Progress in Respiratory Virus Vaccine Development

Abstract

Viral respiratory infections cause significant morbidity and mortality in infants and young children as well as in at-risk adults and the elderly. Although many viral pathogens are capable of causing respiratory disease, vaccine development has to focus on a limited number of pathogens, such as those that commonly cause serious lower respiratory illness (LRI). Whereas influenza virus vaccines have been available for some time (see the review by Clark and Lynch in this issue), vaccines against other medically important viruses such as respiratory syncytial virus (RSV), the parainfluenza viruses (PIVs), and metapneumovirus (MPVs) are not available. This review aims to provide a brief update on investigational vaccines against RSV, the PIVs, and MPV that have been evaluated in clinical trials or are currently in clinical development.

The burden of acute lower respiratory illness (ALRI) caused by viral pathogens is impressive and leaves no doubt that effective and affordable vaccines are urgently needed, especially for the prevention of illness in infants, young children, the elderly, and at-risk adults such as those with cardiopulmonary disease or a compromised immune system.^1–4 Globally, ALRI remains the most important cause of postneonatal mortality in children under 5 years of age, accounting for ~1.6 million deaths every year.⁵ Although bacterial pathogens such as Streptococcus pneumoniae and Haemophilus influenzae type b (Hib) are responsible for ~50% of this under-5 ALRI mortality, respiratory syncytial virus (RSV) is by far the most important viral cause of ALRI, with an estimated 3.4 million episodes of severe ALRI necessitating hospital admission and 66,000 to 199,000 deaths annually in children under age 5 years.^1,6

RSV infection is probably the most frequent viral cause of a child’s first lower respiratory illness (LRI), and in industrialized countries approximately one in a hundred healthy term infants is hospitalized with RSV bronchiolitis or pneumonia, often at an age as young as 1 to 5 months. The parainfluenza viruses (PIVs), metapneumoviruses (MPVs), and influenza viruses are other important causes of LRI in infants, with PIV3 and MPV causing LRI almost as early in life as RSV, whereas PIV1, PIV2, and influenza virus disease is more commonly seen in children over 6 months of age. PIV1 and PIV2 are common causes of upper respiratory illness (URI) and LRI in toddlers and in preschool children, with croup as the signature disease of PIV1. Although RSV is on every pediatrician’s mind, PIV3, MPV, and influenza often go undiagnosed. Conversely, in adults with cardiopulmonary disease and in the elderly, influenza morbidity and mortality are widely recognized, whereas the burden of RSV disease is often underappreciated.^7–9 This overview focuses on investigational vaccines against RSV, MPV, and the PIVs. Influenza virus vaccines are discussed by Clark and Lynch in this issue. Rhinovirus, adenovirus, and coronavirus vaccine development efforts are not covered here.

THE VIRUSES

RSV, MPV, and the PIVs are all members of the Paramyxoviridae family of viruses in the order Monogavirales (i.e., they are enveloped viruses that encode a single-stranded, nonsegmented RNA genome of negative polarity). Compared with large DNA viruses such as the Herpesviridae, Paramyxoviridae genomes are small and simple: the genomes of RSV, MPV, and PIV1, for instance, are ~15.2, 13.2, and 15.5 kilobases long, respectively. They encode between 7 (PIV1) and 11 (RSV) proteins. RSV, MPV, and the PIVs all encode two large surface glycoproteins that are targeted by infectivity-neutralizing antibodies and hence are important antigens in vaccine development. One of the glycoproteins, designated G for RSV/MPV and hemagglutinin-neuraminidase (HN) for the PIVs, mediates attachment of the virus to the cell surface. The other surface glycoprotein, designated F, facilitates fusion of the virus envelope with the cell membrane.

PATHOGENESIS AND CORRELATES OF IMMUNITY

Transmission of RSV, MPV, and PIVs can occur via airborne particles or droplets or by direct contact. Upper respiratory tract (URT) epithelial cells are thought to be the usual port of entry, but virus in airborne particles might also be transmitted directly to the lower respiratory tract (LRT).¹⁰ RSV, MPV, and PIV replication is limited to the respiratory tract throughout the course of disease, and viremia or systemic spread does not occur in an immunocompetent host. Virus replication takes place in polarized epithelial cells, and progeny virus buds from the apical surface of these cells into the lumen of the respiratory tract. ^11–13 Primary RSV infection of the URT is estimated to progress to clinical LRI in 25 to 40% of the infected children. Risk factors associated with severe LRI include young age, underlying lung disease (eg, as a result of premature birth), genetic determinants, ^14,15 environmental factors such as cigarette smoke exposure,¹⁶ and malnutrition.¹⁷ The cytopathic effect (CPE) of viral infection in primary respiratory epithelial cells differs greatly between individual respiratory viruses. Although primary influenza virus pneumonia is characterized by extensive CPE with destruction of the respiratory epithelium, RSV and the PIVs cause only limited CPE in primary human airway epithelial cells.¹² Direct viral effects on the function of epithelial cells (eg, reduced ciliary beat and increased mucus production) have been reported but are not consistently observed.^11,18 In untreated fatal RSV disease, cellular infiltrates dominate histopathology, suggesting that the host’s immune response does contribute significantly to clinical disease.¹⁹ Virus titers in the respiratory tract seem to correlate well with the magnitude of an inflammatory response and with disease severity in experimentally infected healthy adults,²⁰ and RSV titers also correlate with illness severity in infants with naturally acquired RSV infection.²¹ Reactive airway disease (RAD) has been described as a long-term consequence of RSV LRTI in the first 2 years of life, and cohort studies suggest that the more severe the LRTI, the longer these consequences persist.^22–24

