Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein.

Respiratory syncytial virus (RSV) infections remain a major cause of respiratory disease and hospitalizations among infants. Infection recurs frequently and establishes a weak and short-lived immunity. To date, RSV immunoprophylaxis and vaccine research is mainly focused on the RSV fusion (F) protein, but a vaccine remains elusive. The RSV F protein is a highly conserved surface glycoprotein and is the main target of neutralizing antibodies induced by natural infection. Here, we analyzed an internalization process of antigen-antibody complexes after binding of RSV-specific antibodies to RSV antigens expressed on the surface of infected cells. The RSV F protein and attachment (G) protein were found to be internalized in both infected and transfected cells after the addition of either RSV-specific polyclonal antibodies (PAbs) or RSV glycoprotein-specific monoclonal antibodies (MAbs), as determined by indirect immunofluorescence staining and flow-cytometric analysis. Internalization experiments with different cell lines, well-differentiated primary bronchial epithelial cells (WD-PBECs), and RSV isolates suggest that antibody internalization can be considered a general feature of RSV. More specifically for RSV F, the mechanism of internalization was shown to be clathrin dependent. All RSV F-targeted MAbs tested, regardless of their epitopes, induced internalization of RSV F. No differences could be observed between the different MAbs, indicating that RSV F internalization was epitope independent. Since this process can be either antiviral, by affecting virus assembly and production, or beneficial for the virus, by limiting the efficacy of antibodies and effector mechanism, further research is required to determine the extent to which this occurs in vivo and how this might impact RSV replication.IMPORTANCE Current research into the development of new immunoprophylaxis and vaccines is mainly focused on the RSV F protein since, among others, RSV F-specific antibodies are able to protect infants from severe disease, if administered prophylactically. However, antibody responses established after natural RSV infections are poorly protective against reinfection, and high levels of antibodies do not always correlate with protection. Therefore, RSV might be capable of interfering, at least partially, with antibody-induced neutralization. In this study, a process through which surface-expressed RSV F proteins are internalized after interaction with RSV-specific antibodies is described. One the one hand, this antigen-antibody complex internalization could result in an antiviral effect, since it may interfere with virus particle formation and virus production. On the other hand, this mechanism may also reduce the efficacy of antibody-mediated effector mechanisms toward infected cells.

Report of the 2nd "French Clinical Vaccinology Meeting Jean-Gerard Guillet": immunization and respiratory diseases.

The 2nd French Clinical Vaccinology conference held on 20th April 2009 in Paris (France) was a unique opportunity to discuss basic and translational research in vaccinology and its implications for patients for respiratory diseases. This conference is organized by the Clinical Research Center Cochin-Pasteur, that has been involved for several years clinical research in vaccines. We report on here the key findings of the conference, especially the immunization of the chronic respiratory diseases, the clinical effectiveness of vaccines and the development of new vaccines in pulmonology.

Structure-antigenicity relationship studies of the central conserved region of human respiratory syncytial virus protein G.

BBG2Na is a recombinant protein, composed in part of carrier protein BB and of the central conserved domain of the attachment glycoprotein G of human respiratory syncytial virus (HRSV) subgroup A. This protein is a potent vaccine candidate against HRSV. G2Na contains several contiguous B-cell epitopes, occupying sequential positions in the linear sequence of the protein. One of the epitopes contains four cysteines that are completely conserved in known strains of HRSV and form a 'cysteine noose' motif. In this study, we analysed circular dichroism (CD) spectra of BBG2Na and its B-cell epitopes. We also used NMR and molecular dynamics simulations to determine the three-dimensional structure of the cysteine noose domain. We observed significant structural differences related to the length of peptides containing the cysteine noose. These differences show good correlation with the immunogenic activity of the peptides. It is shown that a single Val(171) addition induces a pronounced structure stabilization of the cysteine noose peptide G4a (1-4/2-3) (residues 172-187), which is associated with a 100-fold increase in its antigenicity vis-à-vis a G-protein specific monoclonal antibody.

Safety and immunogenicity of a novel recombinant subunit respiratory syncytial virus vaccine (BBG2Na) in healthy young adults.

