Human parainfluenza virus 3 vaccine candidates attenuated by codon-pair deoptimization are immunogenic and protective in hamsters.

Human parainfluenza virus type 3 (HPIV3) is a major pediatric respiratory pathogen lacking available vaccines or antiviral drugs. We generated live-attenuated HPIV3 vaccine candidates by codon-pair deoptimization (CPD). HPIV3 open reading frames (ORFs) encoding the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and polymerase (L) were modified singly or in combination to generate 12 viruses designated Min-N, Min-P, Min-M, Min-FHN, Min-L, Min-NP, Min-NPM, Min-NPL, Min-PM, Min-PFHN, Min-MFHN, and Min-PMFHN. CPD of N or L severely reduced growth in vitro and was not further evaluated. CPD of P or M was associated with increased and decreased interferon (IFN) response in vitro, respectively, but had little effect on virus replication. In Vero cells, CPD of F and HN delayed virus replication, but final titers were comparable to wild-type (wt) HPIV3. In human lung epithelial A549 cells, CPD F and HN induced a stronger IFN response, viral titers were reduced 100-fold, and the expression of F and HN proteins was significantly reduced without affecting N or P or the relative packaging of proteins into virions. Following intranasal infection in hamsters, replication in the nasal turbinates and lungs tended to be the most reduced for viruses bearing CPD F and HN, with maximum reductions of approximately 10-fold. Despite decreased in vivo replication (and lower expression of CPD F and HN in vitro), all viruses induced titers of serum HPIV3-neutralizing antibodies similar to wt and provided complete protection against HPIV3 challenge. In summary, CPD of HPIV3 yielded promising vaccine candidates suitable for further development.

Safety and Immunogenicity of an mRNA-Based hMPV/PIV3 Combination Vaccine in Seropositive Children.

OBJECTIVES: Human metapneumovirus (hMPV) and parainfluenza virus type 3 (PIV3) are common respiratory illnesses in children. The safety and immunogenicity of an investigational mRNA-based vaccine, mRNA-1653, encoding membrane-anchored fusion proteins of hMPV and PIV3, was evaluated in hMPV/PIV3-seropositive children. METHODS: In this phase 1b randomized, observer-blind, placebo-controlled, dose-ranging study, hMPV/PIV3-seropositive children were enrolled sequentially into 2 dose levels of mRNA-1653 administered 2 months apart; children aged 12 to 36 months were randomized (1:1) to receive 10-µg of mRNA-1653 or placebo and children aged 12 to 59 months were randomized (3:1) to receive 30-µg of mRNA-1653 or placebo. RESULTS: Overall, 27 participants aged 18 to 55 months were randomized; 15 participants received 10-µg of mRNA-1653 (n = 8) or placebo (n = 7), whereas 12 participants received 30-µg of mRNA-1653 (n = 9) or placebo (n = 3). mRNA-1653 was well-tolerated at both dose levels. The only reported solicited local adverse reaction was tenderness at injection site; solicited systemic adverse reactions included grade 1 or 2 chills, irritability, loss of appetite, and sleepiness. A single 10-µg or 30-µg mRNA-1653 injection increased hMPV and PIV3 neutralizing antibody titers (geometric mean fold-rise ratio over baseline: hMPV-A = 2.9-6.1; hMPV-B = 6.2-13.2; PIV3 = 2.8-3.0) and preF and postF binding antibody concentrations (geometric mean fold-rise ratio: hMPV preF = 5.3-6.1; postF = 4.6-6.5 and PIV3 preF = 13.9-14.2; postF = 11.0-12.1); a second injection did not further increase antibody levels in these seropositive children. Binding antibody responses were generally preF biased. CONCLUSIONS: mRNA-1653 was well-tolerated and boosted hMPV and PIV3 antibody levels in seropositive children aged 12 to 59 months, supporting the continued development of mRNA-1653 or its components for the prevention of hMPV and PIV3.

Human parainfluenza virus type 3 expressing the respiratory syncytial virus pre-fusion F protein modified for virion packaging yields protective intranasal vaccine candidates.