The contribution of innate immune responses to the resolution of or susceptibility to viral respiratory illness is better appreciated now than just a few years ago.²⁵ This is due in part to an increasing number of genetic susceptibility studies that suggested a role for surfactant proteins, cytokines, and pattern recognition receptors in protection from severe RSV disease,^26–29 but also due to a much broader interest in and better understanding of innate immunity in general. Cytotoxic T lymphocyte (CTL) responses are widely recognized to play an important role in resolving primary infection, and there is some evidence they can contribute to protection against disease upon reinfection.^30–32 However, virus-specific T cell responses are short lived and CTLs may even contribute to RSV pathogenesis.^33,34 T-helper type 1 (Th1) responses are generally viewed as beneficial, whereas Th2 responses have been implicated in adverse outcomes of RSV disease.^35,36 Recent studies observed an inadequate—rather than an exaggerated—T cell response in fatal RSV disease.³⁷ In lung tissues of infants with fatal RSV bronchiolitis, CD20 positive lymphocytes and immunoglobulin M (IgM-), IgG-, and IgA-positive plasma cells were prominent, but CD4 positive T cells were not.³⁸

A high titer of virus neutralizing serum antibody (directed against the two large surface glycoproteins) is the most widely accepted correlate of long-term protection against disease.^39–42 Mucosal antibody induced during primary infection is also very effective in restricting replication of challenge virus, and some studies suggest that mucosal neutralizing antibody is even a better correlate of protection than serum antibody,^43,44 but mucosal antibody titers wane over a period of months following primary infection, and reinfection is needed for titers to persist.⁴¹ High titer serum neutralizing antibody (IgG) may gain access to the luminal surface of the LRT by transudation and restrict virus replication locally. However, transudation is not an efficient mechanism in the URT, where mucosal IgA antibody plays a dominant role as a correlate of long-term protection, and virus-specific IgA and IgM (but not IgG) in nasopharyngeal secretions are associated with better oxygen saturation in infants with RSV or influenza LRI.³⁸ In agreement with these observations, palivizumab, the parenterally administered monoclonal IgG antibody preparation that is used as RSV prophylaxis in high-risk infants, decreases the titer of infectious RSV in tracheal secretions but not nasal secretions of intubated infants within 1 day of palivizumab treatment initiation.⁴⁵ However, the higher-affinity RSV antibody motavizumab has been shown to reduce RSV infectivity and RSV RNA levels in URT secretions, indicating that some transfer of serum IgG across mucosal URT membranes does occur.⁴⁶

CHALLENGES IN RESPIRATORY VIRUS VACCINE DEVELOPMENT

With the exception of influenza virus vaccines and (temporarily) an adenovirus type 4 and 7 vaccine used in the military, licensed vaccines to prevent viral acute respiratory disease are not available. This is notwithstanding an obvious medical need for such vaccines and in spite of many years of active vaccine research, suggesting that there are formidable obstacles to be overcome (Table 1). The first of these challenges is that primary infection with RSV, PIV, or MPV does not provide durable protection against reinfection, although disease upon reinfection is usually ameliorated and restricted to the URT. In adults who were repeatedly experimentally challenged with RSV, at least 25% could be reinfected within 2 months of the first challenge.⁴⁷ Similarly, PIV-1- and MPV-experienced healthy adults can be reinfected with homologous challenge virus, and both virus shedding and URI are observed in 25 to 50% of volunteers.⁴⁴ In infants and children, disease severity decreases with increasing numbers of reinfections.⁴⁸ Therefore it is not surprising that LRI is more frequently seen after primary infection than following secondary or tertiary infections. A realistic goal of immunization has to be a reduction of severe disease rather than induction of sterilizing immunity, similar to what has been achieved with rotavirus vaccines. Second, although a high titer of neutralizing serum IgG is able to prevent LRI via transudation to the luminal surface of the LRT, mucosal immunity is crucial to protect the URT or to prevent spread to the LRT. However, the induction of persisting protective mucosal immunity requires more than one infection, and more than one dose of vaccine will therefore be needed to induce durable immunity. Third, vaccines are needed to protect young infants and the elderly. Different vaccines and separate approaches are likely needed to protect these two high-risk groups. Clinical evaluation of candidate vaccines has to start in healthy young adults and then progress to younger and/or older age groups, thus increasing the time needed for clinical vaccine development. Live attenuated vaccines that are appropriately attenuated for seronegative infants will certainly be overattenuated for the elderly.⁴⁹ Fourth, young infants are less likely to develop high titer neutralizing antibodies.^50,51 A more limited B cell repertoire,⁵² inefficient antigen presentation, and limited T cell help might contribute to this observation. In addition, in the first few months of life, maternally acquired IgG exerts an immunosuppressive effect, particularly on the humoral immune response.⁵³ The use of live attenuated virus vaccines that infect the URT may circumvent the neutralizing effect of maternally acquired serum antibody but this does not necessarily guarantee the induction of a robust immune response.⁵⁴ Clinical trials with attenuated RSV candidate vaccines emphasize how challenging it can be to determine valid correlates of protection in infants younger than 3 months of age. In an RSV vaccine trial in infants and children, for instance, virus neutralizing serum IgG could only be detected in 19% of infants less than 3 months of age, whereas 88% of children between 6 and 36 months of age developed a greater than fourfold increase in neutralizing antibody titers.⁵⁰ In this trial, an increase in virus-specific serum IgA seemed the best but still imperfect correlate of protection, being detected in ~30% of vaccinees. Despite the inability to define a sensitive and specific in vitro correlate of protection, more than 90% of the young infants were protected against replication of a second dose of vaccine virus, indicating that young infants can develop a protective immune response.⁵⁰

Table 1.

