key: cord-0773732-6r8wvn9s
authors: Chen, Zhenhai
title: Parainfluenza virus 5–vectored vaccines against human and animal infectious diseases
date: 2018-01-05
journal: Rev Med Virol
DOI: 10.1002/rmv.1965
sha: f3e48841747b33e9660801a34de2c13e93482802
doc_id: 773732
cord_uid: 6r8wvn9s

Parainfluenza virus 5 (PIV5), known as canine parainfluenza virus in the veterinary field, is a negative‐sense, nonsegmented, single‐stranded RNA virus belonging to the Paramyxoviridae family. Parainfluenza virus 5 is an excellent viral vector and has been used as a live vaccine for kennel cough for many years in dogs without any safety concern. It can grow to high titers in many cell types, and its genome is stable even in the presence of foreign gene insertions. So far, PIV5 has been used to develop vaccines against influenza virus, respiratory syncytial virus, rabies virus, and Mycobacterium tuberculosis, demonstrating its ability to elicit robust and protective immune responses in preclinical animal models. Parainfluenza virus 5–based vaccines can be administered intranasally, intramuscularly, or orally. Interestingly, prior exposure of PIV5 does not prevent a PIV5‐vectored vaccine from generating robust immunity, indicating that the vector can be used more than once. Here, these encouraging results are reviewed together along with discussion of the desirable advantages of the PIV5 vaccine vector to aid future vaccine design and to accelerate progression of PIV5‐based vaccines into clinical trials.

infect humans and animals including dogs, pigs, cats, hamsters, guinea pigs, cattle, and panda. [1] [2] [3] [4] [5] [6] Parainfluenza virus 5 was first isolated as a contaminant from simian cells in 1956, and thus named simian virus 5. 7 However, there is no sufficient evidence from subsequent studies indicating that PIV5 is a simian virus. Therefore, the virus was subsequently renamed PIV5 by International Committee on Taxonomy of Viruses in 2009. Notably, PIV5 was renamed in 2016 to mammalian rubulavirus 5, but the name, "PIV5," will be used in this review. 8 Parainfluenza virus 5 has been associated with human diseases such as Creutzfeldt-Jakob disease, multiple sclerosis, and the common cold, but subsequent studies have been unable to confirm PIV5 as the etiological agent of these diseases. 1, 2, 9 Parainfluenza virus 5 is thought to contribute to upper respiratory infections in dogs, and it is a common component of vaccines designed to prevent canine infectious respiratory disease, also known as "kennel cough." [10] [11] [12] [13] Infection of dogs with PIV5 does not lead to respiratory illness, indicating that PIV5 alone is not pathogenic in dogs. 14, 15 Veterinary vaccines containing live PIV5 have been used in dogs for at least 40 years without any safety concern, 16 suggesting that PIV5 is safe.

In 1994, the first rescue of rabies virus (RABV) from cloned cDNA marked a major milestone in the field of nonsegmented, negativestrand RNA virus (NNSV) research. 17 Since then, more and more reverse genetics systems of NNSVs have been developed. They have become powerful tools in basic virus research and translational research, including their use as vaccine vectors for prevention of infectious diseases and delivery vectors for gene therapy. In the past decades, NNSV members including Sendai virus, vesicular stomatitis Abbreviations: Ad5, adenovirus 5; CTL, cytotoxic T lymphocyte; GE, gene end; GFP, green fluorescent protein; GM-CSF, Granulocyte-macrophage colonystimulating factor; GS, gene start; HPAI, highly pathogenic avian influenza; i.m., intramuscular; i.n., intranasal; IFN, interferon; NA, neuraminidase; NDV, Newcastle disease virus; NNSV, nonsegmented negative-strand RNA virus; nt, nucleotides; PFU, Plaque-forming unit; PIV5, Parainfluenza virus 5; RABV, Rabies virus; RSV, respiratory syncytial virus; s.c., subcutaneous; TB, tuberculosis; VV, vaccinia virus virus, NDV, and RABV have been extensively explored for these applications. [18] [19] [20] [21] Parainfluenza virus 5 has also emerged as a novel and attractive vector in vaccine studies. Parainfluenza virus 5 is an NNSV member that infects host respiratory epithelium, making it an attractive vector for developing live-vectored vaccines that induce protective mucosal immune responses. So far, a number of PIV5-based vaccine candidates have been successful in protecting against viral and bacterial infections in multiple animal models, suggesting that PIV5 vector is highly worthy of further exploration in the field of vaccine research. This is the first review of PIV5-vectored vaccines against human and animal infectious diseases (Table 1) , along with discussion of the advantages of the PIV5 vaccine vector platform to aid future vaccine design and to accelerate progression of PIV5-based vaccines into clinical trials.

