SEBOV glycoprotein (GP) and ZEBOV GP genes together in a single complex adenovirus-based vaccine (cAdVax) vector. Vaccination of mice with the bivalent cAdVaxE(GPs/z) vaccine led to efficient induction of EBOV-specific antibody and cell-mediated immune responses to both species of EBOV. In addition, the cAdVax technology demonstrated induction of a 100% protective immune response in mice, as all vaccinated C57BL/6 and BALB/c mice survived challenge with a lethal dose of ZEBOV (30,000 times the 50% lethal dose). This study demonstrates the potential efficacy of a bivalent EBOV vaccine based on a cAdVax vaccine vector design.

De novo syntheses of Marburg virus antigens from adenovirus vectors induce potent humoral and cellular immune responses.

Marburg virus (MARV) is an African filovirus that causes a deadly hemorrhagic fever in humans, with up to 90% mortality. Currently, there are no MARV vaccines or therapies approved for human use. We hypothesized that developing a vaccine that induces a de novo synthesis of MARV antigens in vivo will lead to strong induction of both a humoral and cell-mediated immune response against MARV. Here, we develop and characterize three novel gene-based vaccine candidates which express the viral glycoprotein (GP) from either the Ci67, Ravn or Musoke strain of MARV. Immunization of mice with complex adenovirus (Ad)-based vaccine candidates (cAdVax vaccines), led to efficient production of both antibodies and cytotoxic T lymphocytes (CTL) specific to Musoke strain GP and Ci67 strain GP, respectively. Antibody responses were also shown to be cross-reactive across the MARV strains, but not cross-reactive to Ebola virus, a related filovirus. Additionally, three 1 x 10(8)pfu doses of vaccine vector were demonstrated to be safe in mice, as this did not lead to any detectable toxicity in liver or spleen. These promising results indicate that a cAdVax-based vaccine could be effective for induction of both humoral and cell-mediated immune responses to multiple strains of the Marburg virus.