Challenges for RSV, PIV, and MPV Vaccine Development

Immunity/Pathogenesis Primary infection with wild type (wt) virus does not induce durable protection Replication is limited to the respiratory epithelium/no viremia Serum antibody does not protect the upper respiratory tract Mucosal antibody titers wane Live attenuated vaccines are less immunogenic than wt virus Attenuation and immunogenicity are linked/narrow therapeutic window Multiple doses of vaccine are needed Formalin-inactivated RSV vaccine induced enhanced disease Regulatory and safety concerns Association between RSV LRI and reactive airway disease/asthma

Difficult target population Infants <4 months of age: More limited antibody repertoire More difficult to induce neutralizing antibodies Maternal antibodies suppress immune response Enrollment into clinical trials is slow Age group experiences sudden infant death syndrome (SIDS) and apnea Older adults Everybody is seropositive (but not necessarily protected) Difficult to define immunogenicity end point

Virus-specific factors Highly infectious viruses able to infect early in life (RSV >MPV >PIV) Poor growth and limited physical stability (RSV only) Viruses efficiently evade the innate immune response Evade detection by hiding danger signals Suppress type 1 interferon response

No animal model that replicates disease Preclinical development relies on surrogate correlates of protection

History of formalin-inactivated RSV vaccine failure Safety and regulatory concerns

LRI, lower respiratory illness; MPV, metapneumovirus; PIV, parainfluenza virus; RSV, respiratory syncytial virus.

APPROACHES TO RSV, PIV, AND MPV VACCINE DEVELOPMENT

Inactivated and Subunit RSV Vaccines

The first attempt at developing an RSV vaccine in the 1960s employed formalin-inactivated (FI) whole virus (FI-RSV) adjuvanted with alum. This vaccine not only failed to induce a protective immune response but led to enhanced disease upon natural infection with wild-type (wt) RSV. As a result, 80% of the vaccinees needed to be hospitalized following wt RSV infection, and two children died.⁵⁵ Subsequently, there has been great apprehension surrounding clinical development of a whole virus, nonreplicating RSV vaccine for use in RSV seronegative infants and children. Enhanced disease has never been observed with live attenuated vaccines, and it is not seen in seropositive subjects, neither with inactivated nor with subunit RSV vaccines.^56,57

Several purified F protein vaccines (Wyeth, PFP-1, PFP-2, and PFP-3) were evaluated in clinical trials in adults and children, but to the authors knowledge, this program is no longer active.⁵⁸ PFP-3 (purified F protein with aluminum phosphate) was tested in 298 seropositive children 1 to 12 years of age with cystic fibrosis, and although the vaccine was found to be safe and immunogenic, it did not seem to confer a protective effect.⁵⁹ Similarly, a PFP-2 trial in 35 healthy pregnant women and their offspring indicated that PFP-2 was safe, but there was no significant increase in RSV neutralizing IgG titers following vaccination in the third trimester, and a protective effect in the offspring could not be expected.⁶⁰

The candidate vaccine BBG2Na (Pierre Fabre) was developed by fusing the conserved central domain of the RSV G protein to the albumin-binding region of streptococcal protein G, and this vaccine appeared safe and immunogenic in phase 1 and phase 2 studies. However, unexpected side effects in a phase 3 trial (purpura/type III hypersensitivity) in a small number of vaccine recipients halted further development.⁶¹

Live Attenuated Vaccines

Preclinical and clinical development of live attenuated RSV and PIV vaccines for use in infants has a long history, and a few helpful concepts have been developed to guide decision making. First, the severity of illness following wild-type virus infection correlates with the level of virus replication (i.e., peak virus titer) in the respiratory tract. Conversely, restriction of virus replication correlates with attenuation, and below a certain threshold, clinical disease is absent or infrequent. Second, no animal model is as permissive for virus replication as seronegative human infants, and clinical studies in the target population are key to selecting an appropriately attenuated vaccine virus for further development. Nonetheless, seronegative rodents and nonhuman primates as well as primary human airway epithelial cells are helpful in selecting a vaccine virus that has a good chance of being appropriately attenuated for infants. Third, the attenuation phenotype of a live attenuated vaccine can be enhanced through successive importation of individual point mutations or sets of attenuating point mutations from related animal or human viruses. Conserved sequences in homologous proteins can inform the selection of attenuating mutations. Fourth, point mutations can be stabilized to increase genetic and phenotypic stability of vaccines by deletion of amino acid residues at the site of an attenuating point mutation or by codon stabilization.⁶² Amino acid assignments can be stabilized in many instances by identifying codons that would require two or three nucleotide changes to revert to an amino acid that leads to the loss of the attenuation phenotype. Such stabilized codons not only increase genetic and phenotypic stability of vaccine viruses but can also manifest an increased level of temperature sensitivity and attenuation compared with the original mutant, which can help to further attenuate slightly underattenuated candidate vaccines.^62–65