A novel recombinant respiratory syncytial virus (RSV) subunit vaccine, designated BBG2Na, was administered to 108 healthy adults randomly assigned to receive 10, 100, or 300 microg of BBG2Na in aluminum phosphate or saline placebo. Each subject received 1, 2, or 3 intramuscular injections of the assigned dose at monthly intervals. Local and systemic reactions were mild, and no evidence of harmful properties of BBG2Na was reported. The highest ELISA and virus-neutralizing (VN) antibody responses were evident in the 100- and 300-microg groups; second or third injections provided no significant boosts against RSV-derived antigens. BBG2Na induced > or 2-fold and > or =4-fold increases in G2Na-specific ELISA units in up to 100% and 57% of subjects, respectively; corresponding RSV-A-specific responses were 89% and 67%. Furthermore, up to 71% of subjects had > or =2-fold VN titer increases. Antibody responses to 2 murine lung protective epitopes were also highly boosted after vaccination. Therefore, BBG2Na is safe, well tolerated, and highly immunogenic in RSV-seropositive adults.

Differential histopathology and chemokine gene expression in lung tissues following respiratory syncytial virus (RSV) challenge of formalin-inactivated RSV- or BBG2Na-immunized mice.

A BALB/c mouse model of enhanced pulmonary pathology following vaccination with formalin-inactivated alum-adsorbed respiratory syncytial virus (FI-RSV) and live RSV challenge was used to determine the type and kinetics of histopathologic lesions induced and chemokine gene expression profiles in lung tissues. These data were compared and contrasted with data generated following primary and/or secondary RSV infection or RSV challenge following vaccination with a promising subunit vaccine, BBG2Na. Severe peribronchiolitis and perivascularitis coupled with alveolitis and interstitial inflammation were the hallmarks of lesions in the lungs of FI-RSV-primed mice, with peak histopathology evident on days 5 and 9. In contrast, primary RSV infection resulted in no discernible lesions, while challenge of RSV-primed mice resulted in rare but mild peribronchiolitis and perivascularitis, with no evidence of alveolitis or interstitial inflammation. Importantly, mice vaccinated with a broad dose range (20 to 0.02 microg) of a clinical formulation of BBG2Na in aluminium phosphate demonstrated histopathology similar to that observed in secondary RSV infection. At the molecular level, FI-RSV priming was characterized by a rapid and strong up-regulation of eotaxin and monocyte chemotactic protein 3 (MCP-3) relative gene expression (potent lymphocyte and eosinophil chemoattractants) that was sustained through late time points, early but intermittent up-regulation of GRO/melanoma growth stimulatory activity gene and inducible protein 10 gene expression, while macrophage inflammatory protein 2 (MIP-2) and especially MCP-1 were up-regulated only at late time points. By comparison, primary RSV infection or BBG2Na priming resulted in considerably lower eotaxin and MCP-3 gene expression increases postchallenge, while expression of lymphocyte or monocyte chemoattractant chemokine genes (MIP-1beta, MCP-1, and MIP-2) were of higher magnitude and kinetics at early, but not late, time points. Our combined histopathologic and chemokine gene expression data provide a basis for differentiating between aberrant FI-RSV-induced immune responses and normal responses associated with RSV infection in the mouse model. Consequently, our data suggest that BBG2Na may constitute a safe RSV subunit vaccine for use in seronegative infants.

A novel bipolar mode of attachment to aluminium-containing adjuvants by BBG2Na, a recombinant subunit hRSV vaccine.

Human respiratory syncytial virus (hRSV) is a major pathogen responsible for bronchiolitis and severe pulmonary disease in very young children, immunodeficient patients and the elderly. BBG2Na, a recombinant chimeric protein produced in Escherichia coli, is a promising subunit vaccine candidate against this respiratory pathogen, composed of G2Na, the central domain of RSV G glycoprotein, and BB, an albumin binding domain of streptococcal protein G. BBG2Na has a basic isoelectric point (pI 9.3) and as expected, is strongly adsorbed by aluminium phosphate (AP). Surprisingly, BBG2Na is also strongly adsorbed by aluminium hydroxide (AH), which normally binds molecules with acidic isoelectric points. This behaviour was unexpected according to the well established adsorption model of Hem and co-workers. Our observations may be explained by the bipolar two-domain structure of the BBG2Na chimera which is not reflected by the global basic isoelectric point of the whole protein: the BB domain has an acidic isoelectric point (pI 5.5) and the G2Na domain a highly basic one (pI 10.0). Importantly, formulation in either aluminium salt resulted in equally high immunogenicity and protective efficacy against RSV in mice. From a physicochemical point of view, this unique property of BBG2Na makes it eminently suitable for combination to either paediatric or elderly multivalent AH- or AP-containing vaccines already in the market or in development.

