Population-based incidence of human metapneumovirus infection among hospitalized children.

BACKGROUND: Human metapneumovirus (HMPV) is a leading cause of acute respiratory illness (ARI) in children. Population-based incidence rates and comprehensive clinical characterizations of disease have not been established. METHODS: We conducted population-based prospective surveillance for 2 years in 2 US counties of HMPV infection among children <5 years old who were hospitalized with ARI or fever. Nasal and throat specimens obtained with swabs were tested for HMPV by real-time reverse-transcription polymerase chain reaction and genotyped. RESULTS: Forty-two (3.8%) of 1104 children tested positive for HMPV. The overall annual rate of HMPV-associated hospitalizations per 1000 children <5 years old was 1.2 (95% confidence interval [CI], 0.9-1.6). This rate was highest among infants 0-5 months old (4.9 per 1000 [95% CI, 2.9-7.2]), followed by children 6-11 months old (2.9 per 1000 [95% CI, 1.4-4.7]). The annual rate of hospitalization for HMPV infection was less than that for respiratory syncytial virus infection but similar to that for influenza and parainfluenza virus 3 infection in all age groups. The mean age of children hospitalized with HMPV infection was 6 months. Bronchiolitis, pneumonia, and asthma were the most common diagnoses among children with HMPV infection. All 4 HMPV subgroups were detected during both years at both sites. HPMV infection was most prominent from March through May. CONCLUSION: HMPV was detected in 3.8% of children hospitalized with ARI or fever, with a population incidence similar to that of influenza virus and parainfluenza virus 3.

Genetic diversity and evolution of human metapneumovirus fusion protein over twenty years.

BACKGROUND: Human metapneumovirus (HMPV) is an important cause of acute respiratory illness in children. We examined the diversity and molecular evolution of HMPV using 85 full-length F (fusion) gene sequences collected over a 20-year period. RESULTS: The F gene sequences fell into two major groups, each with two subgroups, which exhibited a mean of 96% identity by predicted amino acid sequences. Amino acid identity within and between subgroups was higher than nucleotide identity, suggesting structural or functional constraints on F protein diversity. There was minimal progressive drift over time, and the genetic lineages were stable over the 20-year period. Several canonical amino acid differences discriminated between major subgroups, and polymorphic variations tended to cluster in discrete regions. The estimated rate of mutation was 7.12 x 10(-4) substitutions/site/year and the estimated time to most recent common HMPV ancestor was 97 years (95% likelihood range 66-194 years). Analysis suggested that HMPV diverged from avian metapneumovirus type C (AMPV-C) 269 years ago (95% likelihood range 106-382 years). CONCLUSION: HMPV F protein remains conserved over decades. HMPV appears to have diverged from AMPV-C fairly recently.

Development of a PIV-vectored RSV vaccine: preclinical evaluation of safety, toxicity, and enhanced disease and initial clinical testing in healthy adults.

MEDI-534 is a bivalent live attenuated vaccine candidate against human respiratory syncytial virus (hRSV) and human parainfluenza virus type 3 (hPIV3) that was previously shown to be immunogenic and to protect rodents and African green monkeys from wild-type (wt) hRSV challenge. We performed further preclinical evaluations to address the safety of MEDI-534 prior to human testing. MEDI-534 did not predispose rodents to enhanced RSV disease following wt-RSV challenge, and the tissue tropism of the chimeric virus was confined to the respiratory tract. Representative clinical trial material did not produce toxicity in rats. In adults, MEDI-534 was highly restricted in replication, did not boost RSV and PIV3 antibody titers, and produced no medically significant vaccine-related adverse events thereby warranting further evaluation in pediatric populations.

Deletion of human metapneumovirus M2-2 increases mutation frequency and attenuates growth in hamsters.