Since vaccine virus replication correlates with immunogenicity and reactogenicity, attempts have been made either to increase antigen expression in an infected host cell or to enhance the immune response at a given level of replication. These strategies have been reviewed elsewhere⁶⁵ but should at least be mentioned here. First, paramyxovirus genes that are closer to the 3′ end of the viral genome are expressed more efficiently, and expression of important antigens can be enhanced by shifting the respective genes to a promoter-proximal position. Second, genes that regulate viral RNA synthesis, such as the RSV M2–2 gene that downregulates mRNA synthesis, can be deleted. Third, codon usage of important antigens can be optimized to enhance expression. Fourth, cytokines that may improve immunogenicity can be coexpressed from an additional gene within the recombinant viral genome. Granulocyte-macrophage colony-stimulating factor (GM-CSF), for instance, can be coexpressed to enhance antigen presentation and dendritic cell maturation.^66,67 Fifth, mutations that inactivate viral interferon antagonists can lead to increased type 1 interferon production in the host and thereby increase vaccine immunogenicity.^68–72

Virus-Like Particles

Virus-like particles (VLPs) are formed by the self-assembly of viral structural proteins, but they do not contain a viral genome and thus are not able to replicate. One of the advantages of VLPs is that the viral proteins have a good chance to be presented as in the infectious virus (i.e., viral proteins are arranged in their native conformation and oligomerization state within a virus-size particle).⁷³ The fusion proteins of paramyxoviruses, for instance, are arranged as homotrimers in the viral envelope, and trimerization is important for both function and antigenicity. VLPs are thought to induce strong B cell responses because the highly repetitive surface of the particles is able to cross-link specific B cell receptors.⁷⁴ VLP-based vaccines are a fairly recent development and so far only two pathogens are targeted by approved VLP-based vaccines: hepatitis B virus and human papillomavirus. However, many more VLP-based vaccines are in clinical development. One VLP-based RSV vaccine has just entered clinical trials and will briefly be discussed in this article.

Replication Defective Vectors

Several replication defective vectors have been employed in vaccine development, but only two systems will be mentioned here, alphavirus vectors and adenovirus vectors, because they are currently being used in RSV vaccine development. Alphavirus vectors have been used to express several antigens, including those of influenza virus and cytomegalovirus (CMV). Alphaviruses (eg, Venezuelan equine encephalitis virus and Semliki Forest virus) have a single-stranded positive-sense RNA genome that encodes the viral nonstructural proteins (including the viral polymerase) in the 5′ portion of the genome and the structural proteins in the 3′ portion. Whereas the nonstructural proteins are translated directly from the viral genomic RNA, the structural proteins are translated from a subgenomic RNA that is transcribed from a promoter that is only present on the full-length negative strand RNA replication intermediate.⁷⁵ If the structural protein coding region is deleted from the genome and a gene or genes of interest are inserted in its place, the self-replicating RNA or replicon vector cannot form an alphavirus particle, but it can go through a single replicative cycle that leads to cytoplasmatic expression of the foreign gene(s).^76,77 Alphavirus replicon vaccines are noteworthy for their ability to induce mucosal immune responses following systemic administration,^78,79 and they have been shown to induce protective immunity against RSV in the respiratory tract of cotton rats.⁸⁰ Alphavirus vector-based vaccines against influenza and CMV (Alphavax, Durham, NC) have been evaluated clinically,⁸¹ but clinical data for the RSV vaccine based on this technology are not available yet.

Recombinant replication defective adenovirus (rAd) vectors have been evaluated extensively as candidate HIV vaccines.^82,83 There are more than 50 Ad serotypes, and the fact that these double-stranded DNA viruses can accommodate large foreign gene inserts makes them very valuable as vaccines. rAd serotype 5 (rAd5) stands out as the best characterized Ad serotype, and it is also more immunogenic than many others. Ad5 is a good inducer of cytotoxic T cell immunity, and it initiates a robust antibody response. Although Ad vectors have mostly been used as systemically administered vaccines, more recent results from nonhuman primate studies indicate that Ad5 can also be delivered as an aerosol to induce mucosal immunity as well as CD4 and CD8 T cell responses.⁸⁴ Two concerns are frequently mentioned when considering rAd5 vectors as a vaccine platform. The first is that between 50 and 90% of adults are Ad5 seropositive, and preexisting immunity is known to reduce the immunogenicity of systemically administered rAd5 vectored antigens.^85,86 The second concern resulted from a recent Phase 2b HIV vaccine trial (the STEP trial) in which a rAd5 vector expressing HIV antigens failed to protect vaccinees, and in an exploratory analysis Ad5 seropositive men were found to be at increased risk of HIV acquisition.⁸⁷ The mechanism underlying this observation is still not understood, but rAd serotypes with low seroprevalence in humans are being explored as alternatives. The characteristics of rAd-based RSV vaccine vectors have recently been reviewed elsewhere,⁸⁸ and this platform has potential for both adult and pediatric vaccination. rAd-based RSV vaccines have been evaluated preclinically as part of a collaboration between the National Institute of Allergy and Infectious Diseases (NIAID) Vaccine Research Center and GenVec Inc., and clinical development is planned.⁸⁹

SELECTED INDIVIDUAL VACCINES IN CLINICAL DEVELOPMENT

Subunit RSV Vaccines

The only candidate subunit vaccine that has been evaluated in more recent clinical trials is a subunit vaccine containing the RSV-A F, G, and M proteins (Sanofi Pasteur, Swiftwater, PA). This vaccine was evaluated in a dose-ranging study in 561 adults 65 years of age or older (Table 2). The vaccine’s reactogenicity was similar to that seen with seasonal influenza virus vaccination, with injection site tenderness as the most common local adverse event. Surprisingly, only the nonadjuvanted (but not the adjuvanted) high-dose vaccine induced a ≥fourfold rise in neutralizing antibody titers against RSV in ≥50% of vaccinees.⁹⁰ Similar results (i.e., nonadjuvanted subunit vaccine being more immunogenic than aluminum-phosphate adjuvanted vaccine) were obtained in a trial of this vaccine in 1169 older adults with cardiopulmonary disease (Table 2). In that study, the subunit vaccine appeared safe and immunogenic, and it did not interfere with the humoral response to influenza vaccination, suggesting that this vaccine could potentially offer a benefit to older adults. RSV antibody titers returned to baseline within a year, implying that annual immunization would be necessary.⁹¹

Table 2.

