JGV Papers in Press. Published February 9, 2015 as doi:10.1099/vir.0.000083
Journal of General Virology Recent vaccine development for human metapneumovirus --Manuscript Draft-Manuscript Number:
JGV-D-14-00071R1
Full Title:
Recent vaccine development for human metapneumovirus
Article Type:
Review
Section/Category:
Animal - Negative-strand RNA Viruses
Corresponding Author:
Xiaoyong Bao University of Texas Medical Branch Galveston, TX UNITED STATES
First Author:
Junping Ren
Order of Authors:
Junping Ren Thien Phan Xiaoyong Bao
Abstract:
Human metapneumovirus (hMPV) and its close family member respiratory syncytial virus (RSV) are two major causes of lower respiratory tract infection in the pediatrics population. hMPV is also a common cause of morbidity and mortality worldwide in the immunocompromised patients and older adults. Repeated infections occur often demonstrating a heavy medical burden. However, there is currently no hMPV-specific prevention treatment. This review focuses on the current literatures on hMPV vaccine development. We believe that a better understanding of the role(s) of viral proteins in host responses might lead to efficient prophylactic vaccine development.
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Recent vaccine development for human metapneumovirus
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Junping Rena, Thien Phana & Xiaoyong Baoa,b,c *
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Running Title: hMPV vaccine development
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Author Affiliations
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a
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&Immunity, University of Texas Medical Branch, Galveston, TX.
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*Correspondence should be addressed to Xiaoyong Bao, Ph.D..
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Department of Pediatrics, Division of Clinical and Experimental Immunology & Infectious
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Diseases, 301 University Blvd., Galveston, TX 77555-0372; Fax (409)772-0460;
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Department of Pediatrics, bInstitute for Translational Science, CInstitute for Human Infections
Tel. (409) 772-1777; Email: [email protected]
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Key Words: hMPV, vaccine, and recombinant hMPV.
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Abstract
Human metapneumovirus (hMPV) and its close family member respiratory syncytial
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virus (RSV) are two major causes of lower respiratory tract infection in the pediatrics population.
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hMPV is also a common cause of morbidity and mortality worldwide in the
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immunocompromised patients and older adults. Repeated infections occur often demonstrating a
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heavy medical burden. However, there is currently no hMPV-specific prevention treatment. This
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review focuses on the current literatures on hMPV vaccine development. We believe that a better
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understanding of the role(s) of viral proteins in host responses might lead to efficient
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prophylactic vaccine development.
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Introduction
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Human metapneumovirus (hMPV), a negative sense single-stranded RNA virus, belongs
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to the Paramyxoviridae family that also includes respiratory syncytial virus (RSV) and
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parainfluenza virus (van den Hoogen et al., 2001). Soon after its discovery in 2001, hMPV has
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been quickly recognized as a frequent cause for lower respiratory tract infections in young
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children, the immunocompromised patients and older adults (Edwards et al., 2013;Englund et
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al., 2006;Esper et al., 2004;Falsey et al., 2003). Although hMPV is a clinical important
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pathogen, no vaccine is currently available.
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hMPV comprises two genetic groups, A and B. Each group has two sub-genetic classes,
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i.e. A1 and A2; B1 and B2. Currently, there are five contemporary circulating clades of hMPV,
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which have existed for decades (Gaunt et al., 2011;Papenburg et al., 2013). Its antigenome
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contains nine open reading frames for viral protein expression: 3’-N-P-M-F-M2-1-M2-2-SH-G-
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L-5’. Among them, the nucleoprotein N, phosphoprotein P, and large protein L are essential for
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RNA synthesis, which is comprised of two independent events: viral replication and gene
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transcription. Upon entry, the viral RNA-dependent RNA polymerase (RdRp), formed by the L
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and P proteins, binds the N-encapsidated genome at the leader region, then sequentially
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transcribes each genes by recognizing start and stop signals flanking viral genes. mRNAs are
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capped and polyadenylated during synthesis. Replication presumably starts when enough
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nucleoprotein is present to encapsidate neo-synthetized antigenomes and genomes. The negative-
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sense genome is replicated into a positive-sense antigenome, which serves as a template for
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replication of many copies of the viral genome (Bermingham & Collins, 1999;Schildgen et al.,
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2011;van den Hoogen et al., 2002). In addition to P and L, the M2-2 protein is important for viral
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RNA synthesis as well. Whether hMPV M2-2 is a key regulatory factor involved in the switch of
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the viral RNA polymerase from viral gene transcription to viral genome replication like RSV
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M2-2 protein is still controversial (Buchholz et al., 2005;Kitagawa et al., 2010;Ren et al., 2012).
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The phosphoprotein P, glycoprotein G, small hydrophobic protein SH, and M2-2 protein have
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been shown to modulate host immune responses(Bao et al., 2008a;Bao et al., 2008b;Goutagny et
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al., 2010;Ren et al., 2012;Ren et al., 2014). Recombinant hMPV, lacking G, SH and M-2,
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individually or in combination, or having P replaced with avian P, are attenuated (Biacchesi et
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al., 2005;Pham et al., 2005). Fusion protein F is essential for hMPV entry and also induces a
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strong humoral immune response (Cox et al., 2014). In this review, we will discuss the recent
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status and efforts for hMPV vaccine development based on the functions of these proteins. We
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hope all these studies will eventually decrease the medical burden caused by such a clinical
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important pathogen- hMPV.
