A chimeric human-bovine parainfluenza virus type 3 expressing measles virus hemagglutinin is attenuated for replication but is still immunogenic in rhesus monkeys.

The chimeric recombinant virus rHPIV3-N(B), 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-N(B)HA, replicated like its attenuated rHPIV3-N(B) parent virus in vitro and in the upper and lower respiratory tracts of rhesus monkeys, indicating that the insertion of the measles virus HA did not further attenuate rHPIV3-N(B) in vitro or in vivo. Monkeys immunized with rHPIV3-N(B)HA 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. An attenuated HPIV3 expressing a major protective antigen of measles virus provides a method for immunization against measles by the intranasal route, a route that has been shown with HPIV3 and respiratory syncytial virus vaccines to be relatively refractory to the neutralizing and immunosuppressive effects of maternally derived virus-specific serum antibodies. It should now be possible to induce a protective immune response against measles virus in 6-month-old infants, an age group that in developing areas of the world is not responsive to the current measles virus vaccine.

Construction of a live-attenuated bivalent vaccine virus against human parainfluenza virus (PIV) types 1 and 2 using a recombinant PIV3 backbone.

PIV1 and PIV2 are important agents of pediatric respiratory tract disease. We are developing live-attenuated vaccines against these viruses. We earlier constructed a PIV3/PIV1 antigenic chimeric virus, designated rPIV3-1, in which the hemagglutinin-neuraminidase (HN) and fusion (F) proteins of wild type rPIV3 were replaced by their PIV1 counterparts. In the present study, rPIV3-1 was used as a vector to express the HN protein of PIV2 to generate a single virus capable of inducing immunity to both PIV1 and PIV2. The PIV2 HN open reading frame was expressed from an extra gene cassette, under the control of PIV3 cis-acting transcription signals, inserted between the F and HN genes of rPIV3-1. The recombinant derivative, designated rPIV3-1.2HN, was readily recovered and exhibited a level of temperature sensitivity and in vitro growth similar to that of its parental virus. The rPIV3-1.2HN virus was restricted in replication in both the upper and lower respiratory tracts of hamsters compared with rPIV3-1, identifying an attenuating effect of the PIV2 HN insert in hamsters. rPIV3-1.2HN elicited serum antibodies to both PIV1 and PIV2 and induced resistance against challenge with wild type PIV1 or PIV2. Thus, rPIV3-1.2HN, a virus attenuated solely by the insertion of the PIV2 HN gene, functioned as a live attenuated bivalent vaccine candidate against both PIV1 and PIV2.

Bovine parainfluenza virus type 3 (BPIV3) fusion and hemagglutinin-neuraminidase glycoproteins make an important contribution to the restricted replication of BPIV3 in primates.

This study examines the contribution of the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein genes of bovine parainfluenza virus type 3 (BPIV3) to its restricted replication in the respiratory tract of nonhuman primates. A chimeric recombinant human parainfluenza type 3 virus (HPIV3) containing BPIV3 F and HN glycoprotein genes in place of its own and the reciprocal recombinant consisting of BPIV3 bearing the HPIV3 F and HN genes (rBPIV3-F(H)HN(H)) were generated to assess the effect of glycoprotein substitution on replication of HPIV3 and BPIV3 in the upper and lower respiratory tract of rhesus monkeys. The chimeric viruses were readily recovered and replicated in simian LLC-MK2 cells to a level comparable to that of their parental viruses, suggesting that the heterologous glycoproteins were compatible with the PIV3 internal proteins. HPIV3 bearing the BPIV3 F and HN genes was restricted in replication in rhesus monkeys to a level similar to that of its BPIV3 parent virus, indicating that the glycoprotein genes of BPIV3 are major determinants of its host range restriction of replication in rhesus monkeys. rBPIV3-F(H)HN(H) replicated in rhesus monkeys to a level intermediate between that of HPIV3 and BPIV3. This observation indicates that the F and HN genes make a significant contribution to the overall attenuation of BPIV3 for rhesus monkeys. Furthermore, it shows that BPIV3 sequences outside the F and HN region also contribute to the attenuation phenotype in primates, a finding consistent with the previous demonstration that the nucleoprotein coding sequence of BPIV3 is a determinant of its attenuation for primates. Despite its restricted replication in the respiratory tract of rhesus monkeys, rBPIV3-F(H)HN(H) conferred a level of protection against challenge with HPIV3 that was indistinguishable from that induced by previous infection with wild-type HPIV3. The usefulness of rBPIV3-F(H)HN(H) as a vaccine candidate against HPIV3 and as a vector for other viral antigens is discussed.

Human parainfluenza virus type 3 (PIV3) expressing the hemagglutinin protein of measles virus provides a potential method for immunization against measles virus and PIV3 in early infancy.