Parainfluenza virus 5 has a nonsegmented genome consisting of a single strand of negative-sense RNA that is 15 246 nucleotides (nt) in length. The total length of the virus genome is a multiple of 6 and encapsidated by the N protein, which provides protection from nuclease digestion. The genome, flanked by 3′-leader and 5′-trailer sequences, includes 7 nonoverlapping genes in the order of 3′-NP-V/ P-M-F-SH-HN-L-5′. It encodes the nucleocapsid protein (NP), V protein (V), phosphoprotein (P), matrix protein (M), fusion protein (F), small hydrophobic protein (SH), hemagglutinin-neuraminidase (HN), and RNA polymerase large protein (L). 3, 22 V is encoded by a single gene (V/P) derived from the unedited RNA. P is generated by RNA editing of V/P gene, in which the insertion of two nontemplated guanine nucleotides during transcription results in a frame shift during translation. 23 The RNA-dependent RNA polymerase of PIV5 consists of two proteins: P and L. The L protein is responsible for the majority of enzymatic activities involved in viral RNA replication and transcription, as well as the addition of the 5′ cap structure and 3′ poly(A) sequence. 3 Parainfluenza virus 5 RNA genomes, including negative-sense genome and positivesense antigenome, are encapsidated by NP, forming helical ribonucleoproteins, which are essential for virus assembly and budding.

The genome of PIV5 contains noncoding regions (gene junctions) between each gene that range in sizes of approximately 118 to 256 nt. The noncoding regions involve gene end (GE) transcription signals, intergenic regions, and gene start (GS) signals. These GE and GS signals control transcription termination and reinitiation of upstream and downstream genes. The polar mechanism of PIV5 transcription results in a gradient of mRNA abundance that is highest at the 3′ end of the genome and decreases toward the 5′ end, following the order of NP > V/P > M > F > SH > HN > L ( Figure 1B ). 3, 22 3 | CONSTRUCTION OF RECOMBINANT PIV5 VIRUSES EXPRESSING FOREIGN GENES The reverse genetics system to rescue PIV5 was first established in 1997. 24 The virus is rescued from a cloned cDNA that contains the full genome sequence in the positive-sense orientation flanked by a T7 promoter and hepatitis delta virus ribozyme. To rescue PIV5, cells are infected with a recombinant vaccinia virus expressing T7 RNA polymerase (vTF7.3) and then transfected with the PIV5 molecular clone along with helper plasmids encoding NP, P, and L genes. Viral RNA synthesized by T7 RNA polymerase is encapsidated with NP and associates with the polymerase complex, composed of P and L. The polymerase complex transcribes and replicates the genome, and progeny can be used in place of vTF7.3-infected cells. 25 Another approach, which we prefer to use in our laboratory, involves construction of a eukaryotic plasmid expressingT7 RNA polymerase that is cotransfected with the PIV5 infectious clone plasmid and NP, P, L-encoding plasmids ( Figure 1C ). This method has the added benefit of enabling virus rescue PIV5-G will not be neutralized by anti-rabies antibodies. The attenuated RABV vaccine also has safety concerns when injected into the human brain.

Respiratory syncytial virus is one of the leading causes of respiratory illness that results in mortality and morbidity in young children, immunocompromised individuals, and senior citizens. Thus far, there is no licensed RSV vaccine, and a safe and efficacious RSV vaccine remains an unmet need. Two PIV5-vectored vaccines expressing RSV glycoproteins F (PIV5/F) and G (PIV5/G) were evaluated in animal models for their immunogenicity and efficacy of protection against RSV infection. 52 First, PIV5/F and PIV5/G were examined in mice for proof-ofconcept testing. It was found that serum neutralizing antibodies were generated in PIV5/F-immunized mice but not in PIV5/G-immunized mice. Despite this, reduced viral burden was found in the lungs of PIV5/G-immunized mice presumably because RSV G-specific antibodies are protective independent of conventional neutralization activity in vitro. This work demonstrated that a single-dose immunization with PIV5/F or PIV5/G elicited protective immunity against RSV challenge in mice. PIV5/F and PIV5/G were further evaluated as single-dose inoculations in more relevant preclinical animal models. 53 In cotton rats, both PIV5/F and PIV5/G elicited RSV-specific serum antibodies and conferred complete protection in the lung against RSV challenge.

In African green monkeys, PIV5/F conferred the greatest reduction in post-challenge RSV titers in the respiratory tract, while PIV5/G was relatively less efficacious. The PIV5 vaccines were also able to boost RSV neutralizing antibody responses in African green monkeys with prior exposure to RSV. These studies demonstrate that the PIV5 is a promising vaccine vector for RSV-naïve and RSV-exposed persons (pediatric and elderly) against RSV infection, with PIV5/F as a superior RSV vaccine candidate.

