The effect of 5' and 3' non-translated regions on the expression of a transgene from a Newcastle disease virus vector.

Newcastle disease virus (NDV) is an avian virus and a promising vector for the development of vaccines for veterinary and human use. The optimal vaccine vector performance requires a stable high-level expression of a transgene. The foreign genes are usually incorporated in the genome of NDV as individual transcription units, whose transcription and subsequent translation of the mRNA are regulated by the 5' and 3' untranslated regions (UTRs) flanking the open reading frame of the transgene. Here, we investigated if the UTRs derived from the cognate NDV genes would increase the expression of a model protective antigene from an NDV vector. Our results show that in chicken DF1 cells, none of the UTRs tested significantly outperformed generic short sequences flanking the transgene, while in human HeLa cells, UTRs derived from the M gene of NDV statistically significantly increased the expression of the transgene. The UTRs derived from the HN gene significantly downregulated the transgene expression in both cell cultures. Further experiments demonstrated that NDV UTRs differently affect the mRNA abundance and translation efficacy. While both M and HN UTRs decreased the level of the transgene mRNA in infected cells compared to the mRNA flanked by generic UTRs, M, and particularly, HN UTRs strongly increased the mRNA translation efficacy. The major determinants of translation enhancement are localized in the 5'UTR of HN. Thus, our data reveal a direct role of NDV UTRs in translational regulation, and inform future optimization of NDV vectors for vaccine and therapeutic use.

Encoding of a transgene in-frame with a Newcastle disease virus protein increases transgene expression and stability.

Newcastle disease virus (NDV) has been extensively explored as a vector for vaccine and oncolytic therapeutic development. In conventional NDV-based vectors, the transgene is arranged as a separate transcription unit in the NDV genome. Here, we expressed haemagglutinin protein (HA) of an avian influenza virus using an NDV vector design in which the transgene ORF is encoded in-frame with the ORF of an NDV gene. This arrangement does not increase the number of transcription units in the NDV genome, and imposes a selection pressure against mutations interrupting the transgene ORF. We placed the HA ORF upstream or downstream of N, M, F and HN ORFs of NDV so that both proteins are encoded in-frame and are separated by either a self-cleaving 2A peptide, furin cleavage site or both. Only constructs in which HA was placed downstream of the NDV HN were viable. These constructs expressed the transgene at a higher level compared to the vector encoding the same transgene in the same position in the NDV genome but as a separate transcription unit. Furthermore, the transgene expressed in one ORF with the NDV protein proved to be more stable over multiple passages. Thus, this design may be useful for applications where the stability of the transgene expression is highly important for a recombinant NDV vector.

Intranasal immunization with avian paramyxovirus type 3 expressing SARS-CoV-2 spike protein protects hamsters against SARS-CoV-2.

Current vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are administered parenterally and appear to be more protective in the lower versus the upper respiratory tract. Vaccines are needed that directly stimulate immunity in the respiratory tract, as well as systemic immunity. We used avian paramyxovirus type 3 (APMV3) as an intranasal vaccine vector to express the SARS-CoV-2 spike (S) protein. A lack of pre-existing immunity in humans and attenuation by host-range restriction make APMV3 a vector of interest. The SARS-CoV-2 S protein was stabilized in its prefusion conformation by six proline substitutions (S-6P) rather than the two that are used in most vaccine candidates, providing increased stability. APMV3 expressing S-6P (APMV3/S-6P) replicated to high titers in embryonated chicken eggs and was genetically stable, whereas APMV3 expressing non-stabilized S or S-2P were unstable. In hamsters, a single intranasal dose of APMV3/S-6P induced strong serum IgG and IgA responses to the S protein and its receptor-binding domain, and strong serum neutralizing antibody responses to SARS-CoV-2 isolate WA1/2020 (lineage A). Sera from APMV3/S-6P-immunized hamsters also efficiently neutralized Alpha and Beta variants of concern. Immunized hamsters challenged with WA1/2020 did not exhibit the weight loss and lung inflammation observed in empty-vector-immunized controls; SARS-CoV-2 replication in the upper and lower respiratory tract of immunized animals was low or undetectable compared to the substantial replication in controls. Thus, a single intranasal dose of APMV3/S-6P was highly immunogenic and protective against SARS-CoV-2 challenge, suggesting that APMV3/S-6P is suitable for clinical development.

