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

Vaccine 19 (2001) 3620– 3631 www.elsevier.com/locate/vaccine

Construction of a live-attenuated bivalent vaccine virus against human parainfluenza virus (PIV) types 1 and 2 using a recombinant PIV3 backbone Tao Tao, Mario H. Skiadopoulos, Fatemeh Davoodi, Sonja R. Surman, Peter L. Collins, Brian R. Murphy * Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 7, Rm 106, 7 Center Dri6e MSC 0720, Bethesda, MD 20892, USA Received 13 June 2000; received in revised form 5 March 2001; accepted 7 March 2001

Abstract 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. Published by Elsevier Science Ltd. Keywords: Vaccine; Human parainfluenza virus (PIV); Recombinant PIV3 backbone

1. Introduction The human parainfluenza viruses (PIVs), specifically serotypes 1, 2 and 3 (PIV1, PIV2, and PIV3), cause severe respiratory tract disease that leads to hospitalization of infants and young children [1]. PIV1, PIV2, and PIV3 are distinct serotypes that do not induce significant cross-neutralization or cross-protection [2,3]. PIV3 is second only to respiratory syncytial virus (RSV) as the most common cause of viral pneumonia in infants and young children [4,5]. PIV1 and PIV2 are the principal etiologic agents of laryngotracheobronchitis (croup) and also can cause severe pneumonia and bronchiolitis [1]. In a long term study in infants and children over a 20-year period, PIV1, PIV2, and PIV3 were identified * Corresponding author. Tel.: + 1-301-594-1616; fax: +1-301-4968312. E-mail address: [email protected] (B.R. Murphy). 0264-410X/01/$ - see front matter. Published by Elsevier Science Ltd. PII: S0264-410X(01)00101-3

as etiologic agents responsible for 6.0, 3.2, and 11.5%, respectively, of hospitalizations for respiratory tract diseases [5]. In total, they accounted for 18% of the hospitalizations [5]. Infection with parainfluenza viruses also can lead to otitis media in children [6]. Thus, there is a need to produce vaccines against these viruses that can prevent the serious lower respiratory tract disease and the otitis media that accompanies infections with PIVs. A vaccine has not yet been approved for the prevention of PIV or RSV disease in humans. RSV and PIV3 cause significant illness within the first 4 months of life whereas most of the illness caused by PIV1 and PIV2 comes after 6 months of age [1,7]. A likely immunization sequence employing live-attenuated PIV vaccines would be administration of RSV and PIV3 vaccines together as a combined vaccine which would be given two or more times, with the first dose administered at or before 1 month of age, followed by a bivalent PIV1

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and PIV2 vaccine at 4 and 6 months of age. The most promising paramyxovirus vaccine candidates to date are live-attenuated viruses, since these have been shown to be efficacious in non-human primates even in the presence of passively transferred serum antibodies, an experimental situation simulating that of the young infant with maternally-acquired serum IgG antibodies [8,9]. The present study seeks to develop a bivalent live-attenuated PIV1 and PIV2 candidate vaccine based on the reverse genetics system used to recover wild type and attenuated derivatives of PIV3 [10 – 15]. PIV3 is a member of the Respiro6irus genus of the Paramyxo6iridae family in the order Mononega6irales [1]. Its genome is a single-stranded, negative-sense RNA of 15462 nucleotides (nt) in length [1]. PIV3 is the best characterized human PIV and represents the prototype human PIV. It encodes up to nine proteins: the nucleocapsid protein N, the phosphoprotein P, the C, D and V proteins of unknown functions, the matrix protein M, the fusion glycoprotein F, the hemagglutinin-neuraminidase glycoprotein HN, and the large polymerase protein L [1,11]. The recombinant PIV3 recovery system was earlier used to generate a PIV1 vaccine candidate by replacing the PIV3 HN and F open reading frames (ORFs) with those of PIV1 [14]. This was done using a PIV3 recombinant backbone that contains 12 of the 15 cp45 point mutations, whose attenuation phenotypes have been characterized [13]. This rPIV3-1cp45 chimeric virus is highly attenuated in the upper and lower respiratory tract of hamsters [14] and induces a high level of protection against PIV1 infection. Thus, it represents a promising live-attenuated human PIV1 vaccine candidate. Since many studies have demonstrated that various members of the Mononega6irales, referred to as mononegaviruses, are able to function as vectors of other viral proteins [16– 23], we chose to evaluate the chimeric PIV3-1 virus as a vector to express the major protective antigen of PIV2, the HN protein, as an extra gene. In the present study, a PIV3-1 virus expressing the PIV2 HN protein was generated and shown to be attenuated for hamsters. This virus was able to induce resistance to both PIV1 and PIV2. The usefulness of this reverse genetics system in the development of a live-attenuated bivalent PIV1 and PIV2 vaccine is discussed.