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Inactivated vaccines
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Formalin-inactivated influenza is commonly used for mass immunization because it is in
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good stability, easy for manufacturing, and biologically safe due to the absence of viral
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replication, (http://www.cdc.gov/vaccines/hcp/vis/vis-statements/flu.html). However, the
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vaccination of a formalin-inactivated human RSV vaccine (FI-hRSV) led to enhanced disease
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upon natural infection (Kapikian et al., 1969;Kim et al., 1969). Enhanced disease probably
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resulted from 1) a Th2-biased T cell-memory responses (Boelen et al., 2000;Hussell et al.,
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1997;Openshaw et al., 1992), 2) formaldehyde hypersensitivity (Moghaddam et al., 2006),
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and/or 3) immature antibody production and its associated weak recognition of hRSV epitopes
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from natural infections(Delgado et al., 2009). Recently, decrease in FI-hRSV enhanced disease
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by RSV G glycoprotein peptide was reported, suggesting the antibody specific to RSV G is
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critical for RSV pathogenesis control (Rey et al., 2013). Similarly, vaccine-enhanced pulmonary
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disease and Th2 response following hMPV challenge were also observed in animals vaccinated
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with formalin-inactivated hMPV (Hamelin et al., 2007;Yim et al., 2007), suggesting that
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formalin-inactivated hMPV may not be a suitable vaccine candidate.
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Other inactivation methods are also being investigated for safe vaccine development. For
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example, a nanoemulsion-adjuvanted inactive RSV has been demonstrated to be able to induce
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durable RSV-specific humoral responses, decrease mucus production and increase viral
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clearance, without evidence of Th2 immune mediated pathology (Lindell et al., 2011). In the
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meanwhile, vaccinated mice exhibited an enhanced Th1/Th17 response. Although the safety of
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nanoemulsion-adjuvanted inactive RSV vaccine candidate needs to be further investigated
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(Mukherjee et al., 2011), nanoemulsion inactivation could be applied to hMPV, if nanoemulsion
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inactivated hMPV is immunogenic and protective, and has balanced immune responses.
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Viral protein-based vaccines
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Subunit vaccines are purified or expressed viral proteins, full-length or partial. The
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expressed proteins are usually in a form of virus-like particles (VLPs), nanoparticles, or with
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immune-enhancing adjuvants (Anderson et al., 2013). The most immunogenic protein among
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paramyxoviruses is mainly the fusion protein F. In terms of RSV, a close family member of
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hMPV, its F in a form of nanoparticle is being evaluated in a phase II clinical trial by Novavax
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(Driscoll, 2013).
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Several animal studies using hMPV proteins as subunit vaccine candidates have been
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recently conducted. By using retroviral core particles as a carrier, intraperitoneal injection of
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hMPV F induces a strong humoral immune response against both homologous and heterologous
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strains. Moreover, the induced neutralizing antibodies prevented mortality upon subsequent
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infection of the lungs with both homologous and heterologous viruses, while hMPV glycoprotein
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G vaccination did not induce neutralizing activity (Levy et al., 2013). Similar results were
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observed using an alphavirus replicon- or parainfluenza virus type 3 (PIV3)-based hMPV F
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vaccine (Mok et al., 2008;Tang et al., 2005). It has been also demonstrated that animals
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vaccinated by intramuscular injection of adjuvanted soluble hMPV F proteins develop humoral
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immune response. However, such response diminished rapidly over time (Herfst et al., 2008b).
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Recently, Dr. Williams’ group demonstrated that hMPV VLPs obtained by expressing matrix
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(M) and F protein in suspension-adapted human embryonic kidney epithelial (293-F) cells
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provide immune protection against hMPV replication in the lungs of mice, and are not associated
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with a Th2-skewed cytokine response, Mice immunized with F-M-VLPs mounted an F-specific
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antibody response, and generated CD8+ T cells recognizing an F-protein derived epitope. VLP
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immunization also induced a neutralizing antibody response, which was enhanced by the
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addition of either TiterMax Gold or α-galactosylceramide adjuvant. All of these suggested that
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fusion protein-based vaccine is a potential candidate for hMPV vaccine development (Cox et al.,
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2014).