Human respiratory syncytial virus (RSV) and parainfluenza virus type 3 (HPIV3) are among the most common viral causes of childhood bronchiolitis and pneumonia worldwide, and lack effective antiviral drugs or vaccines. Recombinant (r) HPIV3 was modified to express the RSV fusion (F) glycoprotein, the major RSV neutralization and protective antigen, providing a live intranasal bivalent HPIV3/RSV vaccine candidate. This extends previous studies using a chimeric bovine-human PIV3 vector (rB/HPIV3). One advantage is that rHPIV3 expresses all of the HPIV3 antigens compared to only two for rB/HPIV3. In addition, the use of rHPIV3 as vector should avoid excessive attenuation following addition of the modified RSV F gene, which may occur with rB/HPIV3. To enhance its immunogenicity, RSV F was modified (i) to increase the stability of the prefusion (pre-F) conformation and (ii) by replacement of its transmembrane (TM) and cytoplasmic tail (CT) domains with those of HPIV3 F (H3TMCT) to increase incorporation in the vector virion. RSV F (+/- H3TMCT) was expressed from the first (F/preN) or the second (F/N-P) gene position of rHPIV3. The H3TMCT modification dramatically increased packaging of RSV F into the vector virion and, in hamsters, resulted in significant increases in the titer of high-quality serum RSV-neutralizing antibodies, in addition to the increase conferred by pre-F stabilization. Only F-H3TMCT/preN replication was significantly attenuated in the nasal turbinates by the RSV F insert. F-H3TMCT/preN, F/N-P, and F-H3TMCT/N-P provided complete protection against wt RSV challenge. F-H3TMCT/N-P exhibited the most stable and highest expression of RSV F, providing impetus for its further development.

Safety and infectivity of two doses of live-attenuated recombinant cold-passaged human parainfluenza type 3 virus vaccine rHPIV3cp45 in HPIV3-seronegative young children.

BACKGROUND: Human parainfluenza virus type 3 (HPIV3) is a common cause of upper and lower respiratory tract illness in infants and young children. Live-attenuated cold-adapted HPIV3 vaccines have been evaluated in infants but a suitable interval for administration of a second dose of vaccine has not been defined. METHODS: HPIV3-seronegative children between the ages of 6 and 36 months were randomized 2:1 in a blinded study to receive two doses of 105 TCID50 (50% tissue culture infectious dose) of live-attenuated, recombinant cold-passaged human PIV3 vaccine (rHPIV3cp45) or placebo 6 months apart. Serum antibody levels were assessed prior to and approximately 4-6 weeks after each dose. Vaccine virus infectivity, defined as detection of vaccine-HPIV3 in nasal wash and/or a≥4-fold rise in serum antibody titer, and reactogenicity were assessed on days 3, 7, and 14 following immunization. RESULTS: Forty HPIV3-seronegative children (median age 13 months; range 6-35 months) were enrolled; 27 (68%) received vaccine and 13 (32%) received placebo. Infectivity was detected in 25 (96%) of 26 evaluable vaccinees following doses 1 and 9 of 26 subject (35%) following dose 2. Among those who shed virus, the median duration of viral shedding was 12 days (range 6-15 days) after dose 1 and 6 days (range 3-8 days) after dose 2, with a mean peak log10 viral titer of 3.4 PFU/mL (SD: 1.0) after dose 1 compared to 1.5 PFU/mL (SD: 0.92) after dose 2. Overall, reactogenicity was mild, with no difference in rates of fever and upper respiratory infection symptoms between vaccine and placebo groups. CONCLUSION: rHPIV3cp45 was immunogenic and well-tolerated in seronegative young children. A second dose administered 6 months after the initial dose was restricted in those previously infected with vaccine virus; however, the second dose boosted antibody responses and induced antibody responses in two previously uninfected children.

A replication-incompetent influenza virus bearing the HN glycoprotein of human parainfluenza virus as a bivalent vaccine.