BACKGROUND: Human metapneumovirus (hMPV) infection can cause acute lower respiratory tract illness in infants, the immunocompromised, and the elderly. Currently there are no licensed preventative measures for hMPV infections. Using a variant of hMPV/NL/1/00 that does not require trypsin supplementation for growth in tissue culture, we deleted the M2-2 gene and evaluated the replication of rhMPV/DeltaM2-2 virus in vitro and in vivo. RESULTS: In vitro studies showed that the ablation of M2-2 increased the propensity for insertion of U nucleotides in poly-U tracts of the genomic RNA. In addition, viral transcription was up-regulated although the level of genomic RNA remained comparable to rhMPV. Thus, deletion of M2-2 alters the ratio between hMPV genome copies and transcripts. In vivo, rhMPV/DeltaM2-2 was attenuated compared to rhMPV in the lungs and nasal turbinates of hamsters. Hamsters immunized with one dose of rhMPV/DeltaM2-2 were protected from challenge with 106 PFU of wild type (wt) hMPV/NL/1/00. CONCLUSION: Our results suggest that hMPV M2-2 alters regulation of transcription and influences the fidelity of the polymerase complex during viral genome replication. In the hamster model, rhMPVDeltaM2-2 is attenuated and protective suggesting that deletion of M2-2 may result in a potential live vaccine candidate. A more thorough knowledge of the hMPV polymerase complex and the role of M2-2 during hMPV replication are being studied as we develop a potential live hMPV vaccine candidate that lacks M2-2 expression.

A phase 1 study of 4 live, recombinant human cytomegalovirus Towne/Toledo chimeric vaccines.

BACKGROUND: Human cytomegalovirus (HCMV) infection acquired in utero often results in severe consequences, including mental retardation and deafness. Although not evaluated for this indication, live attenuated HCMV vaccines based on the Towne strain are well-tolerated and have demonstrated moderate efficacy in other clinical settings. METHODS: To produce live HCMV vaccine candidates that retain the excellent safety profile of the Towne strain but are more immunogenic, the genomes of the Towne strain and the unattenuated HCMV Toledo strain were recombined to yield 4 independent chimeric vaccine candidates. These vaccine candidates were evaluated in 20 HCMV-seropositive persons, in a phase 1, double-blinded, placebo-controlled trial. Participants received a single dose of vaccine or placebo, and the safety and tolerability of the vaccine candidates were evaluated. RESULTS: There was no difference in systemic symptoms between the vaccine and placebo recipients. As a group, vaccine recipients experienced more injection-site reactions than did placebo recipients; however, these were generally minor and short-lived. Vaccine virus could not be detected in blood, urine, or saliva samples obtained from any vaccine recipient. CONCLUSIONS: The Towne/Toledo chimeric vaccine candidates were well tolerated and did not cause systemic infection. Additional human trials are warranted to further evaluate the potential of these vaccine candidates as live virus vaccines.

Genetic stability of live, cold-adapted influenza virus components of the FluMist/CAIV-T vaccine throughout the manufacturing process.

FluMist is a live-attenuated, trivalent influenza vaccine (LAIV) recently approved for intranasal administration. To demonstrate genetic stability during manufacture of the vaccine viruses in LAIV and a similar vaccine in development (CAIV-T), full genome consensus sequences were determined at multiple manufacturing stages for four influenza type A and five type B strains. The critical cold-adapted (ca), temperature-sensitive (ts) and attenuated (att) mutations were preserved in the virus manufacturing intermediates. Moreover, sequence identity was observed for all vaccine intermediates of the same strain. Minor sequence differences were noted in the shared gene segments of the vaccine viruses and their common progenitor master donor virus (MDV) and several of the hemagglutinin (HA) and neuraminidase (NA) genes contained nucleotide differences when compared to the wild-type parent. Nonetheless, all vaccine viruses retained the ca, ts, and att phenotypes. Thus, genetic and phenotypic stability of the vaccine viruses is maintained during the manufacture of LAIV/CAIV-T vaccines.

The role of human metapneumovirus in upper respiratory tract infections in children: a 20-year experience.

BACKGROUND: The role that human metapneumovirus (hMPV) plays in the etiology of upper respiratory tract infections (URIs) in children over a period of many years has not been evaluated previously. METHODS: By use of real-time reverse-transcriptase polymerase chain reaction, we retrospectively tested nasal wash (NW) specimens for hMPV that had been obtained from a cohort of 1532 infants and children with URIs who were prospectively followed for an average of 2.4 years during the period from 1982 to 2001. Virus genes were sequenced, and prospectively collected clinical data were analyzed. RESULTS: There were 2710 visits for URIs for which routine cultures did not reveal a viral etiology. Archival NW specimens from 2384 of these visits were available. hMPV RNA was detected in 118 (5%) of 2384 specimens. The mean age of the children with hMPV infection was 20 months, and 78% of illnesses occurred from December through May. Acute otitis media (AOM) was detected in 50% of these children. hMPV circulated each year, but the numbers of isolates detected varied by year. Reinfections with both homologous and heterologous strains occurred. Four distinct genetic lineages were present over the 20 years of surveillance, with several different lineages circulating during some seasons. CONCLUSIONS: hMPV was detected in a substantial number of children with URIs and concomitant AOM.