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Other hMPV proteins which have been used for protein-based vaccine development
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include P and G proteins (Levy et al., 2013;Palavecino et al., 2014). A recombinant bacillus
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Calmette-Guerin (BCG, a carrier to promote immune response against antigens from other
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bacterial, parasitic, and viral pathogens) expressing hMPV P protein is able to confer strong
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effector phenotypes to both CD4+ and CD8+ T cells, which showed protective hMPV immunity
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equivalent to actively immunized animals. However, several groups have suggested that hMPV
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G-based subunit did not develop protective antibodies, suggesting hMPV G is not important for
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immunogenicity (Levy et al., 2013;Ryder et al., 2010;Skiadopoulos et al., 2006). Interestingly,
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studies using recombinant hMPV lacking G protein (rhMPV-G) suggested that G protein plays
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an important role in inducing protective immune responses (Biacchesi et al., 2004b). Although
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the results on the role of G in immunogenicity are still controversial, there are several
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possibilities may contribute to unsuccessful immunogenicity of G during the single protein
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immunization process. One possibility is that hMPV G undergoes certain modification on the
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level of gene and/or protein during the single protein immunization, similar to what has been
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described for RSV F protein(Yang et al., 2013).Another possibility is that same carriers may
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have reduced ability to incorporate G than F (Levy et al., 2013). Overall, whether G is important
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in immunogenicity still needs to be clarified.
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Overall, the immunization using hMPV F-based subunit vaccine is promising; but more
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experiments are needed to determine the combination of inoculation routes, carrier forms, and
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length of F to induce the best immunogenicity efficacy and duration. Since other hMPV proteins
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are also important for immunogenicity and immune balance, subunit immunization requires more
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investigation on the effect of immunization on Th1/Th2/Th17 balance.
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Live attenuated vaccines:
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Live attenuated vaccines can be divided into two groups: non-recombinant and
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recombinant. Non-recombinant live attenuated viruses are usually generated by natural
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mutations/deletions during viral passages in cells with or without experimental stresses such as
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chemical mutagenesis and cold passage (Crowe, Jr. et al., 1995;Juhasz et al., 1997;Whitehead et
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al., 1998).The major risk of non-recombinant live attenuated vaccine is its in vivo reversion and
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recovery of viral pathogenicity and subsequent disease development. Some non-recombinant
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live-attenuated RSV vaccines have been evaluated in clinical trials, but showed some side effects
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and also insufficient attenuation (Wright et al., 2000). Temperature-sensitive hMPV strains have
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been generated recently by the group of Drs. Fouchier and Osterhaus. Immunized hamsters
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showed protective immunity (Herfst et al., 2008a).
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The recombinant live-attenuated viruses are generated from the cells transfected with
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hMPV cDNA genome, with/without gene modification/deletion, along with plasmids encoding
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individual proteins essential for forming RdPp complex (Bao et al., 2008b;Biacchesi et al.,
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2004a;Ren et al., 2012). The attenuation of recombinant hMPV has been achieved by the
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deletion of certain accessory genes. They are recombinant hMPV lacking G (rhMPV-∆G), G and
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SH (rhMPV-∆G/SH), and M2-2 (rhMPV-∆M2-2) (Biacchesi et al., 2004b;Biacchesi et al.,
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2005;Buchholz et al., 2005). In infected hamsters, rhMPV-∆G and rhMPV-∆G/SH were at least
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40-fold and 600-fold restricted in replication in the lower and upper respiratory tract,
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respectively, compared to wild-type rhMPV. However, in rodent model, rhMPV lacking SH
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alone (rhMPV-ΔSH) replicated somewhat more efficiently in hamster lungs when compared to
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wild-type (WT)-rhMPV, indicating that SH is completely dispensable in vivo and that its deletion
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does not confer an attenuating effect. In infected African green monkeys, the attenuation of
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rhMPV-∆M2-2 reached higher level than that of rhMPV-∆G, and had induce comparable
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immunogenicity and protective efficiency (Biacchesi et al., 2005).There is another attenuated
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recombinant hMPV whose P protein was replaced with avian MPV P protein (rhMPV-Pavian).
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Recently, a wild type recombinant hMPV has been approved to be a suitable parent virus for
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development of live-attenuated hMPV vaccine candidates in experimental human infection trial
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(Talaat et al., 2013). Although rhMPV-Pavian was found to be poorly infectious in healthy
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adults (Schmidt, 2011), the mechanisms associated with poor infectivity of rhMPV-Pavian and
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whether other attenuated recombinant viruses have a similar human infectivity issues need to be
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investigated in the future.
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Other factors should be considered in designing future vaccines.
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Although F protein is believed to be a major factor determining the immunogenicity of
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hMPV, identification of viral antigens that activate both protective cytotoxic T-lymphocyte
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(CTL) and humoral responses are still necessary to develop a successful vaccination strategy.
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Indeed, several CTL peptides have been proved to be important for CD8+ CTL responses to
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hMPV challenge. These peptides are 164VGALIFTKL172 from N for H-2b mice, 56CYLENIEII64
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from M2-2 protein for H-2d mice, and 35KLILALLTFL44 from SH protein and 32SLILIGITTL41
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from G protein for HLA-A*0201 transgenic mice. Vaccination with these hMPV CTL epitopes
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upregulates expression of Th1-type cytokines in the lungs and peribronchial lymph nodes of
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hMPV-challenged mice, resulted in reduced viral titers and disease in mouse models (Herd et al.,
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2006). Recently, dominant and subdominant hMPV H-2d epitopes were screened and identified
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(Melendi et al., 2007). Among 12 selected predicted epitopes, 81GYIDDNQSI89 of M2-1 and
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307
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respectively during primary infection. In addition, a wide diversification of H-2d-restricted
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epitopes, in the memory CTL response after secondary and third infection were also identified,
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including a subdominant role for the memory CTL response against 56CYLENIEII64 of M2-2.