Identification and characterisation of multiple linear B cell protectopes in the respiratory syncytial virus G protein.

Respiratory syncytial virus (RSV) is an important respiratory pathogen in man, against which no vaccine is available. However, recent evidence suggests that antibodies to the RSV F and G proteins may play an important role in disease prevention. We previously demonstrated that BBG2Na, a subunit vaccine candidate including residues 130-230 of the Long strain G protein, protects rodents against RSV challenge. Using a panel of monoclonal antibodies (MAb) and synthetic peptides, five linear B cell epitopes were identified that mapped to residues 152-163, 165-172, 171-187 (two over-lapping epitopes) and 196-204. Antibody passive transfer and peptide immunisation studies revealed that all were protective. Pepscan analyses of anti-RSV-A and BBG2Na murine polyclonal sera suggested stronger immunogenicity of some protective epitopes (protectopes) in the context of BBG2Na compared with live virus. However, all the identified murine B cell protectopes were conserved in RSV seropositive humans. Should these protectopes correspond with protection in humans, BBG2Na may constitute a very interesting vaccine candidate against RSV.

Protection against respiratory syncytial virus (RSV) elicited in mice by plasmid DNA immunisation encoding a secreted RSV G protein-derived antigen.

Plasmid vectors encoding two different variants, one cytoplasmic and one secreted version, of a candidate vaccine BBG2Na to respiratory syncytial virus (RSV), were constructed and evaluated in a nucleic acid vaccination study. The two different vectors, which employed the Semliki Forest virus gene amplification system, were found to express BBG2Na appropriately in in vitro cell cultures. Immunisation of mice with the plasmid vectors elicited significant serum anti-BBG2Na IgG responses only in the mice receiving the plasmid encoding the secreted version of BBG2Na. Consistent with antibody induction data, sterilising lung protection against RSV-A challenge was also only observed in this group. These results indicate that the targeting of antigen expression (intracellular versus secreted) would be an important factor to consider in the design of nucleic acid vaccines.

Influence of administration dose and route on the immunogenicity and protective efficacy of BBG2Na, a recombinant respiratory syncytial virus subunit vaccine candidate.

The immunogenicity and protective efficacy of BBG2Na, a novel recombinant respiratory syncytial virus subunit vaccine candidate, was assessed in BALB/c mice under various conditions of dose, administration route and number of immunisations. A single intra-peritoneal (i.p.) dose of 2 microg, or two doses of 0.2 microg, were sufficient to induce elevated RSV-A serum antibodies and sterilising lung protective immunity. Serum antibody titres were significantly boosted following second immunisations, but not a third. Of three routes of immunisation, i.p. induced the highest RSV-A antibody titres, followed in efficacy by the intra-muscular (i. m.) and subcutaneous (s.c.) routes. Nonetheless, all three routes induced comparable and sterilising lung protection. In contrast, upper respiratory tract protection was observed only after i.p. vaccination, although significant viral titre reductions were evident following i.m. or s.c. immunisations. Interestingly, Pepscan analyses indicated that antibody epitope usage was highest in i.p. and lowest in i.m. immunised mice, respectively. Nonetheless, all routes resulted in antibody responses to known lung protective epitopes (protectopes). Thus, the prevention of serious lower respiratory tract disease, the principle goal of a RSV vaccine, but not URT infection, is dose dependent but unlikely to be influenced by the route of BBG2Na administration.

CD4(+) T-cell-mediated antiviral protection of the upper respiratory tract in BALB/c mice following parenteral immunization with a recombinant respiratory syncytial virus G protein fragment.

We analyzed the protective mechanisms induced against respiratory syncytial virus subgroup A (RSV-A) infection in the lower and upper respiratory tracts (LRT and URT) of BALB/c mice after intraperitoneal immunization with a recombinant fusion protein incorporating residues 130 to 230 of RSV-A G protein (BBG2Na). Mother-to-offspring antibody (Ab) transfer and adoptive transfer of BBG2Na-primed B cells into SCID mice demonstrated that Abs are important for LRT protection but have no effect on URT infection. In contrast, RSV-A clearance in the URT was achieved in a dose-dependent fashion after adoptive transfer of BBG2Na-primed T cells, while it was abolished in BBG2Na-immunized mice upon in vivo depletion of CD4(+), but not CD8(+), T cells. Furthermore, the conserved RSV-A G protein cysteines and residues 193 and 194, overlapping the recently identified T helper cell epitope on the G protein (P. W. Tebbey et al., J. Exp. Med. 188:1967-1972, 1998), were found to be essential for URT but not LRT protection. Taken together, these results demonstrate for the first time that CD4(+) T cells induced upon parenteral immunization with an RSV G protein fragment play a critical role in URT protection of normal mice against RSV infection.