An S101P substitution in the putative cleavage motif of the human metapneumovirus fusion protein is a major determinant for trypsin-independent growth in vero cells and does not alter tissue tropism in hamsters.

Human metapneumovirus (hMPV), a recently described paramyxovirus, is a major etiological agent for lower respiratory tract disease in young children that can manifest with severe cough, bronchiolitis, and pneumonia. The hMPV fusion glycoprotein (F) shares conserved functional domains with other paramyxovirus F proteins that are important for virus entry and spread. For other paramyxovirus F proteins, cleavage of a precursor protein (F0) into F1 and F2 exposes a fusion peptide at the N terminus of the F1 fragment, a likely prerequisite for fusion activity. Many hMPV strains have been reported to require trypsin for growth in tissue culture. The majority of these strains contain RQSR at the putative cleavage site. However, strains hMPV/NL/1/00 and hMPV/NL/1/99 expanded in our laboratory contain the sequence RQPR and do not require trypsin for growth in Vero cells. The contribution of this single amino acid change was verified directly by generating recombinant virus (rhMPV/NL/1/00) with either proline or serine at position 101 in F. These results suggested that cleavage of F protein in Vero cells could be achieved by trypsin or S101P amino acid substitution in the putative cleavage site motif. Moreover, trypsin-independent cleavage of hMPV F containing 101P was enhanced by the amino acid substitution E93K. In hamsters, rhMPV/93K/101S and rhMPV/93K/101P grew to equivalent titers in the respiratory tract and replication was restricted to respiratory tissues. The ability of these hMPV strains to replicate efficiently in the absence of trypsin should greatly facilitate the generation, preclinical testing, and manufacturing of attenuated hMPV vaccine candidates.

Evaluation of AD472, a live attenuated recombinant herpes simplex virus type 2 vaccine in guinea pigs.

An attenuated recombinant herpes simplex virus type 2 (HSV-2), designated as AD472, was constructed by deleting both copies of the gamma(1)34.5 gene, UL55-56, UL43.5, and the US10-12 region from HSV-2 strain G. This virus was engineered to be a safe and effective live attenuated HSV-2 vaccine and was tested in the guinea pig model of genital herpes to evaluate its ability to protect from disease upon challenge with the wild type (wt) virus, HSV-2 (G). AD472 administered intramuscularly did not prevent infection or virus replication in the vaginal tract, but did reduce both lesion development and severity in a dose-dependent manner in guinea pigs challenged with the wt virus. Frequency of reactivation from latency was low compared with that of the parent virus, HSV-2 (G). Immunization with AD472 at doses of 1x10(5)PFU generally precluded colonization of the ganglia or establishment of latency by the challenge virus. Results presented here support the concept of a rationally engineered live attenuated vaccine for the prevention of the genital disease associated with infection by HSV-2.

A host-range restricted parainfluenza virus type 3 (PIV3) expressing the human metapneumovirus (hMPV) fusion protein elicits protective immunity in African green monkeys.

Human metapneumovirus (hMPV) infection causes respiratory tract disease similar to that observed during human respiratory syncytial virus infection (hRSV). hMPV infections have been reported across the entire age spectrum although the most severe disease occurs in young children. No vaccines, chemotherapeutics or antibodies are presently available for preventing or treating hMPV infections. In this study, a bovine/human chimeric parainfluenza virus type 3 (b/h PIV3) expressing the human parainfluenza type 3 (hPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) proteins was engineered to express hMPV fusion (F) protein from the second genome position (b/h PIV3/hMPV F2) with the goal of generating a novel hMPV vaccine. b/h PIV3/hMPV F2 was previously shown to protect hamsters from challenge with wt hMPV (Tang RS, Schickli JH, Macphail M, Fernandes F, Bicha L, Spaete J, et al. Effects of human metapneumovirus and respiratory syncytial virus antigen insertion in two 3' proximal genome positions of bovine/human parainfluenza virus type 3 on virus replication and immunogenicity. J Virol 2003;77:10819-28) and is here further evaluated for efficacy and immunogenicity in African green monkeys (AGMs). AGMs immunized intranasally and intratracheally with b/h PIV3/hMPV F2 generated hMPV- and hPIV3-specific humoral and cellular immune responses and were protected from wt hMPV infection. In a separate study, the host-range restriction of b/h PIV3/hMPV F2 replication relative to wt hPIV3 was performed in rhesus monkeys to demonstrate attenuation. These studies showed that b/h PIV3/hMPV F2 was immunogenic, protective and attenuated in non-human primates and warrants further evaluation in humans as a vaccine candidate for prevention of hMPV-associated respiratory tract diseases.