Correction: Generation by Reverse Genetics of an Effective, Stable, Live-Attenuated Newcastle Disease Virus Vaccine Based on a Currently Circulating, Highly Virulent Indonesian Strain.

[This corrects the article DOI: 10.1371/journal.pone.0052751.].

Ebola vaccine-induced protection in nonhuman primates correlates with antibody specificity and Fc-mediated effects.

Although substantial progress has been made with Ebola virus (EBOV) vaccine measures, the immune correlates of vaccine-mediated protection remain uncertain. Here, five mucosal vaccine vectors based on human and avian paramyxoviruses provided nonhuman primates with varying degrees of protection, despite expressing the same EBOV glycoprotein (GP) immunogen. Each vaccine produced antibody responses that differed in Fc-mediated functions and isotype composition, as well as in magnitude and coverage toward GP and its conformational and linear epitopes. Differences in the degree of protection and comprehensive characterization of the response afforded the opportunity to identify which features and functions were elevated in survivors and could therefore serve as vaccine correlates of protection. Pairwise network correlation analysis of 139 immune- and vaccine-related parameters was performed to demonstrate relationships with survival. Total GP-specific antibodies, as measured by biolayer interferometry, but not neutralizing IgG or IgA titers, correlated with survival. Fc-mediated functions and the amount of receptor binding domain antibodies were associated with improved survival outcomes, alluding to the protective mechanisms of these vaccines. Therefore, functional qualities of the antibody response, particularly Fc-mediated effects and GP specificity, rather than simply magnitude of the response, appear central to vaccine-induced protection against EBOV. The heterogeneity of the response profile between the vaccines indicates that each vaccine likely exhibits its own protective signature and the requirements for an efficacious EBOV vaccine are complex.

Recovery of Recombinant Avian Paramyxovirus Type-3 Strain Wisconsin by Reverse Genetics and Its Evaluation as a Vaccine Vector for Chickens.

A reverse genetic system for avian paramyxovirus type-3 (APMV-3) strain Wisconsin was created and the infectious virus was recovered from a plasmid-based viral antigenomic cDNA. Green fluorescent protein (GFP) gene was cloned into the recombinant APMV-3 genome as a foreign gene. Stable expression of GFP by the recovered virus was confirmed for at least 10 consecutive passages. APMV-3 strain Wisconsin was evaluated against APMV-3 strain Netherlands and APMV-1 strain LaSota as a vaccine vector. The three viral vectors expressing GFP as a foreign protein were compared for level of GFP expression level, growth rate in chicken embryo fibroblast (DF-1) cells, and tissue distribution and immunogenicity in specific pathogen-free (SPF) day-old chickens. APMV-3 strain Netherlands showed highest growth rate and GFP expression level among the three APMV vectors in vitro. APMV-3 strain Wisconsin and APMV-1 strain LaSota vectors were mainly confined to the trachea after vaccination of day-old SPF chickens without any observable pathogenicity, whereas APMV-3 strain Netherlands showed wide tissue distribution in different body organs (brain, lungs, trachea, and spleen) with mild observable pathogenicity. In terms of immunogenicity, both APMV-3 strain-vaccinated groups showed HI titers two to three fold higher than that induced by APMV-1 strain LaSota vaccinated group. This study offers a novel paramyxovirus vector (APMV-3 strain Wisconsin) which can be used safely for vaccination of young chickens as an alternative for APMV-1 strain LaSota vector.