Influenza virus and human parainfluenza virus (HPIV) are major etiologic agents of acute respiratory illness in young children. Inactivated and live attenuated influenza vaccines are approved in several countries, yet no vaccine is licensed for HPIV. We previously showed that a replication-incompetent PB2-knockout (PB2-KO) virus that possesses a reporter gene in the coding region of the PB2 segment can serve as a platform for a bivalent vaccine. To develop a bivalent vaccine against influenza and parainfluenza virus, here, we generated a PB2-KO virus possessing the hemagglutinin-neuraminidase (HN) glycoprotein of HPIV type 3 (HPIV3), a major surface antigen of HPIV, in its PB2 segment. We confirmed that this virus replicated only in PB2-expressing cells and expressed HN. We then examined the efficacy of this virus as a bivalent vaccine in a hamster model. High levels of virus-specific IgG antibodies in sera and IgA, IgG, and IgM antibodies in bronchoalveolar lavage fluids against both influenza virus and HPIV3 were detected from hamsters immunized with this virus. The neutralizing capability of these serum antibodies was also confirmed. Moreover, the immunized hamsters were completely protected from virus challenge with influenza virus or HPIV3. These results indicate that PB2-KO virus expressing the HN of HPIV3 has the potential to be a novel bivalent vaccine against influenza and human parainfluenza viruses.

Evaluation of two chimeric bovine-human parainfluenza virus type 3 vaccines in infants and young children.

Human parainfluenza virus type 3 (HPIV3) is an important cause of lower respiratory tract illness in children, yet a licensed vaccine or antiviral drug is not available. We evaluated the safety, tolerability, infectivity, and immunogenicity of two intranasal, live-attenuated HPIV3 vaccines, designated rHPIV3-N(B) and rB/HPIV3, that were cDNA-derived chimeras of HPIV3 and bovine PIV3 (BPIV3). These were evaluated in adults, HPIV3 seropositive children, and HPIV3 seronegative children. A total of 112 subjects participated in these studies. Both rB/HPIV3 and rHPIV3-N(B) were highly restricted in replication in adults and seropositive children but readily infected seronegative children, who shed mean peak virus titers of 10(2.8) vs. 10(3.7)pfu/mL, respectively. Although rB/HPIV3 was more restricted in replication in seronegative children than rHPIV3-N(B), it induced significantly higher titers of hemagglutination inhibition (HAI) antibodies against HPIV3. Taken together, these data suggest that the rB/HPIV3 vaccine is the preferred candidate for further clinical development.

The cDNA-derived investigational human parainfluenza virus type 3 vaccine rcp45 is well tolerated, infectious, and immunogenic in infants and young children.

BACKGROUND: Human parainfluenza virus type 3 (HPIV3) is an important yet underappreciated cause of lower respiratory tract illness in children, and a licensed vaccine is not yet available. METHODS: A live-attenuated investigational HPIV3 vaccine virus designated rcp45 was derived from cDNA by using reverse genetics. rcp45 is genetically similar to the biologically derived cp45 vaccine virus and contains all of the known attenuating mutations of cp45, but has the advantage of a short, well-characterized passage history. We evaluated the tolerability, infectivity, and immunogenicity of 2 intranasal doses of rcp45 administered 4 to 10 weeks apart in a placebo-controlled, double-blind trial. A total of 45 infants and children between 6 and 36 months of age participated in this study. Tolerability and antibody responses to vaccine or placebo were assessed in all recipients. Infectivity was assessed by quantitation of vaccine virus shedding in a subset of vaccinated children. RESULTS: rcp45 was well tolerated and highly infectious in HPIV3-seronegative children. A second dose of vaccine administered 4 to 10 weeks after the first dose was restricted in replication and did not boost serum antibody responses. The stability of 9 cp45 mutations, including the 6 major attenuating mutations, was examined and confirmed for viral isolates from 10 children. CONCLUSIONS: The level of attenuation and immunogenicity of cDNA-derived rcp45 is comparable to what was previously observed with the biologically derived cp45 vaccine, and preliminary data suggest that the attenuating mutations in this vaccine virus are genetically stable. Continued clinical development of rcp45 is warranted.

Progress in the development of human parainfluenza virus vaccines.

In children under 5 years of age, human parainfluenza viruses (HPIVs) as a group are the second most common etiology of acute respiratory illness leading to hospitalization, surpassed only by respiratory syncytial virus but ahead of influenza viruses. Using reverse genetics systems for HPIV serotypes 1, 2 and 3 (HPIV1, 2 and 3), several live-attenuated HPIVs have been generated and evaluated as intranasal vaccines in adults and in children. Two vaccines against HPIV3 were found to be well tolerated, infectious and immunogenic in Phase I trials in HPIV3-seronegative infants and children and should progress to proof-of-concept trials. Vaccines against HPIV1 and HPIV2 are less advanced and have just entered pediatric trials.