Human cytomegalovirus plasmid-based amplicon vector system for gene therapy.

We have constructed and evaluated the utility of a helper-dependent virus vector system that is derived from Human Cytomegalovirus (HCMV). This vector is based on the herpes simplex virus (HSV) amplicon system and contains the HCMV orthologs of the two cis-acting functions required for replication and packaging of HSV genomes, the complex HCMV viral DNA replication origin (oriLyt), and the cleavage packaging signal (the a sequence). The HCMV amplicon vector replicated independently and was packaged into infectious virions in the presence of helper virus. This vector is capable of delivering and expressing foreign genes in infected cells including progenitor cells such as human CD34+ cells. Packaged defective viral genomes were passaged serially in fibroblasts and could be detected at passage 3; however, the copy number appeared to diminish upon serial passage. The HCMV amplicon offers an alternative vector strategy useful for gene(s) delivery to cells of the hematopoietic lineage.

Parainfluenza virus type 3 expressing the native or soluble fusion (F) Protein of Respiratory Syncytial Virus (RSV) confers protection from RSV infection in African green monkeys.

Respiratory syncytial virus (RSV) causes respiratory disease in young children, the elderly, and immunocompromised individuals, often resulting in hospitalization and/or death. After more than 40 years of research, a Food and Drug Administration-approved vaccine for RSV is still not available. In this study, a chimeric bovine/human (b/h) parainfluenza virus type 3 (PIV3) expressing the human PIV3 (hPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) proteins from an otherwise bovine PIV3 (bPIV3) genome was employed as a vector for RSV antigen expression with the aim of generating novel RSV vaccines. b/h PIV3 vaccine candidates expressing native or soluble RSV F proteins were evaluated for efficacy and immunogenicity in a nonhuman primate model. b/h PIV3 is suited for development of pediatric vaccines since bPIV3 had already been evaluated in clinical studies in 1- and 2-month-old infants and was found to be safe, immunogenic, and nontransmissible in a day care setting (Karron et al., Pediatr. Infect. Dis. J. 15:650-654, 1996; Lee et al., J. Infect. Dis. 184:909-913, 2001). African green monkeys immunized with b/h PIV3 expressing either the native or soluble RSV F protein were protected from challenge with wild-type RSV and produced RSV neutralizing and RSV F-protein specific immunoglobulin G serum antibodies. The PIV3-vectored RSV vaccines evaluated here further underscore the utility of this vector system for developing safe and immunogenic pediatric respiratory virus vaccines.

Recovery of human metapneumovirus genetic lineages a and B from cloned cDNA.

Human metapneumovirus (hMPV) is a newly discovered pathogen associated with respiratory tract illness, primarily in young children, immunocompromised individuals, and the elderly. The genomic sequence of the prototype hMPV isolate NL/1/00 without the terminal leader and trailer sequences has been reported previously. Here we describe the leader and trailer sequences of two hMPV isolates, NL/1/00 and NL/1/99, representing the two main genetic lineages of hMPV. Minigenome constructs in which the green fluorescent protein or chloramphenicol acetyltransferase genes are flanked by the viral genomic ends derived from both hMPV lineages and transcribed using a T7 RNA polymerase promoter-terminator cassette were generated. Cotransfection of minigenome constructs with plasmids expressing the polymerase complex components L, P, N, and M2.1 in 293T or baby hamster kidney cells resulted in expression of the reporter genes. When the minigenome was replaced by a sense or antisense full-length cDNA copy of the NL/1/00 or NL/1/99 viral genomes, recombinant virus was recovered from transfected cells. Viral titers up to 10(7.2) and 10(5.7) 50% tissue culture infective dose/ml were achieved with the sense and antisense plasmids, respectively. The recombinant viruses replicated with kinetics similar to those of the parental viruses in Vero cells. This reverse genetics system provides an important new tool for applied and fundamental research.