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Given the importance of CTL epitopes in the immunogenicity, the deficiency of such epitope(s)
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by complete gene deletion in live attenuated rhMPV may lead to the reduced ability of rhMPV to
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induce the immunogenicity. To prolong immunogenicity of F protein-based vaccination or to
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enhance the immunogenicity of deletion mutants of rhMPV, co-immunizing the host with
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peptides containing CTL epitopes may be a good option.
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SPKAGLLSL315 of N were identified to be dominant and subdominant H-2d epitope
In addition, since the epitopes identified in the N and M2-2 proteins are completely
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conserved, protection afforded by a CTL epitope vaccine is expected to extend to both hMPV
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strains. Identifying viral proteins which are important for antiviral signaling regulation is also
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critical in vaccine design. Recently, we identified that some viral proteins, such as G and M2-2,
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play significant roles in suppressing hMPV-induced host innate immunity (Bao et al., 2008b;Bao
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et al., 2013;Kolli et al., 2011;Ren et al., 2012). Regarding M2-2 protein, we and others found
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that it is a protein with multiple functions. It not only regulates the viral gene transcription and
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viral RNA replication(Buchholz et al., 2005;Ren et al., 2012), but also contains a CTL epitope
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and targets central adaptors for RIG-I and TLRs (Herd et al., 2006;Kitagawa et al., 2010;Ren et
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al., 2012;Ren et al., 2014). In addition, M2-2 also plays a significant role in regulating the
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expression of miRNAs, some of which are important for the expression of immune related genes
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(Deng et al., 2014). The multi-functions of viral protein(s) raise the need to identify the domains
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respectively responsible for their function, as it is important for rational design of live attenuated
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recombinant virus. Recently, we identified that the regulatory domains of M2-2 for viral gene
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and genome replication are different (Ren et al., 2012). We also identified M2-2 motifs which
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are responsible for their inhibition on antiviral signaling (manuscript in preparation). All these
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pieces of information on M2-2 might provide a foundation to design M2-2-based live attenuated
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vaccine candidates. For example, mutants containing mutations on 1) M2-2’s viral replication
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domain for replication attenuation purpose, and 2) protein interactive motifs to abolish M2-2’s
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suppression on antiviral signaling for immunogenicity enhancement. On the other hand, the
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domains which are important for the transcription of viral genes should not be modified in order
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to 1) minimize frequent mutations of other viral proteins (Schickli et al., 2008), 2) prevent
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skewedTh1/Th2 balance (Becker, 2006), and 3) maintain all naïve CTL epitopes for
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immunogenicity purpose (Herd et al., 2006), Overall, dissecting the functional domains of viral
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protein is highly desirable for vaccine development.
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Discussion
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An efficient vaccine candidate should ideally be safe and more immunogenic and protective than
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natural hMPV infection, which only launches incomplete immune protection. Studies in cotton
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rats revealed that immunization with FI-hMPV enhanced pathology in the lungs of animals after
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subsequent infection with hMPV (Yim et al., 2007), excluding it as a promising candidate.
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Subunit vaccines seem to induce short protective in response to primary infection (Herfst et al.,
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2008b). However, they may be useful to boost the immune response in individuals that have
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previously been exposed to hMPV. In addition, subunit vaccines are promising and safe,
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especially in the form of non-infectious carrier, to provide certain duration of protection for the
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risk groups such as premature infants, immunocompromised individuals and the elderly. Current
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live attenuated hMPV vaccines are promising especially to those infants and young children.
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However, the balance between a satisfactory degree of attenuation and a satisfactory level of
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immunogenicity may be difficult to obtain. We are currently exploring the possibilities to
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identify major immune regulatory protein(s) and associated functional motifs with an aim to
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develop vaccine candidates carrying decent attenuation and immunogenicity. Overall, a variety
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of vaccination strategies have been explored and tested in rodent models and non-human primate
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models. However, none has yet been tested in human volunteers. Recent experimental infection
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of adults with recombinant wild-type hMPV have been performed, demonstrating possibilities to
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test the immune efficiency of live attenuated recombinant hMPV in human in the future
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Acknowledgements
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All authors concur there are no conflicts of interest associated in this published work. This work
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was supported by grants from the National Institutes of Health-National Institute of Allergy and
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Infectious DiseasesR56 AI107033-01A1, Research Pilot Grant from John Sealy Memorial
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Foundation, UTMB, the American Lung Association RG232529N, and American Heart
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Association 12BGIA12060008 to X.B.