A novel human parainfluenza virus type 1 (HPIV1) with separated P and C genes is useful for generating C gene mutants for evaluation as live-attenuated virus vaccine candidates.

A novel recombinant human parainfluenza virus type 1 (rHPIV1), rHPIV1-C+P, in which the overlapping open reading frames of the C and P genes were separated in order to introduce mutations into the C gene without affecting P, was generated. Infectious rHPIV1-C+P was readily recovered and replicated as efficiently as HPIV1 wild type (wt) in vitro and in African green monkeys (AGMs). rHPIV1-C+P expressed increased levels of C protein and, surprisingly, activated the type I IFN and apoptosis responses more strongly than HPIV1 wt. rHPIV1-C+P provided a useful backbone for recovering an attenuated P/C gene mutation (Delta 84-85), which was previously unrecoverable, likely due to detrimental effects of the deletion on the P protein. rHPIV1-C(Delta 84-85)+P and an additional mutant, rHPIV1-C(Delta 169-170)+P, were found to replicate to similar titers in vitro and to activate the type I IFN and apoptosis responses to a similar degree as rHPIV1-C+P. rHPIV1-C(Delta 84-85)+P was found to be highly attenuated in AGMs, and all viruses were immunogenic and effective in protecting AGMs against challenge with HPIV1 wt. rHPIV1-C(Delta 84-85)+P will be investigated as a potential live-attenuated vaccine candidate for HPIV1.

Current status of vaccines for parainfluenza virus infections.

Parainfluenza viruses (PIV) have been generally disregarded as pathogens in spite of their importance in pediatric lower respiratory illness. Because PIVs account for 17% of hospitalized illness associated virus isolation, the development of PIV vaccine would be a major advance in preventing lower respiratory tract infection in infants and young children. We will review in detail several PIV vaccine candidates and recent newer approaches to PIV vaccine development. Intranasally administered bovine PIV3 (bPIV3) vaccine and cold-adapted PIV3 vaccine have been evaluated throughout the pediatric age spectrum. BPIV3 does not give a robust response to the heterotypic human strain although seroconversion rate to bPIV3 is 57-65%. However, bPIV3 vaccine is being used as an attenuated backbone for insertion of human PIV3 hemagglutinin-neuraminidase and fusion (F) proteins and a surface protein, F, of respiratory syncytial virus. The effectiveness of this vaccine against both PIV3 and RSV challenge has been demonstrated in African green monkeys. The cold-adapted PIV3 vaccine has been extensively evaluated and is safe and immunogenic in seronegative children with a seroconversion rate of 79%. These promising candidates deserve to enter into efficacy trials both for their ability to prevent PIV3 disease and as a model of protection against respiratory illness by mucosal vaccination.

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.

Sendai virus recombinant vaccine expressing hPIV-3 HN or F elicits protective immunity and combines with a second recombinant to prevent hPIV-1, hPIV-3 and RSV infections.

The human parainfluenza viruses (hPIVs) and respiratory syncytial virus (RSV) are the leading causes of serious respiratory illness in the human pediatric population. Despite decades of research, there are currently no licensed vaccines for either the hPIV or RSV pathogens. Here we describe the testing of hPIV-3 and RSV candidate vaccines using Sendai virus (SeV, murine PIV-1) as a vector. SeV was selected as the vaccine backbone, because it has been shown to elicit robust and durable immune activities in animal studies, and has already advanced to human safety trials as a xenogenic vaccine for hPIV-1. Two new SeV-based hPIV-3 vaccine candidates were first generated by inserting either the fusion (F) gene or hemagglutinin-neuraminidase (HN) gene from hPIV-3 into SeV. The resultant rSeV-hPIV3-F and rSeV-hPIV3-HN vaccines expressed their inserted hPIV-3 genes upon infection. The inoculation of either vaccine into cotton rats elicited binding and neutralizing antibody activities, as well as interferon-gamma-producing T cells. Vaccination of cotton rats resulted in protection against subsequent challenges with either homologous or heterologous hPIV-3. Furthermore, vaccination of cotton rats with a mixture of rSeV-hPIV3-HN and a previously described recombinant SeV expressing the F protein of RSV resulted in protection against three different challenge viruses: hPIV-3, hPIV-1 and RSV. Results encourage the continued development of the candidate recombinant SeV vaccines to combat serious respiratory infections of children.