Inter-Institutional Partnerships to Develop Veterinarian-Investigators through the NIH Comparative Biomedical Scientist Training Program Benefit One Health Goals.

Limitations in workforce size and access to resources remain perennial challenges to greater progress in academic veterinary medicine and engagement between human and veterinary medicine (One Health). Ongoing resource constraints occur in part due to limited public understanding of the role veterinarians play in improving human health. One Health interactions, particularly through interdisciplinary collaborations in biomedical research, present constructive opportunities to inform resource policies and advance health care. To this end, inter-institutional partnerships between individual veterinary medical education programs (VMEPs) and several National Institutes of Health (NIH) intramural research programs have created synergies beyond those provided by individual programs. In the NIH Comparative Biomedical Scientist Training Program (CBSTP), interdisciplinary cross-training of veterinarians consisting of specialty veterinary medicine coupled with training in human disease research leading to a PhD, occurs collaboratively on both VMEP and NIH campuses. Pre-doctoral veterinary student research opportunities have also been made available. Through the CBSTP, NIH investigators and national biomedical science policy makers gain access to veterinary perspective and expertise, while veterinarians obtain additional opportunities for NIH-funded research training. CBSTP Fellows serve as de facto ambassadors enhancing visibility for the profession while in residence at NIH, and subsequently through a variety of university, industry, and government research appointments, as graduates. Thus, the CBSTP represents an inter-institutional opportunity that not only addresses critical needs for veterinarian-scientists in the biomedical workforce, but also simultaneously exposes national policy makers to veterinarian-scientists' specialized training, leading to more effective realization of One Health goals to benefit human and animal health.

Newcastle Disease Virus as a Vaccine Vector for SARS-CoV-2.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 16 million infections and more than 600,000 deaths worldwide. There is an urgent need to develop a safe and effective vaccine against SARS-CoV-2. Currently, several strategies are being pursued to develop a safe and effective SARS-CoV-2 vaccine. However, each vaccine strategy has distinct advantages and disadvantages. Therefore, it is important to evaluate multiple vaccine platforms to select the most efficient vaccine platform for SARS-CoV-2. In this regard, Newcastle disease virus (NDV), an avian virus, has several well-suited properties for development of a vector vaccine against SARS-CoV-2. Here, we elaborate on the idea of considering NDV as a vaccine vector for SARS-CoV-2.

Comparative Protective Efficacies of Novel Avian Paramyxovirus-Vectored Vaccines against Virulent Infectious Bronchitis Virus in Chickens.

Viral vectored vaccines are desirable alternatives for conventional infectious bronchitis virus (IBV) vaccines. We have recently shown that a recombinant Newcastle disease virus (rNDV) strain LaSota expressing the spike (S) protein of IBV strain Mass-41 (rLaSota/IBV-S) was a promising vaccine candidate for IBV. Here we evaluated a novel chimeric rNDV/avian paramyxovirus serotype 2 (rNDV/APMV-2) as a vaccine vector against IBV. The rNDV/APMV-2 vector was chosen because it is much safer than the rNDV strain LaSota vector, particularly for young chicks and chicken embryos. In order to determine the effectiveness of this vector, a recombinant rNDV/APMV-2 expressing the S protein of IBV strain Mass-41 (rNDV/APMV-2/IBV-S) was constructed. The protective efficacy of this vector vaccine was compared to that of the rNDV vector vaccine. In one study, groups of one-day-old specific-pathogenic-free (SPF) chickens were immunized with rLaSota/IBV-S and rNDV/APMV-2/IBV-S and challenged four weeks later with the homologous highly virulent IBV strain Mass-41. In another study, groups of broiler chickens were single (at day one or three weeks of age) or prime-boost (prime at day one and boost at three weeks of age) immunized with rLaSota/IBV-S and/or rNDV-APMV-2/IBV-S. At weeks six of age, chickens were challenged with a highly virulent IBV strain Mass-41. Our challenge study showed that novel rNDV/APMV-2/IBV-S provided similar protection as rLaSota/IBV-S in SPF chickens. However, compared to prime-boost immunization of chickens with chimeric rNDV/APMV-2, rLaSota/IBV-S and/or a live IBV vaccine, single immunization of chickens with rLaSota/IBV-S, or live IBV vaccine provided better protection against IBV. In conclusion, we have developed the novel rNDV/APMV-2 vector expressing S protein of IBV that can be a safer vaccine against IB in chickens. Our results also suggest a single immunization with a LaSota vectored IBV vaccine candidate provides better protection than prime-boost immunization regimens.