Recombinant human parainfluenza virus type 3 (PIV3) was used as a vector to express the major protective antigen of measles virus, the hemagglutinin (HA) glycoprotein, in order to create a bivalent PIV3-measles virus that can be administered intranasally. The measles virus HA open reading frame (ORF) was inserted as an additional transcriptional unit into the N-P, P-M, or HA-neuraminidase (HN)-L gene junction of wild-type PIV3 or into the N-P or P-M gene junction of an attenuated derivative of PIV3, termed rcp45L. The recombinant PIV3 (rPIV3) viruses bearing the HA inserts replicated more slowly in vitro than their parental viruses but reached comparable peak titers of >/=10(7.5) 50% tissue culture infective doses per ml. Each of the wild-type or cold-passaged 45L (cp45L) PIV3(HA) chimeric viruses replicated 5- to 10-fold less well than its respective parent virus in the upper respiratory tract of hamsters. Thus, insertion of the approximately 2-kb ORF itself conferred attenuation, and this attenuation was additive to that conferred by the cp45L mutations. The attenuated cp45L PIV3(HA) recombinants induced a high level of resistance to replication of PIV3 challenge virus in hamsters and induced very high levels of measles virus neutralizing antibodies (>1:8,000) that are well in excess of those known to be protective in humans. rPIV3s expressing the HA gene in the N-P or P-M junction induced about 400-fold more measles virus-neutralizing antibody than did the rPIV3 with the HA gene in the HN-L junction, indicating that the N-P or P-M junction appears to be the preferred insertion site. Previous studies indicated that the PIV3 cp45 virus, a more attenuated version of rcp45L, replicates efficiently in the respiratory tract of monkeys and is immunogenic and protective even when administered in the presence of very high titers of passively transferred PIV3 antibodies (A. P. Durbin, C. J. Cho, W. R. Elkins, L. S. Wyatt, B. Moss, and B. R. Murphy, J. Infect. Dis. 179:1345-1351, 1999). This suggests that this intranasally administered PIV3(HA) chimeric virus can be used to immunize infants with maternally acquired measles virus antibodies in whom the current parenterally administered live measles virus vaccine is ineffective.

Long nucleotide insertions between the HN and L protein coding regions of human parainfluenza virus type 3 yield viruses with temperature-sensitive and attenuation phenotypes.

Recombinant parainfluenza virus 3 (rPIV3) is being developed as a vector to express foreign genes as a bivalent or multivalent live attenuated virus vaccine. In the present study, we examined the effect of inserted foreign sequence on virus replication in vitro and in vivo, focusing on the parameter of insert length. In one type of construct, foreign sequence of increasing length was flanked by PIV3 transcription signals and inserted as an additional gene unit (GU insert) between the HN and L genes, so that one additional mRNA would be made. In a second type of construct, foreign sequence was inserted into the downstream NCR (NCR insert) of the HN gene, so that the number of encoded mRNAs remained unchanged. In each case, the foreign sequence was designed to lack any significant open reading frame, which permitted an evaluation of the effect of insert length on replication independent of an effect of an expressed protein. The GU or NCR insert sizes ranged from 168 nucleotides (nt) to 3918 nt. rPIV3s containing GU insertions of up to 3918 nt in length, the largest size tested, were viable and replicated efficiently at permissive temperatures in vitro, but a reduction in plaque size was seen at 39 degrees C and 40 degrees C. The rPIV3 with a 3918-nt GU insertion was restricted in replication in the upper (fivefold) and lower (25-fold) respiratory tracts of hamsters. Although a 1908-nt GU insertion did not significantly modify replication of wild-type PIV3 in vitro or in vivo, its introduction significantly augmented the level of temperature sensitivity (ts) and attenuation (att) specified by three mutations in the L protein of a cold-passaged attenuated PIV3 vaccine virus. rPIV3s bearing a 3126- or 3894-nt NCR insertion exhibited in vitro and in vivo phenotypes like those of the rPIV3s bearing similar-sized GU insertions. These findings indicate that rPIV3s whose genome length has been increased by more than 3000 nt by either a GU or an NCR insertion exhibit an unexpected host-range phenotype, that is, efficient replication in vitro but restricted replication in hamsters, especially in the lower respiratory tract. Furthermore, these effects were greatly enhanced when the rPIV3 backbone contained other ts or att mutations. The implications of these findings for the use of single-stranded, negative-sense RNA viruses as vectors for vaccines are discussed.

Generation of a parainfluenza virus type 1 vaccine candidate by replacing the HN and F glycoproteins of the live-attenuated PIV3 cp45 vaccine virus with their PIV1 counterparts.

