Laboratory And Pre-clinical Studies Of Parainfluenza Vir

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Abstract

Progress has been made toward the development of a parainfluenza (PIV)type 1 vaccine (PIV1). Human parainfluenza virus type 1 (HPIV1), a major cause of croup in infants and young children, accounts for 6% of hospitalizations for pediatric respiratory tract disease. A complete consensus sequence was determined for the genomic RNA of human parainfluenza virus type 1 (HPIV1) strain Washington/20993/1964, a clinical isolate that previously was shown to be virulent in adults. This consensus sequence was used to generate a full?length antigenomic cDNA and to recover a recombinant wild type HPIV1 (rHPIV1). The replication of rHPIV1 in vitro and in the respiratory tract of hamsters was similar to that of its biologically derived parent virus. The similar biological properties of rHPIV1 and HPIV1 WASH/64 in vitro and in vivo authenticates the rHPIV1 sequence as that of a wild type virus. This rHPIV1 can now be used as a substrate to introduce attenuating mutations for the generation of live?attenuated HPIV1 vaccine candidates. Sendai virus, referred to here as murine PIV1 (MPIV1), is antigenically related toHPIV1 and is being considered for use as a live attenuated vaccine to protect against HPIV1 and also as a recombinant vaccine vector expressing antigens to protect against viral disease in humans. The level of replication of MPIV1 and HPIV1 was compared in African green monkeys and chimpanzees. Surprisingly, MPIV1 replicated as efficiently as HPIV1 in the upper and lower respiratory tract of African green monkeys at doses of 104 and 106, and replicated only slightly less efficiently at both sites in chimpanzees. The high level of replication of MPIV1 observed in the upper and lower respiratory tract of these primates suggests that MPIV1 likely would require significant attenuation before it could be given to humans as a vaccine against HPIV1 or as a vaccine vector. A recombinant live-attenuated chimeric 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. 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. Progress has been made toward the development of a PIV3 vaccine based on the importation of genes from bovine PIV3 that attenuate HPIV3 for no-human primates. The Kansas strain of bovine parainfluenza virus type 3 (BPIV3) is 100-1000 fold restricted in replication in the respiratory tract of non-human primates compared to human PIV3 (HPIV3), an important pathogen of infants and young children. We have examined the genetic basis for the host-range attenuation phenotype of BPIV3 by exchanging each open reading frame (ORF) of a recombinant wild type HPIV3 with the analogous ORF from BPIV3. Recombinant chimeric HPIV3s bearing the BPIV3 N or P ORF were highly attenuated in the upper and lower respiratory tract of monkeys, whereas those bearing the BPIV3 M or L ORF or the F and HN genes were only moderately attenuated. This indicates that the genetic determinants of the host-range restriction of replication of BPIV3 for primates is polygenic in nature, with the major determinants being the N and P ORFs. Monkeys immunized with these bovine-human chimeric viruses, including the more highly attenuated ones, developed higher HPIV3 hemagglutination-inhibiting (HAI) serum antibodies than did monkeys immunized with BPIV3 and were protected from challenge with wild type HPIV3. The ability of PIVs to serve as vectors of foreign proteins is still being actively evaluated. The level of replication and immunogenicity of recombinant parainfluenza virus type 3 (rHPIV3) bearing one, two or three gene insertions expressing foreign protective antigens was examined. Viruses with inserts were restricted for replication in the respiratory tract of hamsters, and the level of restriction was a function of the total number of genes inserted, the nature of the insert, and the position of the inserted gene in the gene order. A single insert of HPIV2 HN or measles virus HA reduced the in vivo replication of rHPIV3 up to 25-fold, whereas the HPIV1 HN insert decreased replication almost 1000-fold. This indicates that the HPIV1 HN insert has an attenuating effect in addition to that of the extra gene insert itself, presumably because it is incorporated into the virus particle. Viruses containing two inserts were generally more attenuated than those with a single insert, and viruses with three inserts were over-attenuated for replication in hamsters. Inserts between the N and P genes were slightly more attenuating than those between the P and M genes. A recombinant HPIV3 bearing both the HPIV1 and HPIV2 HN genes (r1HN 2HN) was attenuated, immunogenic and protected immunized hamsters from challenge with HPIV1, HPIV2 and HPIV3. Thus, it is possible to use a single HPIV vector expressing two foreign gene inserts to protect infants and young children from the severe lower respiratory tract disease caused by the three major human PIV pathogens. The chimeric recombinant virus rHPIV3-NB, a version of human parainfluenza virus type 3 (HPIV3) that is attenuated due to the presence of the bovine PIV3 nucleocapsid (N) protein open reading frame (ORF) in place of the HPIV3 ORF, was modified to encode the measles virus hemagglutinin (HA) inserted as an additional, supernumerary gene between the HPIV3 P and M genes. This recombinant, designated rHPIV3-NBHA, replicated like its attenuated rHPIV3-NB parent virus in vitro and in the upper and lower respiratory tract of rhesus monkeys indicating that the insertion of the measles virus HA did not further attenuate rHPIV3-NB in vitro or in vivo. Monkeys immunized with rHPIV3-NBHA developed a vigorous immune response to both measles virus and HPIV3, with serum antibody titers to both measles virus (neutralizing antibody) and HPIV3 (hemagglutination inhibiting antibody) of over 1:500. Recombinant bovine/human parainfluenza virus type 3 (rB/HPIV3), a recombinant bovine PIV3 (rBPIV3) in which the F and HN genes were replaced with their HPIV3 counterparts, was used to express the major protective antigens of respiratory syncytial virus (RSV) in order to create a bivalent mucosal vaccine against RSV and HPIV3. The attenuation of rB/HPIV3 is provided by the host range restriction of the BPIV3 backbone in primates. The recombinant PIV3 expressing the RSV G ORF (rB/HPIV3-G1) was not restricted in its replication in vitro whereas the virus expressing the RSV F ORF (rB/HPIV3-F1) was 8-fold restricted compared to its rB/HPIV3 parent. Both viruses replicated efficiently in the respiratory tract of hamsters, and each induced RSV serum antibody titers similar to those induced by RSV infection, and anti-HPIV3 titers similar to those induced by HPIV3 infection. These results describe a vaccine strategy that obviates the technical challenges associated with a live attenuated RSV vaccine, providing, against the two leading viral agents of pediatric respiratory tract disease, a bivalent vaccine whose attenuation phenotype is based on the extensive host range sequence differences of BPIV3.