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Journal of General Virology Recent vaccine development for human metapneumovirus --Manuscript Draft-Manuscript Number:
JGV-D-14-00071R1
Full Title:
Recent vaccine development for human metapneumovirus
Article Type:
Review
Section/Category:
Animal - Negative-strand RNA Viruses
Corresponding Author:
Xiaoyong Bao University of Texas Medical Branch Galveston, TX UNITED STATES
First Author:
Junping Ren
Order of Authors:
Junping Ren Thien Phan Xiaoyong Bao
Abstract:
Human metapneumovirus (hMPV) and its close family member respiratory syncytial virus (RSV) are two major causes of lower respiratory tract infection in the pediatrics population. hMPV is also a common cause of morbidity and mortality worldwide in the immunocompromised patients and older adults. Repeated infections occur often demonstrating a heavy medical burden. However, there is currently no hMPV-specific prevention treatment. This review focuses on the current literatures on hMPV vaccine development. We believe that a better understanding of the role(s) of viral proteins in host responses might lead to efficient prophylactic vaccine development.
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1
Recent vaccine development for human metapneumovirus
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Junping Rena, Thien Phana & Xiaoyong Baoa,b,c *
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Running Title: hMPV vaccine development
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Author Affiliations
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a
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&Immunity, University of Texas Medical Branch, Galveston, TX.
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*Correspondence should be addressed to Xiaoyong Bao, Ph.D..
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Department of Pediatrics, Division of Clinical and Experimental Immunology & Infectious
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Diseases, 301 University Blvd., Galveston, TX 77555-0372; Fax (409)772-0460;
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Department of Pediatrics, bInstitute for Translational Science, CInstitute for Human Infections
Tel. (409) 772-1777; Email: [email protected]
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Key Words: hMPV, vaccine, and recombinant hMPV.
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20
Abstract
Human metapneumovirus (hMPV) and its close family member respiratory syncytial
21
virus (RSV) are two major causes of lower respiratory tract infection in the pediatrics population.
22
hMPV is also a common cause of morbidity and mortality worldwide in the
23
immunocompromised patients and older adults. Repeated infections occur often demonstrating a
24
heavy medical burden. However, there is currently no hMPV-specific prevention treatment. This
25
review focuses on the current literatures on hMPV vaccine development. We believe that a better
26
understanding of the role(s) of viral proteins in host responses might lead to efficient
27
prophylactic vaccine development.
28
Introduction
29
Human metapneumovirus (hMPV), a negative sense single-stranded RNA virus, belongs
30
to the Paramyxoviridae family that also includes respiratory syncytial virus (RSV) and
31
parainfluenza virus (van den Hoogen et al., 2001). Soon after its discovery in 2001, hMPV has
32
been quickly recognized as a frequent cause for lower respiratory tract infections in young
33
children, the immunocompromised patients and older adults (Edwards et al., 2013;Englund et
34
al., 2006;Esper et al., 2004;Falsey et al., 2003). Although hMPV is a clinical important
35
pathogen, no vaccine is currently available.
36
hMPV comprises two genetic groups, A and B. Each group has two sub-genetic classes,
37
i.e. A1 and A2; B1 and B2. Currently, there are five contemporary circulating clades of hMPV,
38
which have existed for decades (Gaunt et al., 2011;Papenburg et al., 2013). Its antigenome
39
contains nine open reading frames for viral protein expression: 3’-N-P-M-F-M2-1-M2-2-SH-G-
40
L-5’. Among them, the nucleoprotein N, phosphoprotein P, and large protein L are essential for
41
RNA synthesis, which is comprised of two independent events: viral replication and gene
42
transcription. Upon entry, the viral RNA-dependent RNA polymerase (RdRp), formed by the L
43
and P proteins, binds the N-encapsidated genome at the leader region, then sequentially
44
transcribes each genes by recognizing start and stop signals flanking viral genes. mRNAs are
45
capped and polyadenylated during synthesis. Replication presumably starts when enough
46
nucleoprotein is present to encapsidate neo-synthetized antigenomes and genomes. The negative-
47
sense genome is replicated into a positive-sense antigenome, which serves as a template for
48
replication of many copies of the viral genome (Bermingham & Collins, 1999;Schildgen et al.,
49
2011;van den Hoogen et al., 2002). In addition to P and L, the M2-2 protein is important for viral
50
RNA synthesis as well. Whether hMPV M2-2 is a key regulatory factor involved in the switch of
51
the viral RNA polymerase from viral gene transcription to viral genome replication like RSV
52
M2-2 protein is still controversial (Buchholz et al., 2005;Kitagawa et al., 2010;Ren et al., 2012).
53
The phosphoprotein P, glycoprotein G, small hydrophobic protein SH, and M2-2 protein have
54
been shown to modulate host immune responses(Bao et al., 2008a;Bao et al., 2008b;Goutagny et
55
al., 2010;Ren et al., 2012;Ren et al., 2014). Recombinant hMPV, lacking G, SH and M-2,
56
individually or in combination, or having P replaced with avian P, are attenuated (Biacchesi et
57
al., 2005;Pham et al., 2005). Fusion protein F is essential for hMPV entry and also induces a
58
strong humoral immune response (Cox et al., 2014). In this review, we will discuss the recent
59
status and efforts for hMPV vaccine development based on the functions of these proteins. We
60
hope all these studies will eventually decrease the medical burden caused by such a clinical
61
important pathogen- hMPV.