Identification of small-animal and primate models for evaluation of vaccine candidates for human metapneumovirus (hMPV) and implications for hMPV vaccine design.

Human metapneumovirus (hMPV), a recently identified paramyxovirus, is the causative agent of respiratory tract disease in young children. Epidemiological studies have established the presence of hMPV in retrospective as well as current clinical samples in Europe, USA, Canada, Hong Kong and Australia. The hMPV disease incidence rate varied from 7 to 12 %. This rate of disease attack places hMPV in severity between respiratory syncytial virus and human parainfluenza virus type 3, two common respiratory pathogens of young children, the elderly and immunosuppressed individuals. To evaluate the effectiveness and safety of future hMPV antiviral drugs, therapeutic and prophylactic monoclonal antibodies (mAbs), and vaccine candidates, it was necessary to identify small-animal and primate models that efficiently supported hMPV replication in the respiratory tract and produced neutralizing serum antibodies, commonly a clinical correlate of protection in humans. In this study, various rodents (mice, cotton rats, hamsters and ferrets) and two primate species, rhesus macaques and African green monkeys (AGMs), were evaluated for hMPV replication in the respiratory tract. The results showed that hamsters, ferrets and AGMs supported hMPV replication efficiently and produced high levels of hMPV-neutralizing antibody titres. Hamsters vaccinated with subgroup A hMPV were protected from challenge with subgroup A or subgroup B hMPV, which has implications for hMPV vaccine design. Although these animal models do not mimic human hMPV disease signs, they will nevertheless be invaluable for the future evaluation of hMPV antivirals, mAbs and vaccines.

Real-time reverse transcriptase PCR assay for detection of human metapneumoviruses from all known genetic lineages.

The discovery of human metapneumovirus and its implications for respiratory tract disease have emphasized the need for a sensitive, specific, and rapid assay to detect this virus in a clinical setting. It recently became clear that human metapneumovirus can be grouped into at least four genetic lineages. Previously described assays for the detection of human metapneumovirus were developed by using limited sequence information and failed to detect viruses from all four genetic lineages with comparable sensitivities. Here we describe the development and evaluation of a real-time reverse transcriptase PCR assay that detects human metapneumovirus from the four known genetic lineages with equal specificities and sensitivities.

Effects of human metapneumovirus and respiratory syncytial virus antigen insertion in two 3' proximal genome positions of bovine/human parainfluenza virus type 3 on virus replication and immunogenicity.

A live attenuated bovine parainfluenza virus type 3 (PIV3), harboring the fusion (F) and hemagglutinin-neuraminidase (HN) genes of human PIV3, was used as a virus vector to express surface glycoproteins derived from two human pathogens, human metapneumovirus (hMPV) and respiratory syncytial virus (RSV). RSV and hMPV are both paramyxoviruses that cause respiratory disease in young children, the elderly, and immunocompromised individuals. RSV has been known for decades to cause acute lower respiratory tract infections in young children, which often result in hospitalization, while hMPV has only been recently identified as a novel human respiratory pathogen. In this study, the ability of bovine/human PIV3 to express three different foreign transmembrane surface glycoproteins and to induce a protective immune response was evaluated. The RNA-dependent RNA polymerase of paramyxoviruses binds to a single site at the 3' end of the viral RNA genome to initiate transcription of viral genes. The genome position of the viral gene determines its level of gene expression. The promoter-proximal gene is transcribed with the highest frequency, and each downstream gene is transcribed less often due to attenuation of transcription at each gene junction. This feature of paramyxoviruses was exploited using the PIV3 vector by inserting the foreign viral genes at the 3' terminus, at position 1 or 2, of the viral RNA genome. These locations were expected to yield high levels of foreign viral protein expression stimulating a protective immune response. The immunogenicity and protection results obtained with a hamster model showed that bovine/human PIV3 can be employed to generate bivalent PIV3/RSV or PIV3/hMPV vaccine candidates that will be further evaluated for safety and efficacy in primates.