Attenuation and efficacy of human parainfluenza virus type 1 (HPIV1) vaccine candidates containing stabilized mutations in the P/C and L genes.

BACKGROUND: Two recombinant, live attenuated human parainfluenza virus type 1 (rHPIV1) mutant viruses have been developed, using a reverse genetics system, for evaluation as potential intranasal vaccine candidates. These rHPIV1 vaccine candidates have two non-temperature sensitive (non-ts) attenuating (att) mutations primarily in the P/C gene, namely CR84GHNT553A (two point mutations used together as a set) and CDelta170 (a short deletion mutation), and two ts att mutations in the L gene, namely LY942A (a point mutation), and LDelta1710-11 (a short deletion), the last of which has not been previously described. The latter three mutations were specifically designed for increased genetic and phenotypic stability. These mutations were evaluated on the HPIV1 backbone, both individually and in combination, for attenuation, immunogenicity, and protective efficacy in African green monkeys (AGMs). RESULTS: The rHPIV1 mutant bearing the novel LDelta1710-11 mutation was highly ts and attenuated in AGMs and was immunogenic and efficacious against HPIV1 wt challenge. The rHPIV1-CR84G/Delta170HNT553ALY942A and rHPIV1-CR84G/Delta170HNT553ALDelta1710-11 vaccine candidates were highly ts, with shut-off temperatures of 38 degrees C and 35 degrees C, respectively, and were highly attenuated in AGMs. Immunization with rHPIV1-CR84G/Delta170HNT553ALY942A protected against HPIV1 wt challenge in both the upper and lower respiratory tracts. In contrast, rHPIV1-CR84G/Delta170HNT553ALDelta1710-11 was not protective in AGMs due to over-attenuation, but it is expected to replicate more efficiently and be more immunogenic in the natural human host. CONCLUSION: The rHPIV1-CR84G/Delta170HNT553ALY942A and rHPIV1-CR84G/Delta170HNT553ALDelta1710-11 vaccine candidates are clearly highly attenuated in AGMs and clinical trials are planned to address safety and immunogenicity in humans.

Attenuating mutations in the P/C gene of human parainfluenza virus type 1 (HPIV1) vaccine candidates abrogate the inhibition of both induction and signaling of type I interferon (IFN) by wild-type HPIV1.

Recombinant human parainfluenza virus type 1 (HPIV1) and mutants containing point and deletion (Delta) mutations in the P/C gene (r-CDelta10-15HNT553A, r-CR84G, r-CF170S and r-CDelta170), which have previously been evaluated as HPIV1 vaccine candidates, were evaluated for their effect on the type I interferon (IFN) response in vitro. HPIV1 wt infection inhibited the IFN response by inhibiting IFN regulatory factor-3 (IRF-3) activation and IFN production in A549 cells and IFN signaling in Vero cells. In contrast, r-CR84G, r-CF170S and r-CDelta170 were defective for inhibition of IRF-3 activation and IFN production and r-CF170S and r-CDelta170 did not inhibit IFN signaling. Thus, HPIV1 antagonizes the IFN response at both the level of induction and signaling, and antagonism at both levels was disrupted by mutations in the P/C gene. Because CF170S affects C and not P, the anti-IFN function can be attributed to the C proteins. These data, in the context of previous in vivo studies, suggest that the loss of antagonism of the IFN response at both the level of induction and signaling, observed with the P/C mutants, r-CF170S and r-CDelta170, was necessary for significant attenuation in African green monkeys (AGMs).

Introducing point and deletion mutations into the P/C gene of human parainfluenza virus type 1 (HPIV1) by reverse genetics generates attenuated and efficacious vaccine candidates.