Parainfluenza virus type 1 (PIV1) is a major cause of croup in infants and young children, and a vaccine is needed to prevent the serious disease caused by this virus. In the present study, a live attenuated PIV1 vaccine candidate was generated by modification of the extensively-studied PIV3 cold-passaged (cp) cp45 vaccine candidate using the techniques of reverse genetics. The HN and F glycoproteins of the PIV3 cp45 candidate vaccine virus were replaced with those of PIV1. This created a live attenuated PIV1 vaccine candidate, termed rPIV3-1 cp45, which contained the attenuated background of the PIV3 cp45 vaccine virus together with the HN and F protective antigens of PIV1. Three of the 15 mutations of cp45 lie within the HN and F genes, and those in the F gene are attenuating. Thus, some attenuation might be lost by the HN and F glycoprotein replacement. To address this issue we also constructed a derivative of PIV3 cp45, designated rPIV3 cp45 (F(wt)HN(wt)), that possessed wild type PIV3 HN and F glycoproteins but retained the 12 other cp45 mutations. rPIV3 cp45 (F(wt)HN(wt)) replicated in the respiratory tract of hamsters to a level three- to four-fold higher than rPIV3 cp45, indicating that loss of the two attenuating mutations in the cp45 F gene effected a slight reduction in the overall attenuation of cp45 for hamsters. However, the chimeric rPIV3-1 cp45 virus was about 5-fold more restricted in replication in hamsters than rPIV3 cp45 and about 15- to 20-fold more restricted than rPIV3 cp45 (F(wt)HN(wt)). This suggests that two components contribute to the attenuation of the new chimeric rPIV3-1 cp45 PIV1 vaccine candidate: one being the 12 cp45 mutations, which provide most of the observed attenuation, and the other resulting from the introduction of the heterologous PIV1 HN and F proteins into PIV3 (i.e., a chimerization effect). rPIV3-1 cp45 was observed to be immunogenic and protective against challenge with wild type PIV1 in hamsters. This virus shows sufficient promise that it should be evaluated further as a candidate live attenuated vaccine strain for preventing severe lower respiratory tract PIV1 disease in infants and young children.

Attenuation of the recombinant human parainfluenza virus type 3 cp45 candidate vaccine virus is augmented by importation of the respiratory syncytial virus cpts530 L polymerase mutation.

A phenylalanine to leucine mutation at position 521 in the L polymerase of cpts530, a live-attenuated respiratory syncytial virus (RSV) cold-passaged (cp), temperature-sensitive (ts) candidate vaccine, specifies the ts and attenuation (att) phenotypes. Sequence alignment of this region in the L proteins of several distantly related paramyxoviruses revealed that this phenylalanine is conserved. Using reverse genetics, the analogous phenylalanine at position 456 in the L protein of wild-type PIV3 was mutagenized to leucine (F456L). The resulting virus, designated r456(L), was ts (40 degrees C shut-off temperature of plaque formation), and its replication in the upper, but not the lower, respiratory tract of hamsters was 10-fold reduced compared with that of the recombinant wild-type PIV3 (rwt). Thus the phenylalanine to leucine mutation specified a similar level of temperature sensitivity and attenuation in two distantly related paramyxoviruses. We next sought to determine whether the addition of this mutation to the L protein of two rPIV3 candidate vaccine viruses, one bearing the three cp45 ts missense mutations in the L protein (rcp45(L)) and the other bearing all 15 cp45 mutations (rcp45), would further attenuate the viruses in vivo. Each rcp45 derivative to which the F456L mutation was added exhibited an increased level of temperature sensitivity. Furthermore rcp45(L)-456 and rcp45-456 were 100- to 1000-fold more restricted in replication in hamsters than their rcp45(L) and rcp45 parents. Despite the high level of restriction of replication in hamsters, immunization with rcp45-456 induced a moderate level of resistance to replication of PIV3 challenge virus. In contrast to the highly restricted replication observed in hamsters, rcp45-456 was only fivefold more restricted in the respiratory tract of chimpanzees than rcp45 and induced a comparable, moderate to high level of PIV3-specific serum antibodies. rcp45 and rcp45-456 viruses isolated from chimpanzees throughout the 2-week course of replication maintained the level of temperature sensitivity of their respective input viruses, illustrating their phenotypic stability. Thus the acquisition of the F456L mutation by the cp45 virus resulted in a small, incremental increase in its level of attenuation, indicating its possible usefulness in the fine tuning of the level of attenuation of the cp45 vaccine candidate. The ability to transfer mutations identified in heterologous paramyxoviruses, which in this case represent different subfamilies, greatly enhances our ability to rapidly develop novel parainfluenza virus candidate vaccines.

Human immunodeficiency virus type 1 neutralizing antibody serotyping using serum pools and an infectivity reduction assay.

Classification of human immunodeficiency virus type 1 (HIV-1) by neutralization serotype may be important for the design of active and passive immunization strategies. Neutralizing antibody serotyping is hindered by the lack of standard reagents and assay format, and by the weak activity of many individual sera. To facilitate cross-clade neutralization analysis, we used an infectivity reduction assay (IRA) and selected clade-specific serum (or plasma) pools from subjects infected with clade B and E HIV-1, respectively. Several serum pools were utilized; some were selected for strong neutralizing activity against intraclade viruses and others were derived from conveniently available samples. Against a panel of 51 clade B and E viruses, serum pools displayed strong neutralization of most intraclade viruses and significantly diminished cross-clade neutralization. Results were confirmed against a blinded panel of 20 viruses. The data indicate that the phylogenetic classification of virus subtypes B and E corresponds to two distinct neutralization serotypes. This approach to neutralizing antibody serotyping may be useful in defining the antigenic relationship among viruses from other clades.