Ongoing and Recently Completed RSV, MPV, and PIV Vaccine Trials

Target	Vaccine	ClincalTrials.gov ID	Sponsor	N	Current Serostatus Enrolling	Current Age Group (moa)
RSV	F, G, M (subunit)	NA	Sanofi Pasteur	561	Unscreened	Adults >64 years
	F, G, M (subunit)	NA	Sanofi Pasteur	1169	Unscreened	Adults >64 years
	MEDI-559 (live att RSV)	NCT00767416	MedImmune	120	Seroneg	5–23
	RSV-F (VLP)	NCT01290419	Novavax	100	Unscreened	Adults
	MEDI-534 (live att rB/HPIV3)	NCT00686075   NCT00493285	MedImmune	80	Seroneg	6–23
PIV3	rHPIV3cp45	NCT00308412	NIAID	24	Unscreened	6–12
	rHPIV3cp45	NCT00308412	NIAID	21	Seroneg	6–36
	rHPIV3cp45	NCT01021397   NCT01254175	NIAID	30	Seroneg	6–36
	MEDI-560 (rHPIV3cp45)	NCT01150799	MedImmune	30	Unscreened	1–12
	rB/HPIV3	NCT00366782	NIAID	21	Seroneg	6–36
	MEDI-534 (rB/HPIV3)	NCT00686075   NCT00493285	MedImmune	80	Seroneg	6–23
MPV	HMPV-Pa	NCT01255410	NIAID	15	Seropos	15–59
PIV1	Live-att PIV1	NCT00641017	NIAID	21	Seroneg	6–36
	Sendai Virus	NCT00186927	St. Jude	18	Seropos	12–59
PIV2	Live-att PIV2	NCT01139437	NIAID	21	Seropos	15–59

NA, not available; NIAID, National Institute of Allergy and Infectious Diseases.

Live Attenuated RSV Vaccines

Several live attenuated RSV vaccines were developed by serial passage of RSV at increasingly lower temperatures (cold passage, cp) and by chemical mutagenesis, and temperature-sensitive (ts) and non-ts attenuating mutations were identified. Several of these biologically derived candidate vaccines went into clinical trials, but it proved difficult to find an appropriate balance between immunogenicity and attenuation, particularly in infants. The RSV cpts248/404 vaccine, for instance, replicated to an acceptable titer in 1- to 2-month-old infants, and replication of the second dose of vaccine given 4 to 6 weeks after the first dose was highly restricted, indicating that protective immunity was induced. However, 15 of 18 infants who shed more than 10³ PFU/mL after dose 1 experienced nasal congestion, fussiness while trying to sleep, and mild difficulty with feeding, which lasted for approximately 1 day. Although none of the infants developed profuse rhinorrhea, otitis media, fever, or LRI, this vaccine was considered too reactogenic to continue clinical development.⁹²

The availability of a reverse genetics system for RSV since the mid-1990s enabled a more directed and more rational RSV vaccine development approach.⁹³ This system permitted the generation of infectious virus from a cDNA copy of the negative-sense, single-stranded virus genome; thus site-directed mutagenesis could be used to introduce specific mutations into the RSV genome and to evaluate the contribution of each mutation to the attenuation (att) phenotype. Using this methodology, the genetic basis of the attenuation phenotype of cpRSV and cptsRSV mutants was determined. In addition, reverse genetics allowed the targeted deletion of the interferon antagonist genes NS1 and/or NS2, as well as deletion of the small hydrophobic SH gene and the M2–2 regulator of transcription and replication. The NS2 deletion mutant (ΔNS2) turned out to be insufficiently attenuated for use in infants, but the more attenuated M2–2 and NS1 deletion mutants (ΔM2–2 and ΔNS1, respectively) are considered promising RSV candidate vaccines and are being prepared for clinical trials.⁶⁵ The M2–2 deletion mutant is especially attractive because deletion of the M2–2 protein increases viral mRNA transcription while simultaneously decreasing genome replication; thus the vaccine virus is expected to express more antigen (especially F and G) than other live attenuated RSV vaccines.^94,95

Apart from the aforementioned ΔM2–2 and ΔNS1 deletion mutants that have not yet entered clinical trials, a mutant designated RSVcp248/404/1030ΔSH seems to bear great potential. This mutant RSV contains all 11 mutations identified in the biologically derived RSVcpts248/404, but two additional attenuating mutations were introduced (i.e., the SH gene was deleted and one amino acid substitution was generated in the large polymerase protein L—the 1030 mutation). cp248/404/1030ΔSH was evaluated in a phase 1 trial in 1- to 2-month-old infants, the target population for a pediatric RSV vaccine, and was found to be well tolerated and immunogenic.⁵⁰ As with the slightly underattenuated cpts248/404 mutant, neither systemic nor mucosal antibody responses were consistently observed in 1- to 2-month-old infants, but replication of a second dose of vaccine given 4 weeks after the first dose was highly restricted, indicating that protective immunity was induced. MedImmune, LLC (Gaithersburg, MD) is currently evaluating the RSVcp248/404/1030ΔSH vaccine, referred to as MEDI-559, in ~120 children 5 to 23 months of age. In this trial, children were randomized 1:1 and receive three doses of vaccine at 0, 2, and 4 months, and although enrollment is complete, data analysis for this trial is pending (Table 2, NCT00767416).