Contributions of HA1 and HA2 Subunits of Highly Pathogenic Avian Influenza Virus in Induction of Neutralizing Antibodies and Protection in Chickens.

Highly pathogenic avian influenza virus (HPAIV) subtype H5N1 causes a devastating disease in poultry. Vaccination is an effective method of controlling avian influenza virus (AIV) infection in poultry. The hemagglutinin (HA) protein is the major determinant recognized by the immune system of the host. Cleavage of the HA precursor HA0 into HA1 and HA2 subunits is required for infectivity of the AIV. We evaluated the individual contributions of HA1 and HA2 subunits to the induction of HPAIV serum neutralizing antibodies and protective immunity in chickens. Using reverse genetics, recombinant Newcastle disease viruses (rNDVs) were generated, each expressing HA1, HA2, or HA protein of H5N1 HPAIV. Chickens were immunized with rNDVs expressing HA1, HA2, or HA. Immunization with HA induced high titers of serum neutralizing antibodies and prevented death following challenge. Immunization with HA1 or HA2 alone neither induced serum neutralizing antibodies nor prevented death following challenge. Our results suggest that interaction of HA1 and HA2 subunits is necessary for the display of epitopes on HA protein involved in the induction of neutralizing antibodies and protection. These epitopes are lost when the two subunits are separated. Therefore, vaccination with either a HA1 or HA2 subunit may not provide protection against HPAIV.

Author Correction: A recombinant avian paramyxovirus serotype 3 expressing the hemagglutinin protein protects chickens against H5N1 highly pathogenic avian influenza virus challenge.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A recombinant avian paramyxovirus serotype 3 expressing the hemagglutinin protein protects chickens against H5N1 highly pathogenic avian influenza virus challenge.

Highly pathogenic avian influenza (HPAI) is a devastating disease of poultry and a serious threat to public health. Vaccination with inactivated virus vaccines has been applied for several years as one of the major policies to control highly pathogenic avian influenza virus (HPAIV) infections in chickens. Viral-vectored HA protein vaccines are a desirable alternative for inactivated vaccines. However, each viral vector possesses its own advantages and disadvantages for the development of a HA-based vaccine against HPAIV. Recombinant Newcastle disease virus (rNDV) strain LaSota expressing HA protein vaccine has shown promising results against HPAIV; however, its replication is restricted only to the respiratory tract. Therefore, we thought to evaluate avian paramyxovirus serotype 3 (APMV-3) strain Netherlands as a safe vaccine vector against HPAIV, which has high efficiency replication in a greater range of host organs. In this study, we generated rAPMV-3 expressing the HA protein of H5N1 HPAIV using reverse genetics and evaluated the induction of neutralizing antibodies and protection by rAPMV3 and rNDV expressing the HA protein against HPAIV challenge in chickens. Our results showed that immunization of chickens with rAPMV-3 or rNDV expressing HA protein provided complete protection against HPAIV challenge. However, immunization of chickens with rAPMV-3 expressing HA protein induced higher level of neutralizing antibodies compared to that of rNDV expressing HA protein. These results suggest that a rAPMV-3 expressing HA protein might be a better vaccine for mass-vaccination of commercial chickens in field conditions.