Identification of multiple protective epitopes (protectopes) in the central conserved domain of a prototype human respiratory syncytial virus G protein.

A recombinant fusion protein (BBG2Na) comprising the central conserved domain of the respiratory syncytial virus subgroup A (RSV-A) (Long) G protein (residues 130 to 230) and an albumin binding domain of streptococcal protein G was shown previously to protect mouse upper (URT) and lower (LRT) respiratory tracts against intranasal RSV challenge (U. F. Power, H. Plotnicky-Gilquin, T. Huss, A. Robert, M. Trudel, S. Stahl, M. Uhlén, T. N. Nguyen, and H. Binz, Virology 230:155-166, 1997). Panels of monoclonal antibodies (MAbs) and synthetic peptides were generated to facilitate dissection of the structural elements of this domain implicated in protective efficacy. All MAbs recognized native RSV-A antigens, and five linear B-cell epitopes were identified; these mapped to residues 152 to 163, 165 to 172, 171 to 187 (two overlapping epitopes), and 196 to 204, thereby covering the highly conserved cysteine noose domain. Antibody passive-transfer and peptide immunization studies revealed that all epitopes were implicated in protection of the LRT, but not likely the URT, against RSV-A challenge. Pepscan analyses of anti-RSV-A and anti-BBG2Na murine polyclonal sera revealed lower-level epitope usage within the central conserved region in the former, suggesting diminished immunogenicity of the implicated epitopes in the context of the whole virus. However, Pepscan analyses of RSV-seropositive human sera revealed that all of the murine B-cell protective epitopes (protectopes) that mapped to the central conserved domain were recognized in man. Should these murine protectopes also be implicated in human LRT protection, their clustering around the highly conserved cysteine noose region will have important implications for the development of RSV vaccines.

Protective efficacy against respiratory syncytial virus following murine neonatal immunization with BBG2Na vaccine: influence of adjuvants and maternal antibodies.

Alum-adsorbed BBG2Na, a recombinant vaccine derived in part from the respiratory syncytial virus (RSV) subgroup A G protein, induced moderate antibody titers after 1 immunization in 1-week-old mice but conferred complete lung protection upon RSV challenge. The anti-BBG2Na IgG1-IgG2a neonatal isotype profile was suggestive of dominant Th2 responses compared with those in adults. Formulation of BBG2Na with a Th1-driving adjuvant efficiently shifted neonatal responses toward a more balanced and adultlike IgG1-IgG2a profile without compromising its protective efficacy. BBG2Na-induced protective immunity was maintained even after early life immunization in the presence of high titers of maternal antibodies. Under these conditions, the protective efficacy (86%-100%) reflected the high capacity of the nonglycosylated G2Na immunogen to escape inhibition by RSV-A-induced maternal antibodies. Thus, immunization with BBG2Na protected against viral challenge despite neonatal immunologic immaturity and the presence of maternal antibodies, two major obstacles to neonatal RSV vaccine development.

Absence of lung immunopathology following respiratory syncytial virus (RSV) challenge in mice immunized with a recombinant RSV G protein fragment.

The relative immunopathogenic potential of a recombinant fusion protein incorporating residues 130-230 of respiratory syncytial virus (RSV-A) G protein (BBG2Na), formalin-inactivated RSV-A (FI-RSV), and phosphate-buffered saline (PBS) was investigated in mice after immunization and RSV challenge. FI-RSV priming resulted in massive infiltration of B cells and activated CD4(+) and CD8(+) T lymphocytes in mediastinal lymph nodes (MLN) and lungs, where eosinophilia and elevated IFN-gamma, IL-2, -4, -5, -10, and -13 mRNA transcripts were also detected. PBS-primed mice showed only elevated pulmonary IL-2 and IFN-gamma mRNAs, while an activated CD8(+) T cell peak was detected in MLN and lungs. Cell infiltration also occurred in MLN of BBG2Na-immunized mice. However, there was no evidence of T cell, B cell, or granulocyte infiltration or activation in lungs, while transient transcription of Th1-type cytokine genes was evident. The absence of pulmonary infiltration is unlikely due to insufficient viral antigen. Thus, this recombinant fusion RSV G fragment does not prime for adverse pulmonary immunopathologic responses.