The P/C gene of human parainfluenza virus type 1 (HPIV1) encodes a nested set of related accessory C proteins, C'/C/Y1/Y2, which have been shown in other paramyxoviruses to have a role in evasion of the type I interferon (IFN) response following virus infection. We previously demonstrated that a set of two amino acid substitutions, CR84G/HNT553A, and a separate amino acid substitution, CF170S, are independently attenuating for HPIV1 in African green monkeys (AGMs). However, in each case the attenuation (att) phenotype is vulnerable to reversion by a single nucleotide change back to wild type. Using reverse genetics, recombinant HPIV1 (rHPIV1) vaccine candidates were generated that were designed for increased genetic and phenotypic stability by: (i) creating a two-amino acid deletion and substitution at the site of the CF170S mutation, yielding CDelta170; (ii) introducing a six amino acid deletion in the N-terminal region of C, CDelta10-15; and (iii) combining these stable deletion mutations with the att CR84G/HNT553A mutation. The resulting rHPIV1 vaccine candidates were evaluated for attenuation in hamsters and AGMs and for immunogenicity and protective efficacy in AGMs. The CDelta10-15 mutation was attenuating in hamsters but not in AGMs, and likely will be of limited value for an HPIV1 vaccine. Conversely, the CR84G/HNT553A mutation set was attenuating in AGMs but not in hamsters. Thus, these two mutations demonstrated reciprocal host range phenotypes involving different regions of C. The CDelta170 mutation conferred a significant level of attenuation in hamsters and AGMs that closely resembled that of CF170S and will be of particular utility for vaccine development because it involves a deletion of six nucleotides rendering it highly refractory to reversion. The combination of the CR84G/HNT553A mutation set and the CDelta170 deletion mutation yielded a virus, rCR84G/Delta170 HNT553A, that exhibited a satisfactory level of attenuation in hamsters and AGMs and was immunogenic and highly protective against HPIV1 wt challenge. This virus will be evaluated clinically as a live intranasal HPIV1 vaccine, one that can be further attenuated as necessary by the introduction of additional stabilized att mutations previously developed in the L protein.

Human parainfluenza virus type I (HPIV1) vaccine candidates designed by reverse genetics are attenuated and efficacious in African green monkeys.

A set of recombinant, live attenuated human parainfluenza virus type 1 (rHPIV1) vaccine candidates was evaluated for attenuation, immunogenicity, and protective efficacy in African green monkeys (AGMs). Temperature sensitive (ts) and non-ts attenuating (att) mutations in the P/C and L genes were introduced individually or in various combinations into rHPIV1, including the C(R84G) and HN(T553A) mutations identified in the present work and the C(F170S), L(Y942A), and L(L992C) mutations identified previously. The rHPIV1 vaccine candidates exhibited a spectrum of attenuation in AGMs. One genetically and phenotypically stable vaccine candidate, rC(R84G/F170S)L(Y942A/L992C), was attenuated and efficacious in AGMs and is a promising live attenuated intranasal HPIV1 vaccine candidate suitable for clinical evaluation.

Live bivalent vaccine for parainfluenza and influenza virus infections.

Influenza and human parainfluenza virus infections are of both medical and economical importance. Currently, inactivated vaccines provide suboptimal protection against influenza, and vaccines for human parainfluenza virus infection are not available, underscoring the need for new vaccines against these respiratory diseases. Furthermore, to reduce the burden of vaccination, the development of multivalent vaccines is highly desirable. Thus, to devise a single vaccine that would elicit immune responses against both influenza and parainfluenza viruses, we used reverse genetics to generate an influenza A virus that possesses the coding region for the hemagglutinin/neuraminidase ectodomain of parainfluenza virus instead of the influenza virus neuraminidase. The recombinant virus grew efficiently in eggs but was attenuated in mice. When intranasally immunized with the recombinant vaccine, all mice developed antibodies against both influenza and parainfluenza viruses and survived an otherwise lethal challenge with either of these viruses. This live bivalent vaccine has obvious advantages over combination vaccines, and its method of generation could, in principle, be applied in the development of a "cocktail" vaccine with efficacy against several different infectious diseases.

Generation of recombinant human parainfluenza virus type 1 vaccine candidates by importation of temperature-sensitive and attenuating mutations from heterologous paramyxoviruses.