62
Inactivated vaccines
63
Formalin-inactivated influenza is commonly used for mass immunization because it is in
64
good stability, easy for manufacturing, and biologically safe due to the absence of viral
65
replication, (http://www.cdc.gov/vaccines/hcp/vis/vis-statements/flu.html). However, the
66
vaccination of a formalin-inactivated human RSV vaccine (FI-hRSV) led to enhanced disease
67
upon natural infection (Kapikian et al., 1969;Kim et al., 1969). Enhanced disease probably
68
resulted from 1) a Th2-biased T cell-memory responses (Boelen et al., 2000;Hussell et al.,
69
1997;Openshaw et al., 1992), 2) formaldehyde hypersensitivity (Moghaddam et al., 2006),
70
and/or 3) immature antibody production and its associated weak recognition of hRSV epitopes
71
from natural infections(Delgado et al., 2009). Recently, decrease in FI-hRSV enhanced disease
72
by RSV G glycoprotein peptide was reported, suggesting the antibody specific to RSV G is
73
critical for RSV pathogenesis control (Rey et al., 2013). Similarly, vaccine-enhanced pulmonary
74
disease and Th2 response following hMPV challenge were also observed in animals vaccinated
75
with formalin-inactivated hMPV (Hamelin et al., 2007;Yim et al., 2007), suggesting that
76
formalin-inactivated hMPV may not be a suitable vaccine candidate.
77
Other inactivation methods are also being investigated for safe vaccine development. For
78
example, a nanoemulsion-adjuvanted inactive RSV has been demonstrated to be able to induce
79
durable RSV-specific humoral responses, decrease mucus production and increase viral
80
clearance, without evidence of Th2 immune mediated pathology (Lindell et al., 2011). In the
81
meanwhile, vaccinated mice exhibited an enhanced Th1/Th17 response. Although the safety of
82
nanoemulsion-adjuvanted inactive RSV vaccine candidate needs to be further investigated
83
(Mukherjee et al., 2011), nanoemulsion inactivation could be applied to hMPV, if nanoemulsion
84
inactivated hMPV is immunogenic and protective, and has balanced immune responses.
85
Viral protein-based vaccines
86
Subunit vaccines are purified or expressed viral proteins, full-length or partial. The
87
expressed proteins are usually in a form of virus-like particles (VLPs), nanoparticles, or with
88
immune-enhancing adjuvants (Anderson et al., 2013). The most immunogenic protein among
89
paramyxoviruses is mainly the fusion protein F. In terms of RSV, a close family member of
90
hMPV, its F in a form of nanoparticle is being evaluated in a phase II clinical trial by Novavax
91
(Driscoll, 2013).
92
Several animal studies using hMPV proteins as subunit vaccine candidates have been
93
recently conducted. By using retroviral core particles as a carrier, intraperitoneal injection of
94
hMPV F induces a strong humoral immune response against both homologous and heterologous
95
strains. Moreover, the induced neutralizing antibodies prevented mortality upon subsequent
96
infection of the lungs with both homologous and heterologous viruses, while hMPV glycoprotein
97
G vaccination did not induce neutralizing activity (Levy et al., 2013). Similar results were
98
observed using an alphavirus replicon- or parainfluenza virus type 3 (PIV3)-based hMPV F
99
vaccine (Mok et al., 2008;Tang et al., 2005). It has been also demonstrated that animals
100
vaccinated by intramuscular injection of adjuvanted soluble hMPV F proteins develop humoral
101
immune response. However, such response diminished rapidly over time (Herfst et al., 2008b).
102
Recently, Dr. Williams’ group demonstrated that hMPV VLPs obtained by expressing matrix
103
(M) and F protein in suspension-adapted human embryonic kidney epithelial (293-F) cells
104
provide immune protection against hMPV replication in the lungs of mice, and are not associated
105
with a Th2-skewed cytokine response, Mice immunized with F-M-VLPs mounted an F-specific
106
antibody response, and generated CD8+ T cells recognizing an F-protein derived epitope. VLP
107
immunization also induced a neutralizing antibody response, which was enhanced by the
108
addition of either TiterMax Gold or α-galactosylceramide adjuvant. All of these suggested that
109
fusion protein-based vaccine is a potential candidate for hMPV vaccine development (Cox et al.,
110
2014).