Most recently, additional work was published on improved PIV5/F vaccine candidates containing PIV5 vector or RSV-F antigen modifications. 54 

Efforts in using viruses as delivery vectors for vaccines have been fraught with difficulty in the fields of human and veterinary medicine.

If humans or animals have preexisting immunity (especially neutralizing antibodies) to viral vectors, it will theoretically inhibit virus entry into host cells, thereby reducing the dose and antigenicity of vectored antigens. 56 To determine if the presence of preexisting immunity is detrimental to the efficacy of a PIV5-vectored vaccine, dogs with prior exposure to PIV5 were inoculated with PIV5-H3, and efficacy of PIV5-H3 (HA of influenza virus subtype H3) was measured. 16 Dogs seroconverted 2 weeks postinoculation, and the hemaggluitination inhibition antibody titers against an H3N2 virus were greater than 40, which is considered protective in immunological standards, by 3 weeks postinoculation. These results indicate that prior exposure to PIV5 does not prevent a PIV5-vectored vaccine from generating protective immunity. These results are consistent with the previous findings that anti-PIV5 antibodies do not prevent PIV5 infection in mice. 57 The exposure of PIV5 in human populations has also been investigated. Neutralizing antibodies against PIV5 were detected in 29% of human serum samples, but the titers were lower than those in dogs with prior exposure to PIV5. 16 These results suggest that PIV5 vaccines may be able to overcome preexisting immunity to induce immunogenic and protective immune responses against pathogen infections in humans.

Safety is always a critical concern in vaccine research and development. As previously mentioned, PIV5 has a highly stable genome and replicates in the cytoplasm, eliminating the possibility of viral genome integration into the DNA of host cells. Parainfluenza virus 5 is considered nonpathogenic, or very low virulence, to multiple animal species and humans. Therefore, there is no concern for virulence reversion or residual virulence for PIV5 when used as a vaccine vector, unlike with some live-attenuated pathogen vectors. 27 There is concern that expressing a foreign viral envelope protein using a vaccine vector may expand tropism or pathogenicity of the viral vector. Thus far, there is no evidence indicating that this has occurred in PIV5 vaccine research. For example, a PIV5 vaccine expressing influenza virus HA protein (PIV5-H3) has been tested in nude mice to address the issue of potentially enhanced pathogenicity. 38 There were no signs of illness or weight loss observed in these immune-deficient mice when they were infected with PIV5 or PIV5-H3. Consistent with above findings, recombinant PIV5-vaccinated ferrets and mice did not display any clinical signs of disease or discernable pathology. 57 

An ideal virus-vectored vaccine should not only elicit robust B cellmediated protective humoral immune responses but also generate antigen-specific CD8 + T cells and CD4 + T cells. As previously discussed, PIV5 vector is able to induce strong humoral immunity and protection for a variety of vaccine targets. Induction of cellular immunity is also critical for protection against some pathogens, but the induction of cellular immunity by PIV5 has not been investigated in depth. The ability of PIV5 to induce a cellular immune response was tested using PIV5 expressing a model antigen, chicken ovalbumin.

In this experiment, mice were inoculated intranasally and T-cell responses were assessed. 60 Vaccination elicited a strong and long-lasting cytotoxic T lymphocyte (CTL) response with high avidity against ovalbumin. This result suggests that PIV5 is a good vaccine vector for viral antigens, since a high avidity CTL response is optimal for virus clearance. 61 Since PIV5 replication primarily occurs in the respiratory tract, this quality makes it an attractive vaccine vector for generating high avidity CTL responses against respiratory or mucosal pathogen infection.

Tremendous advancements in viral vaccine vector development have been made during the past decades. These advancements rely on improved understanding of viral biology and updated insight into reciprocal interactions between viruses and the host immune system. 62, 63 Currently, viral vectored vaccines remain one of the best strategies for the induction of robust humoral and cellular immunity against human and animal infectious diseases. Parainfluenza virus 5 has become an attractive vector in the field of vaccine research, particularly to develop vaccines that require induction of a protective mucosal immune response. The use of PIV5 as a vector appears to pose no major risk to animal and human health because there is no concern of virulence reversion, residual virulence, or virus recombination. In the future, the design and immunization strategy of the PIV5 vector will be further optimized to induce more potent and complete protective immunity in animals and humans to reduce disease and defend against infectious pathogens.

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How to cite this article: Chen Z. Parainfluenza virus 5-vectored vaccines against human and animal infectious diseases

The author has no competing interest.

Zhenhai Chen http://orcid.org/0000-0002-6704-7546