Protective immunity against respiratory syncytial virus in early life after murine maternal or neonatal vaccination with the recombinant G fusion protein BBG2Na.

Maternal and neonatal immunization were evaluated for their capacity to induce protective immunity against respiratory syncytial virus (RSV) lower respiratory tract infections in early life. Murine models were studied by use of a novel recombinant vaccine candidate, designated BBG2Na, which was derived in part from the RSV (Long) G protein. Maternal immunization resulted in the passive transfer of high levels of RSV-A antibodies to the offspring, which protected them from RSV challenge for up to 14 weeks. Indeed, protection correlated with the detection of RSV antibodies in the serum. Neonatal immunization with BBG2Na induced significant antibody responses even in the first week of life. Most importantly, these neonatal responses were not inhibited by the presence of RSV maternal antibodies. Consequently, the combination of maternal and neonatal immunization with BBG2Na resulted in the continual presence of protective levels of antibodies in the offspring.

Challenge of BALB/c mice with respiratory syncytial virus does not enhance the Th2 pathway induced after immunization with a recombinant G fusion protein, BBG2NA, in aluminum hydroxide.

The polypeptide of aa 130-230 of the G protein (G2Na) of respiratory syncytial virus (RSV) was fused to BB, the albumin-binding region of streptococcal G protein, producing BBG2Na, which induced protective immune responses in rodent models. Evaluation of the immune response in mice immunized with BBG2Na in the adjuvant alhydrogel revealed high amounts of interleukin (IL)-5 and some IL-4 in splenocytes restimulated in vitro. This is compatible with a Th2 response. The activation of the Th2 pathway in such mice was further supported by the detection of IL-5 and G2Na-specific IgE in vivo. Of interest, in contrast to immunization with formalin-inactivated RSV, immunization of mice with BBG2Na followed by intranasal RSV challenge did not lead to increased production of IL-5- or G2Na-specific IgE. However, IgG1- and IgG2a-specific antibodies were boosted. These results demonstrate that the Th2 pathway is not enhanced by RSV challenge in BBG2Na-immunized mice.

Induction of protective immunity in rodents by vaccination with a prokaryotically expressed recombinant fusion protein containing a respiratory syncytial virus G protein fragment.

A subunit approach to the development of a respiratory syncytial virus (RSV) vaccine was investigated. It involved the production, in Escherichia coli, of an RSV (Long) G protein fragment (G2Na) as a C-terminal fusion partner to an albumin binding region (BB) of streptococcal protein G. G2Na incorporated amino acid residues 130-230 and was specifically recognized by murine anti-RSV-A polyclonal serum. In mice, intraperitoneal immunization with BBG2Na induced high anti-RSV-A serum ELISA titers and low to moderate neutralization activity. The immune response induced by BBG2Na demonstrated a potent protective efficacy against upper and lower respiratory tract RSV-A infection. The immunogenicity and protective efficacy of BBG2Na was maintained for at least 47 and 48 weeks, respectively, and was as potent and durable as live RSV-A administered in a similar fashion. Intramuscular immunization of cotton rats with BBG2Na protected lungs from both homologous and heterologous virus challenge. In contrast to mice, however, cotton rat nasal tracts were not protected after BBG2Na immunization. Consistent with antibody-mediated protection, virus was cleared within 24 hr from the lungs of BBG2Na-immunized mice. The anti-RSV-A antibodies induced in mice were exclusively of the IgG1 isotype and were detected in the serum, lungs, and nasal tracts. Passive transfer of these antibodies prevented acute, and eliminated chronic, RSV-A lung infection in normal and immunodeficient mice, respectively, confirming that such antibodies are important and sufficient for BBG2Na-induced pulmonary protection. Our results clearly demonstrate that BBG2Na contains an important immunogenic domain of the RSV G protein. The prokaryotic origin of this protein indicates that glycosylation of the RSV G protein is not necessary for protective efficacy. Thus, BBG2Na has potential as an RSV subunit vaccine.

Selective interference with P protein binding to paramyxovirus nucleocapsids.