111
Other hMPV proteins which have been used for protein-based vaccine development
112
include P and G proteins (Levy et al., 2013;Palavecino et al., 2014). A recombinant bacillus
113
Calmette-Guerin (BCG, a carrier to promote immune response against antigens from other
114
bacterial, parasitic, and viral pathogens) expressing hMPV P protein is able to confer strong
115
effector phenotypes to both CD4+ and CD8+ T cells, which showed protective hMPV immunity
116
equivalent to actively immunized animals. However, several groups have suggested that hMPV
117
G-based subunit did not develop protective antibodies, suggesting hMPV G is not important for
118
immunogenicity (Levy et al., 2013;Ryder et al., 2010;Skiadopoulos et al., 2006). Interestingly,
119
studies using recombinant hMPV lacking G protein (rhMPV-G) suggested that G protein plays
120
an important role in inducing protective immune responses (Biacchesi et al., 2004b). Although
121
the results on the role of G in immunogenicity are still controversial, there are several
122
possibilities may contribute to unsuccessful immunogenicity of G during the single protein
123
immunization process. One possibility is that hMPV G undergoes certain modification on the
124
level of gene and/or protein during the single protein immunization, similar to what has been
125
described for RSV F protein(Yang et al., 2013).Another possibility is that same carriers may
126
have reduced ability to incorporate G than F (Levy et al., 2013). Overall, whether G is important
127
in immunogenicity still needs to be clarified.
128
Overall, the immunization using hMPV F-based subunit vaccine is promising; but more
129
experiments are needed to determine the combination of inoculation routes, carrier forms, and
130
length of F to induce the best immunogenicity efficacy and duration. Since other hMPV proteins
131
are also important for immunogenicity and immune balance, subunit immunization requires more
132
investigation on the effect of immunization on Th1/Th2/Th17 balance.
133
Live attenuated vaccines:
134
Live attenuated vaccines can be divided into two groups: non-recombinant and
135
recombinant. Non-recombinant live attenuated viruses are usually generated by natural
136
mutations/deletions during viral passages in cells with or without experimental stresses such as
137
chemical mutagenesis and cold passage (Crowe, Jr. et al., 1995;Juhasz et al., 1997;Whitehead et
138
al., 1998).The major risk of non-recombinant live attenuated vaccine is its in vivo reversion and
139
recovery of viral pathogenicity and subsequent disease development. Some non-recombinant
140
live-attenuated RSV vaccines have been evaluated in clinical trials, but showed some side effects
141
and also insufficient attenuation (Wright et al., 2000). Temperature-sensitive hMPV strains have
142
been generated recently by the group of Drs. Fouchier and Osterhaus. Immunized hamsters
143
showed protective immunity (Herfst et al., 2008a).
144
The recombinant live-attenuated viruses are generated from the cells transfected with
145
hMPV cDNA genome, with/without gene modification/deletion, along with plasmids encoding
146
individual proteins essential for forming RdPp complex (Bao et al., 2008b;Biacchesi et al.,
147
2004a;Ren et al., 2012). The attenuation of recombinant hMPV has been achieved by the
148
deletion of certain accessory genes. They are recombinant hMPV lacking G (rhMPV-∆G), G and
149
SH (rhMPV-∆G/SH), and M2-2 (rhMPV-∆M2-2) (Biacchesi et al., 2004b;Biacchesi et al.,
150
2005;Buchholz et al., 2005). In infected hamsters, rhMPV-∆G and rhMPV-∆G/SH were at least
151
40-fold and 600-fold restricted in replication in the lower and upper respiratory tract,
152
respectively, compared to wild-type rhMPV. However, in rodent model, rhMPV lacking SH
153
alone (rhMPV-ΔSH) replicated somewhat more efficiently in hamster lungs when compared to
154
wild-type (WT)-rhMPV, indicating that SH is completely dispensable in vivo and that its deletion
155
does not confer an attenuating effect. In infected African green monkeys, the attenuation of
156
rhMPV-∆M2-2 reached higher level than that of rhMPV-∆G, and had induce comparable
157
immunogenicity and protective efficiency (Biacchesi et al., 2005).There is another attenuated
158
recombinant hMPV whose P protein was replaced with avian MPV P protein (rhMPV-Pavian).
159
Recently, a wild type recombinant hMPV has been approved to be a suitable parent virus for
160
development of live-attenuated hMPV vaccine candidates in experimental human infection trial
161
(Talaat et al., 2013). Although rhMPV-Pavian was found to be poorly infectious in healthy
162
adults (Schmidt, 2011), the mechanisms associated with poor infectivity of rhMPV-Pavian and
163
whether other attenuated recombinant viruses have a similar human infectivity issues need to be
164
investigated in the future.
165
Other factors should be considered in designing future vaccines.
166
Although F protein is believed to be a major factor determining the immunogenicity of
167
hMPV, identification of viral antigens that activate both protective cytotoxic T-lymphocyte
168
(CTL) and humoral responses are still necessary to develop a successful vaccination strategy.
169
Indeed, several CTL peptides have been proved to be important for CD8+ CTL responses to
170
hMPV challenge. These peptides are 164VGALIFTKL172 from N for H-2b mice, 56CYLENIEII64
171
from M2-2 protein for H-2d mice, and 35KLILALLTFL44 from SH protein and 32SLILIGITTL41
172
from G protein for HLA-A*0201 transgenic mice. Vaccination with these hMPV CTL epitopes
173
upregulates expression of Th1-type cytokines in the lungs and peribronchial lymph nodes of
174
hMPV-challenged mice, resulted in reduced viral titers and disease in mouse models (Herd et al.,
175
2006). Recently, dominant and subdominant hMPV H-2d epitopes were screened and identified
176
(Melendi et al., 2007). Among 12 selected predicted epitopes, 81GYIDDNQSI89 of M2-1 and
177
307
178
respectively during primary infection. In addition, a wide diversification of H-2d-restricted
179
epitopes, in the memory CTL response after secondary and third infection were also identified,
180
including a subdominant role for the memory CTL response against 56CYLENIEII64 of M2-2.