For any live respiratory vaccine that is attenuated by a combination of point mutations it will be important to assess the vaccine’s genetic and phenotypic stability because transmission of a less attenuated revertant of a vaccine virus is a concern. To achieve phenotypic stability, vaccine viruses can be generated to encode multiple attenuating mutations so that the loss of one or two mutations does not result in a virus that replicates as well as wt virus. Alternatively, gene deletion mutants can be used given that they cannot easily revert to wt sequence. The NS2 deletion mutant mentioned earlier is an example of such a vaccine, and it was evaluated clinically for vaccination of the elderly and other at-risk adults. However, even at a dose as high as 10⁷ PFU the vaccine virus was not shed by adults, and only 13% of vaccinees developed a significant antibody response.⁴⁹ Although this virus was overattenuated for adults, seropositive children shed high titers of vaccine virus, and shedding was temporally associated with URI in seronegative infants, indicating that this vaccine was underattenuated for this cohort and that single live attenuated vaccine for the two target populations was not feasible. Two NS2 gene deletion mutants that encoded additional attenuating point mutations, rA2cp248/404ΔNS2 and rA2cp530/1009ΔNS2, were also evaluated in RSV-seronegative children. Deletion of the NS2 gene from RSVcpts248/404 and RSVcpts530/1009 decreased the infectivity of these vaccine viruses for seronegative children 6 to 24 months of age to 50% and 20%, respectively, and restricted replication to levels (10^1.3 to 10^2.3 PFU/mL) that were no longer associated with upper respiratory illness.⁴⁹ However, these two vaccine candidates appeared overattenuated and insufficiently immunogenic.

Bivalent RSV/PIV-3 vaccines will be discussed at the end of the PIV-3 section.

Virus-Like Particle–Based RSV Vaccines

The generation of enveloped VLPs composed of RSV proteins was reported to be fairly inefficient. However, enveloped VLPs composed of Newcastle disease virus (NDV) nucleocapsid and matrix proteins, as well as chimeric RSV/NDV glycoproteins, can be generated efficiently. These chimeric glycoproteins consist of the ectodomain of RSV F or G protein and the transmembrane and cytoplasmic domains of NDV F and HN, respectively.^96,97 The cytoplasmic domains of NDV F and HN were shown to interact with the NDV M protein, and this interaction is thought to support VLP formation via budding from the cell surface.⁹⁸ VLPs based on this technology were shown to induce high titer RSV-neutralizing serum antibodies following intramuscular vaccination of mice, with the IgG subclass ratio suggesting that a strong Th1 response was initiated. Mice were protected against wt RSV challenge 7 weeks postimmunization.⁹⁶ A VLP expressing the RSV F has entered a phase 1 dose escalation study in healthy adults, but details on the VLP design are not yet available (Table 2, NCT01290419).

PIV3 Vaccines

PIV3 is second only to RSV as a cause of viral bronchiolitis and pneumonia in infants and children (although in some studies MPV competes with PIV3). As with RSV, live attenuated vaccine candidates were developed by serial tissue culture passage of wt virus at suboptimal temperatures. One of these candidate vaccines, HPIV3cp45, was cold-passaged (cp) 45 times and acquired a set of 15 mutations that conferred the cold adaptation (ca), temperature sensitivity (ts), and attenuation (att) phenotypes.⁹⁹ HPIV3cp45 was evaluated in a phase 1 trial in infants as young as 1 month of age and was found to be appropriately attenuated and immunogenic in this target population.⁵¹ Infants received two doses of vaccine either 1 or 3 months apart. The first dose of vaccine infected 94% of the infants, and vaccine virus was shed in low titer for 17 days (mean). Replication of the second dose of vaccine was restricted, indicating that immunization of young infants with HPIV3cp45 resulted in the induction of some protective immunity. Nonetheless, two or three doses of vaccine will likely be needed to induce durable immunity. Serum IgG antibody responses to PIV3 were only observed in a minority of young infants, possibly due to the presence of maternal PIV3-specific IgG, and serum IgA antibody directed against the PIV3 HN glycoprotein seemed the most sensitive correlate of immunity.⁵¹ In a phase 2 trial in 380 children 6 to 18 months of age, including 226 infants and children seronegative for PIV-3, the HPIV-3cp45 vaccine was well tolerated, and no difference was observed in the frequency of URI, cough, fever or otitis media between vaccine and placebo recipients. Eighty-four percent of seronegative vaccine recipients developed a fourfold or greater increase in antibody titer, indicating satisfactory infectivity and immunogenicity.¹⁰⁰ In addition, intranasal vaccination with a single bivalent dose (10⁵ PFU per virus) of RSVcpts248/404 and HPIV3cp45 was evaluated in 6- to 18-month-old seronegative children. Although a slight reduction in HPIV3cp45 infectivity was observed with the combined vaccine (76% vs 92% in the monovalent vaccine), the combined vaccine was immunogenic, and antibody responses were comparable to the monovalent groups.¹⁰¹