Human parainfluenza virus type 1 (HPIV1) is a significant cause of respiratory tract disease in infants and young children for which a vaccine is needed. In the present study, we sought to attenuate HPIV1 by the importation of one or more known attenuating point mutations from heterologous paramyxoviruses into homologous sites in HPIV1. The introduced mutations were derived from three attenuated paramyxoviruses: (i) HPIV3cp45, a live-attenuated HPIV3 vaccine candidate containing multiple attenuating mutations; (ii) the respiratory syncytial virus cpts530 with an attenuating mutation in the L polymerase protein; and (iii) a murine PIV1 (MPIV1) attenuated by a mutation in the accessory C protein. Recombinant HPIV1 (rHPIV1) mutants bearing a single imported mutation in C, any of three different mutations in L, or a pair of mutations in F exhibited a 100-fold or greater reduction in replication in the upper or lower respiratory tract of hamsters. Both temperature-sensitive (ts) (mutations in the L and F proteins) and non-ts (the mutation in the C protein) attenuating mutations were identified. rHPIV1 mutants containing a combination of mutations in L were generated that were more attenuated than viruses bearing the individual mutations, showing that the systematic accretion of mutations can yield progressive increases in attenuation. Hamsters immunized with rHPIV1 mutants bearing one or two mutations developed neutralizing antibodies and were resistant to challenge with wild-type HPIV1. Thus, importation of attenuating mutations from heterologous viruses is an effective means for rapidly identifying mutations that attenuate HPIV1 and for generating live-attenuated HPIV1 vaccine candidates.

Codon substitution mutations at two positions in the L polymerase protein of human parainfluenza virus type 1 yield viruses with a spectrum of attenuation in vivo and increased phenotypic stability in vitro.

The Y942H and L992F temperature-sensitive (ts) and attenuating amino acid substitution mutations, previously identified in the L polymerase of the HPIV3cp45 vaccine candidate, were introduced into homologous positions of the L polymerase of recombinant human parainfluenza virus type 1 (rHPIV1). In rHPIV1, the Y942H mutation specified the ts phenotype in vitro and the attenuation (att) phenotype in hamsters, whereas the L992F mutation specified neither phenotype. Each of these codon mutations was generated by a single nucleotide substitution and therefore had the potential to readily revert to a codon specifying the wild-type amino acid residue. We introduced alternative amino acid assignments at codon 942 or 992 as a strategy to increase genetic stability and to generate mutants that exhibit a range of attenuation. Twenty-three recombinants with codon substitutions at position 942 or 992 of the L protein were viable. One highly ts and att mutant, the Y942A virus, which had a difference of three nucleotides from the codon encoding a wild-type tyrosine, also possessed a high level of genetic and phenotypic stability upon serial passage in vitro at restrictive temperatures compared to that of the parent Y942H virus, which possessed a single nucleotide substitution. We obtained mutants with substitutions at position 992 that, in contrast to the L992F virus, possessed the ts and att phenotypes. These findings identify the use of alternative codon substitution mutations as a method that can be used to generate candidate vaccine viruses with increased genetic stability and/or a modified level of attenuation.

The recombinant chimeric human parainfluenza virus type 1 vaccine candidate, rHPIV3-1cp45, is attenuated, immunogenic, and protective in African green monkeys.

A recombinant live-attenuated chimeric human parainfluenza virus type 1 (HPIV1) candidate vaccine was previously generated by replacing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein open reading frames (ORFs) of the HPIV3 candidate vaccine, rHPIV3cp45, with those of wild-type HPIV1. Previously, this recombinant chimeric virus, designated rHPIV3-1cp45, exhibited a greater level of the temperature sensitivity of replication in vitro and a greater level of attenuation of replication in the respiratory tract of immunized hamsters when compared to its HPIV3cp45 parent virus. In the present study, rHPIV3-1cp45 was evaluated for its level of attenuation and efficacy in African green monkeys (Cercopithecus aethiops), a primate in which both HPIV1 and HPIV3 wild-type viruses replicate efficiently. The rHPIV3-1cp45 candidate vaccine was as restricted in replication in the upper and lower respiratory tract as its thoroughly characterized rHPIV3cp45 parent indicating that the attenuating mutations present in the rHPIV3cp45 backbone specified an appropriate level of attenuation of rHPIV3-1cp45 for primates. The level to which rHPIV3-1cp45 replicated in the respiratory tract of African green monkeys was also sufficient to induce a strong immune response to HPIV1 and provided protection against challenge with wild-type HPIV1. These results provide a basis for further evaluation of this HPIV1 candidate vaccine in humans.