181
Given the importance of CTL epitopes in the immunogenicity, the deficiency of such epitope(s)
182
by complete gene deletion in live attenuated rhMPV may lead to the reduced ability of rhMPV to
183
induce the immunogenicity. To prolong immunogenicity of F protein-based vaccination or to
184
enhance the immunogenicity of deletion mutants of rhMPV, co-immunizing the host with
185
peptides containing CTL epitopes may be a good option.
186
SPKAGLLSL315 of N were identified to be dominant and subdominant H-2d epitope
In addition, since the epitopes identified in the N and M2-2 proteins are completely
187
conserved, protection afforded by a CTL epitope vaccine is expected to extend to both hMPV
188
strains. Identifying viral proteins which are important for antiviral signaling regulation is also
189
critical in vaccine design. Recently, we identified that some viral proteins, such as G and M2-2,
190
play significant roles in suppressing hMPV-induced host innate immunity (Bao et al., 2008b;Bao
191
et al., 2013;Kolli et al., 2011;Ren et al., 2012). Regarding M2-2 protein, we and others found
192
that it is a protein with multiple functions. It not only regulates the viral gene transcription and
193
viral RNA replication(Buchholz et al., 2005;Ren et al., 2012), but also contains a CTL epitope
194
and targets central adaptors for RIG-I and TLRs (Herd et al., 2006;Kitagawa et al., 2010;Ren et
195
al., 2012;Ren et al., 2014). In addition, M2-2 also plays a significant role in regulating the
196
expression of miRNAs, some of which are important for the expression of immune related genes
197
(Deng et al., 2014). The multi-functions of viral protein(s) raise the need to identify the domains
198
respectively responsible for their function, as it is important for rational design of live attenuated
199
recombinant virus. Recently, we identified that the regulatory domains of M2-2 for viral gene
200
and genome replication are different (Ren et al., 2012). We also identified M2-2 motifs which
201
are responsible for their inhibition on antiviral signaling (manuscript in preparation). All these
202
pieces of information on M2-2 might provide a foundation to design M2-2-based live attenuated
203
vaccine candidates. For example, mutants containing mutations on 1) M2-2’s viral replication
204
domain for replication attenuation purpose, and 2) protein interactive motifs to abolish M2-2’s
205
suppression on antiviral signaling for immunogenicity enhancement. On the other hand, the
206
domains which are important for the transcription of viral genes should not be modified in order
207
to 1) minimize frequent mutations of other viral proteins (Schickli et al., 2008), 2) prevent
208
skewedTh1/Th2 balance (Becker, 2006), and 3) maintain all naïve CTL epitopes for
209
immunogenicity purpose (Herd et al., 2006), Overall, dissecting the functional domains of viral
210
protein is highly desirable for vaccine development.
211
Discussion
212
An efficient vaccine candidate should ideally be safe and more immunogenic and protective than
213
natural hMPV infection, which only launches incomplete immune protection. Studies in cotton
214
rats revealed that immunization with FI-hMPV enhanced pathology in the lungs of animals after
215
subsequent infection with hMPV (Yim et al., 2007), excluding it as a promising candidate.
216
Subunit vaccines seem to induce short protective in response to primary infection (Herfst et al.,
217
2008b). However, they may be useful to boost the immune response in individuals that have
218
previously been exposed to hMPV. In addition, subunit vaccines are promising and safe,
219
especially in the form of non-infectious carrier, to provide certain duration of protection for the
220
risk groups such as premature infants, immunocompromised individuals and the elderly. Current
221
live attenuated hMPV vaccines are promising especially to those infants and young children.
222
However, the balance between a satisfactory degree of attenuation and a satisfactory level of
223
immunogenicity may be difficult to obtain. We are currently exploring the possibilities to
224
identify major immune regulatory protein(s) and associated functional motifs with an aim to
225
develop vaccine candidates carrying decent attenuation and immunogenicity. Overall, a variety
226
of vaccination strategies have been explored and tested in rodent models and non-human primate
227
models. However, none has yet been tested in human volunteers. Recent experimental infection
228
of adults with recombinant wild-type hMPV have been performed, demonstrating possibilities to
229
test the immune efficiency of live attenuated recombinant hMPV in human in the future
230
231
Acknowledgements
232
All authors concur there are no conflicts of interest associated in this published work. This work
233
was supported by grants from the National Institutes of Health-National Institute of Allergy and
234
Infectious DiseasesR56 AI107033-01A1, Research Pilot Grant from John Sealy Memorial
235
Foundation, UTMB, the American Lung Association RG232529N, and American Heart
236
Association 12BGIA12060008 to X.B.
237
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