Following the foregoing trials, the vaccine virus was re-derived from cDNA and referred to as recombinant (r)HPIV3cp45, to further reduce the risk of potential biological contamination of the vaccine seed virus. In addition, this technology also permits re-derivation of vaccine seed virus from cDNA at any time. Clinical development of rHPIV3cp45 is conducted in a cooperative research and development agreement between NIAID and MedImmune, LLC. Currently, two phase 1 trials are being sponsored by NIAID. The first protocol enrolled a total of 45 children 6 to 36 months of age into two cohorts, both randomized 2:1 to receive two doses of rHPIV3cp45 (10⁵ TCID₅₀) or placebo 4 to 10 weeks apart (Table 2, NCT00308412). In the first cohort of 24 unscreened infants 6 to 12 months of age, frequent nasal washes were performed for quantitative virology. In this unscreened cohort, all 10 seronegative vaccinees had vaccine virus detected in nasal washes for approximately 2 weeks, with a mean peak titer of 10^3.6 TCID₅₀/mL, whereas only two out of five seropositive vaccinees had vaccine virus detected for a single day each, with a mean peak titer of 10^0.9 TCID₅₀/mL. Only one of the 15 vaccinees shed vaccine following the second dose, again suggesting that a protective immune response restricted replication of the vaccine virus. An additional 21 seronegative children 6 to 36 months of age were enrolled to expand safety and immunogenicity data, and the findings from the first cohort with regard to safety, infectivity and immunogenicity were confirmed. Although rHPIV3cp45 was found to be safe, well tolerated, immunogenic, and indistinguishable in this regard from biologically derived HPIV3cp45, the dosing interval of 1 to 2 months was clearly insufficient to permit reinfection and boosting of the immune response. Two additional studies in seronegative children 6 to 36 months of age were initiated to evaluate two doses of vaccine given 6 months apart (Table 2, NCT01021397 and NCT01254175). MedImmune has initiated a trial evaluating three doses of rHPIV3cp45 given 2 months apart, and this regimen was immunogenic and well tolerated (Table 2, NCT01150799).¹⁰²

A second approach to developing an HPIV3 vaccine is the use of host range mutants. Bovine PIV3 (BPIV3) is a virus of cattle that is antigenically related to human PIV3 (HPIV3) but restricted in replication in nonhuman primates.¹⁰³ BPIV3 was tested in a phase 1 study in infants and children and proved to be infectious, well tolerated, and immunogenic. However, because of antigenic differences between BPIV3 and HPIV3 glycoproteins, antibody titers were lower against HPIV3 than against BPIV3.¹⁰⁴ A four-dose regimen of BPIV3 was evaluated in a phase 2 trial in 192 infants (2 months old) receiving 10⁵ 50% tissue culture infective dose (TCID₅₀), 10⁶ TCID₅₀, or placebo at 2, 4, 6, and 12 to 15 months of age.¹⁰⁵ Either dose was well tolerated, and adverse events occurred at similar frequency in vaccine and placebo recipients, with the exception of fever >38°C after the second dose, which was more common in vaccine recipients. As observed in the phase 1 study already mentioned, seroconversion rates were satisfactory against BPIV3 but only modest against HPIV3.¹⁰⁵ A chimeric recombinant bovine/human PIV3 virus (B/HPIV3), bearing the HPIV3 HN and F genes (encoding the glycoproteins that are targeted by neutralizing antibodies) in place of the BPIV3 genes, was found to be more immunogenic against HPIV3 in rhesus monkeys than the unmodified BPIV3 while retaining the attenuation phenotype of BPIV3.¹⁰⁶ This candidate vaccine was recently evaluated in healthy adults, as well as in seropositive and seronegative children (Table 2, NCT00366782). In adults and seropositive children, B/HPIV3 infected a minority of subjects and was poorly immunogenic, as expected. In seronegative children, the vaccine virus was highly infectious at a dose of 10⁵ TCID₅₀, and more than 90% of the vaccine recipients developed a fourfold or greater rise in HPIV3 HAI (hemagglutination-inhibiting) antibody titers. The mean peak titer of vaccine virus shed was 3.1 log₁₀ TCID₅₀, similar to the mean peak titers we observed for HPIV3cp45 in seronegative children. Although URI and fever were fairly frequent during the 4-week follow-up, rates were similar in vaccinees and in placebo recipients (unpublished results).

Combined PIV3 and RSV Vaccines

The chimeric rB/HPIV3 viruses noted earlier were modified to express either the RSV F protein alone^107,108 or both the G and F proteins^109,110 from additional genes inserted into the B/HPIV3 genome. Each of these constructs functions as a bivalent vaccine virus, expressing the major protective antigens of both RSV and HPIV3. These constructs were developed by MedImmune and NIAID, respectively. The MedImmune construct expressing the RSV F protein only, referred to as MEDI-534, was well tolerated in 1- to 9-year-old seropositive children.¹¹¹ In a separate study in children 6 to 23 months of age (seronegative for both RSV and HPIV3), 67% and 100% of subjects who received 10⁶ TCID₅₀ of MEDI-534 seroconverted to RSV and HPIV3, respectively (Table 2, NCT00686075 and NCT00493285). The incidence of adverse events and solicited events was similar between the MED-534 and placebo groups.¹¹² The related NIAID construct has not entered clinical trials yet.