We previously observed that some anti-nucleoprotein monoclonal antibodies were able to displace P protein from Sendai virus (SV) nucleocapsid cores. The current work extends that observation by showing that such antibody-mediated P displacement is not unique to Sendai virus nucleocapsids, but can also occur with nucleocapsids of a related human respiratory pathogen. Anti-NP antibody prevents binding of SV P protein to nucleocapsids from human parainfluenza virus type 1 (PIV1) or from SV. Antibody also prevents binding of PIV1 P protein to PIV1 nucleocapsids, but not to SV nucleocapsids. We have also examined the stoichiometry of antibody interference with P binding, to determine how large a nucleocapsid region can be protected from P binding by a single antibody molecule. We found that approximately 40 antibody molecules per nucleocapsid complex can block attachment of most P protein. This indicates that a single antibody molecule can prevent P binding to a region representing about 65 nucleoprotein monomers on the nucleocapsid core.

Viral cross-reactivity and antigenic determinants recognized by human parainfluenza virus type 1-specific cytotoxic T-cells.

To obtain information relevant to vaccination against human parainfluenza virus type 1 (hPIV-1), cytotoxic T-lymphocyte (CTL) responses to individual viral components were tested. The CD8-positive T-cell fraction was first enriched from human, adult PBL and grown for several passages in the presence of hPIV-1-infected stimulator cells. T-cell lines were then tested for CTL activity toward hPIV-1 and toward the related viruses hPIV-3 and Sendai virus (the murine parainfluenza type 1 virus). All tested cultures which responded to hPIV-1 also responded to hPIV-3 and Sendai virus, demonstrating sequence conservation between all three viruses among major antigenic determinants for CTL. Specificity for particular viral components was defined using recombinant vaccinia viruses expressing individual proteins from either mouse or human parainfluenza type 1 viruses. Strong CTL responses toward hemagglutinin-neuraminidase, phosphoprotein, and nucleoprotein (NP) were demonstrated. The testing of vaccinia constructs expressing truncated proteins then showed that there were multiple CTL determinants within NP. Several T-cell lines from one donor recognized an NP peptide (amino acids 321-336) conserved between the hPIV-1 and Sendai virus. In total, the results demonstrated that the human CTL response is directed to multiple determinants within several distinct hPIV-1 proteins.

Sequence characterization and expression of the matrix protein gene of human parainfluenza virus type 1.

The nucleotide sequence of the M gene of human parainfluenza virus type 1 (hPIV1) was determined from genomic RNA and cDNA copies of the entire gene. The M gene contained 1173 nucleotides. It had one large open reading frame capable of encoding a protein of 348 amino acids (M(r) = 38,404). The predicted amino acid sequence of the hPIV1 M protein is highly basic (+20 at neutral pH). A pGEM-1 expression vector containing the M gene was used for cell-free transcription and translation. The resultant protein was confirmed to be M by electrophoretic mobility and immunoprecipitation. Among other paramyxoviridae the hPIV1 M amino acid sequence was most closely related to the Sendai virus M sequence (87% identity). The pattern of M gene relatedness observed from the alignment of 16 paramyxoviridae M protein amino acid sequences was not predicted by the viruses' taxonomic classification.

The P genes of human parainfluenza virus type 1 clinical isolates are polycistronic and microheterogeneous.

The nucleotide sequence of the P gene of human parainfluenza virus type 1 (hPIV1) strain C35 was determined directly from genomic viral RNA and by molecular cloning. The gene contained 1893 nucleotides. Four open reading frames (ORF) capable of encoding a P protein (568 amino acids; M(r) = 64,784), a C' protein (219 amino acids; M(r) = 25,997), a C protein (204 amino acids; M(r) = 24,237), and a Y1 protein (182 amino acids; M(r) = 21,471) were identified. The latter three ORFs are in a +1 reading frame relative to P. The sequencing data are consistent with the hPIV1 C' protein being initiated at a GUG codon (nt 68-70), in contrast to the ACG initiation of the Sendai virus (SV) C' protein. Unlike SV, there is no evidence of a hPIV1 ORF capable of encoding a cysteine-rich V protein. Also, there is no ORF capable of encoding a protein analogous to the SV Y2 protein. In vitro transcription, translation, and immunoprecipitation showed that the hPIV1 P gene is polycistronic. Comparison of the P gene with those of two other distinct clinical isolates confirmed the coding potential of the hPIV1 P gene but also revealed genetic heterogeneity among the isolates. Our results indicate that the hPIV1 P gene uses some coding strategies similar to and others that are different from those of other paramyxovirus P genes.