Paramyxoviruses infect the respiratory tract and induce a high level of local and systemic immunity. In addition, we have extensive experience in producing attenuated derivatives of some of these viruses, such as human parainfluenza virus type 3 (HPIV3), using recombinant methods, and in evaluating these in the clinical setting as potential vaccines. Paramyxoviruses, like all non-segmented negative strand RNA viruses, have the advantage of a negligible incidence of recombination, which essentially eliminates the concern of genetic exchange with circulating viruses. This is a major advantage compared to live vaccines based on recombinogenic viruses such as coronaviruses and influenza viruses. Thus, paramyxoviruses have the potential to serve as vaccine vectors for expressing protective antigens of highly pathogenic agents.? HPIV3 replicates in the superficial cells of the respiratory tract and does not spread significantly beyond that site, reducing concerns about infection of distal tissues. Direct intranasal immunization would be an advantage against the numerous pathogens that use the respiratory tract as a portal for entry and egress. We previously used a derivative of HPIV3 as a vector to express the structural proteins of the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV). This identified the spike S glycoprotein as the only significant neutralization and protective antigen among the SARS structural proteins and showed that this PIV3-vectored vaccine was immunogenic and protective against SARS-CoV in African green monkeys.? Next, we evaluated whether intranasal inoculation with an HPIV3-vectored vaccine can induce protective immunity against Ebola virus (EV), a highly contagious and lethal agent of systemic infection and severe viral hemorrhagic fever. We engineered HPIV3 to express the EV glycoprotein (GP) (HPIV3EboGP) alone or in combination with the EV nucleocapsid protein (HPIV3EboGP-NP). Interestingly, GP was incorporated into the vector particle and was functional in that setting. However, guinea pigs infected with a single intranasal inoculation of 100,000 PFU of HPIV3EboGP or HPIV3EboGP-NP showed no apparent signs of disease. This animal model is highly sensitive to EV infection, such that a single PFU contains 400 lethal dose 50% units. Thus, the lack of disease associated with this high dose indicates that EV GP did not confer increased pathogenesis to the HPIV3 vector.? Guinea pigs immunized with a single vaccine dose developed a strong humoral response to the two EV proteins. Upon challenge with a highly lethal intraperitoneal injection of 1000 PFU of EV, there was no disease, no viremia, and no evidence of infection in the spleen, liver, and lungs. In contrast, all of the control animals died of severe EV disease following challenge. Thus, vaccines based on recombinant respiratory viruses can be effective in inducing protective responses against severe systemic infections.? Next, we evaluated the two HPIV3-Ebo constructs in non-human primates in parallel with a third construct, namely one engineered to express EboGP in combination with the cytokine adjuvant granulocyte macrophage colony stimulating factor (GM-CSF). These were administered by the respiratory route to rhesus monkeys, for which HPIV3 infection is mild and asymptomatic and thus mimics an attenuated virus. The constructs were evaluated for immunogenicity and protective efficacy against a highly lethal intraperitoneal challenge with EV. A single immunization with any construct expressing GP was moderately immunogenic against EV and protected 88% of the animals against severe hemorrhagic fever and death caused by EV. Two doses were highly immunogenic, and all of the animals survived challenge and were free of disease signs and of detectable EV challenge virus. The immunogenicity and protective efficacy of the vectored vaccine did not appear to be enhanced by co-expression of NP or GM-CSF. These data illustrate the feasibility of immunization via the respiratory tract against the hemorrhagic fever caused by EV.? The vaccines described above are candidates for further evaluation as pediatric vaccines. However, it was unclear whether they would be effective in adults because the high seroprevalence against HPIV3 in that population might restrict the replication and immunogenicity of an HPIV3 vector. To evaluate this possibility, guinea pigs were infected with HPIV3 and, 40 days later, were immunized by infection with HPIV3EboGP. Replication of HPIV3EboGP was not detected in any of the animals, indicating that immunity to HPIV3 indeed strongly restricted the replication of the vector despite the presence of functional GP in the vector particle. Surprisingly, however, the immune response to EV GP was almost equivalent to that achieved in control animals that had not been previously infected with HPIV3. Thus, it may be possible to achieve immunization in HPIV3-immune animals, although this remains to be investigated in primates.? A second strategy to overcome seroprevalence is to use a vector that does not usually infect humans and is antigenically distinct from common human viruses. Newcastle disease virus (NDV) is an avian paramyxovirus that fits these criteria. There is serological evidence that bird handlers can be infected, but infection apparently does not cause significant disease. NDV exists naturally in a variety of strains that exhibit a wide spectrum of virulence in birds, ranging from highly virulent (velogenic), to moderate virulence (mesogenic), to low virulence (lentigenic). Recombinant versions of the lentigenic vaccine strain LaSota (NDV-LS) and the mesogenic strain Beaudette C (NDV-BC) were engineered to express the HPIV3 hemagglutinin-neuraminidase (HN) protein. When inoculated into African green and rhesus monkeys by the combined intranasal and intratracheal routes, both viruses were highly attenuated and were restricted to the respiratory tract. The serum antibody response to the foreign HN protein induced by a single immunization with either NDV vector was somewhat less than that observed following a wild type HPIV3 infection, whereas the titer following two doses exceeded that observed with HPIV3 infection even though HPIV3 replicates much more efficiently than NDV in these animals.? Next, we engineered NDV to express the SARS-CoV S protein. African green monkeys immunized via the respiratory tract with two doses of the vaccine developed a titer of SARS-CoV-neutralizing antibodies comparable to the robust secondary response observed in animals that have been immunized with a different experimental SARS-CoV vaccine and challenged with SARS-CoV. When animals immunized with NDV-S were challenged with a high dose of SARS-CoV, direct viral assay of lung tissues taken by necropsy at the peak of viral replication demonstrated a 236- or 1,102-fold (depending on the NDV vector construct) mean reduction in pulmonary SARS-CoV titer compared to control animals.? We also engineered NDV to express the hemagglutinin HA glycoprotein of H5N1 highly pathogenic avian influenza virus (HPAIV) (NDV-HA). The NDV-HA virus was highly attenuated in African green monkeys as well as in chickens. In African green monkeys, two doses of NDV-HA induced a titer of H5N1 HPAIV-neutralizing serum antibodies that, based on historic controls, would be consistent with substantial protection against morbidity and mortality caused by H5N1 HPAIV. Moreover, a substantial respiratory mucosal immunoglobulin A response was induced following one and two doses, which would be particularly important in controlling a respiratory pathogen. NDV has potential for further development as a highly attenuated vaccine vector for use in humans against highly pathogenic agents.
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