MPV Vaccines

MPV is similar to RSV not only in its genome organization but also in its epidemiology and the spectrum of disease. Naturally, vaccine development for MPV has followed the example of RSV vaccine development. A recombinant system to generate virus from cDNA was set up within a few years of the initial isolation of MPV, and gene deletion experiments quickly defined nonessential genes. ^113,114 Deletion of the SH gene did little to attenuate the virus in African green monkeys (AGMs), but viruses lacking the attachment glycoprotein G or the M2–2 protein were restricted in replication.¹¹³ A second approach to developing live attenuated MPV candidate vaccines followed the example of B/HPIV3 (i.e., a chimeric virus was derived from cDNA in which a gene of a nonhuman virus substituted for the homologous gene of a human MPV). Avian MPV (AMPV), the causative agent of respiratory disease in poultry, was used here because AMPV is restricted in replication in AGMs, and there were no reports of human disease caused by AMPV, although many employees in the poultry industry are exposed to the virus. The N and P genes of AMPV were substituted individually for the respective gene of HMPV in a recombinant chimeric virus, and the chimeric virus expressing the AMPV P protein (HMPV-Pa) was restricted in the LRT of AGMs while the chimeric virus expressing the AMPV N protein was not significantly attenuated.¹¹⁵ HMPV-Pa therefore appears to be an attractive live attenuated MPV vaccine candidate. Prior to initiating clinical trials with HMPV-Pa, a wt-like cDNA-derived HMPV was evaluated in a clinical trial in healthy adults to establish a benchmark with regard to infectivity and replicative ability. 10⁶ PFU of the wt-like HMPV were given to 22 adults in an inpatient isolation facility, and subjects were observed for a minimum of 12 days prior to discharge (NCT01109329). The wt-like virus infected approximately half of the subjects, and ~40% of the adults shed detectable virus and developed a mild URI. Subsequently, HMPV-Pa was administered to 15 adults, and it was found, as expected, to be poorly infectious and not immunogenic in adults. Studies in seropositive children have been initiated (Table 2, NCT01255410), but we will likely have to wait for studies in seronegative children to learn whether this virus is appropriately attenuated and immunogenic in its target population.

HPIV1 and HPIV2 Vaccines

Live attenuated HPIV1 and HPIV2 candidate vaccines have been generated by “importation” of one or more known attenuating point mutations from heterologous paramyxoviruses. Mutations found in conserved regions of HPIV3cp45, RSVcpts530, and murine PIV1 (MPIV1, Sendai virus) were introduced in the P/C, F, HN, and/or the L gene of HPIV1 and HPIV2, and mutants were evaluated in hamsters and nonhuman primates.^68,69,116 One HPIV1 vaccine candidate that contains two substitutions in the P/C gene (R84G/F170S) and one substitution in the L gene (LY942A) was found to be highly attenuated in AGMs yet conferred protection against wt HPIV1 challenge and induced a robust antibody response.⁶⁹ The codon 170 mutation in the C protein of this vaccine virus is particularly interesting because it abrogates the ability of HPIV1 to evade the host’s immune response. Unlike wt HPIV1, the F170S mutant stimulates interferon β expression and permits interferon signaling,¹¹⁷ thus inducing an earlier (and likely more potent) innate immune response than wt HPIV1.¹¹⁸ As part of this defect, this mutant cannot inhibit double-stranded RNA accumulation as well as wt HPIV1, resulting in activation of cytoplasmic viral sensors such as PKR and MDA5.⁷² To stabilize the identified mutations, the F170S mutation in the C protein was changed to a deletion of codons 169 and 170, and the original Y942H mutation in the L protein was modified to generate a Y942A mutant virus that possessed a similar ts phenotype but that would require three nucleotide substitutions to revert to a virus with a wt growth phenotype in vivo.⁶⁴ This stabilized vaccine virus is currently in a phase 1 trial in HPIV1 seronegative children (Table 2, NCT00641017). Although this study is not completely enrolled yet, preliminary data indicate that vaccine virus replication is highly restricted in seronegative children.

To develop a live attenuated HPIV2 vaccine, five missense mutations found in the large polymerase (L) proteins of attenuated RSV and HPIV3 mutants and in BPIV3 were imported into homologous positions in the HPIV2 L protein.¹¹⁹ Three of these mutations conferred temperature sensitivity in vitro and restriction of replication in vivo.^63,120 The attenuating mutations in L were then either stabilized (codon 948) or deleted (codon 1724) to increase genetic and phenotypic stability. An additional spontaneous T to C nucleotide substitution within the 3′ extragenic leader region of the genome also contributed to HPIV2 attenuation and was included in the candidate vaccine.¹²⁰ This investigational HPIV2 vaccine is referred to as rHPIV2–15C/948L/D1724, and it includes the 15C leader mutation as well as the amino acid substitution 948L and the deletion of codon 1724 (D1724) in the L protein. This virus is highly attenuated in AGMs and provides protection against wt HPIV2 challenge.¹²⁰ Its replication is also restricted in fully differentiated primary human airway epithelium, suggesting that this in vitro model might be useful for the identification of live attenuated respiratory vaccines.¹³ The ongoing phase 1 trial is currently enrolling sero-positive children and will then progress into seronegative infants and children (Table 2, NCT01139437).

CONCLUSION

Several promising candidate vaccines have been evaluated in clinical trials over the past few years, among them live attenuated RSV and PIV vaccines for use in seronegative infants, subunit RSV vaccines for use in high-risk adults, and VLP-based RSV vaccines. For many years, only a few companies were interested in RSV vaccine development, but this has changed dramatically and now several additional companies and new platforms compete to develop these vaccines. No one can tell whether live attenuated, subunit, VLP, replicating, or nonreplicating vectored vaccines will turn out to be successful in phase 3, but hopefully this new competition will help to bring much needed respiratory vaccines to the market sooner. Every year without them will be a lost year, especially for those who don’t receive the care they need.