2. Materials and methods

2.1. Cells, 6iruses, and antibodies to PIV HEp-2 (ATCC CCL 23) cells were maintained in MEM (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS) and 50

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mg/ml gentamicin. LLC-MK2 (ATCC CCL 7.1) cells were maintained in OptiMEM (Life Technologies) supplemented with 10% FBS and 50 mg/ml gentamicin. WHO certified Vero cells [24] below passage 150 were maintained in VP-SFM (Formula No. 960353SA, Life Technologies) supplemented with 2 mM glutamine and 50 mg/ml gentamicin. PIV2/V9412-6 (PIV2), a wild type strain of PIV2, was described earlier [3]. Wild type PIV1/Wash64 (PIV1) and rPIV3, a wild type recombinant version of the JS strain of human PIV3, were described earlier [10,25]. rPIV3-1, a recombinant chimeric PIV3 with its PIV3 F and HN glycoprotein ORFs replaced by their PIV1 counterparts, was described earlier [26]. rPIV3-1cp45, a derivative of rPIV3-1 carrying the complete set of cp45 mutations except those in PIV3 F and HN genes, was described elsewhere [14]. All viruses were propagated in the presence of trypsin (0.5 mg/ml for liquid overlay and 1.0 mg/ml for semisolid overlay) as described [26], except for PIV2 and rPIV3, which replicate in the absence of trypsin. Modified vaccinia virus Ankara strain expressing the T7 RNA polymerase (MVA-T7) was kindly provided by Dr Wyatt and Dr Moss [27]. Antibodies against PIV1 and PIV2 were prepared by immunizing three rabbits each in subcutaneously implanted chambers as described [28]. The immunization chambers were inoculated with 256 HA units of sucrose gradient purified PIV1 or PIV2. The rabbits were boosted three times with 128 HA units per chamber of purified PIV1 or six times with PIV2. Chamber fluid was harvested at 3 weeks intervals, and the fluids from each immunized group were combined to make a PIV1- or a PIV2-specific antibody preparation. The PIV1 and PIV2 antibodies had neutralization titers of 1:1024 and 1:480, respectively.

2.2. Viral RNA extraction, re6erse transcription (RT), polymerase chain reaction (PCR), and DNA sequencing For viral gene cloning or marker verification, amplified viruses were concentrated from culture supernatant fluid [29], and viral RNAs were extracted with Trizol (Life Technologies). RT was performed with random oligonucleotide primers and the Preamplification system (Life Technologies) using the protocol recommended by the manufacturer. RT-PCR amplification was performed using the Advantage cDNA kit (ClonTech, Palo Alto, CA) and PIV-specific primer pairs, while PCR mutagenesis was done using Deep Vent DNA polymerase (New England Biolabs, Beverly, MA). DNA sequence determination was done using dRhodamine dye terminator kit and ABI 310 automated sequencer (Perkin– Elmer, Foster City, CA).

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2.3. Construction of the chimeric antigenomic cDNA of rPIV3 -1 carrying the PIV2 HN ORF as an extra transcriptional cassette We earlier constructed pLit.PIV32HNhc, in which the PIV2 HN ORF is under the control of the PIV3 gene start and gene end transcription signals [30]. The upstream end of the PIV3 L gene also is present in the subclone. PCR mutagenesis was used to introduce an extra PpuMI site corresponding to nt 27– 33 upstream of the start codon of the L gene. The sequences for those PCR primers are, for upstream, 5%-GCCATGGGCCCGAGGAAGGACCCAATAGACA-3% and, for downstream, 5%-CCCGGGTCCTGATTTCCCGAGCACGCTTTG-3%. The resulting DNA fragment contained, in a 5%–3% direction, a naturally occurring PpuMI site, the PIV3 HN 5%-untranslated region, the PIV2 HN ORF, the PIV3 HN 3%-untranslated region including its gene-end, the conserved intergenic trinucleotide, the L gene start, the PIV3 L 5%-untranslated region, and the newly introduced PpuMI site. This PCR fragment was digested with PpuMI and was inserted into PpuMI-digested p38%DPIV31hc [14] to generate p38%DPIV31hc.2HN. The insert from p38%DPIV31hc.2HN was isolated as an 8.5 kb BspEISphI fragment and was then introduced into the BspEISphI window of pFLC.2G + .hc [26] to generate pFLC31hc.2HN (Fig. 1).

2.4. Reco6ery of recombinant chimeric 6iruses HEp-2 cells were transfected with pFLC31hc.2HN plus the support plasmids and were infected with MVA-T7 as earlier described [26] to yield the recombinant virus rPIV3-1.2HN. The transfection harvest containing rPIV3-1.2HN was passaged twice on LLC-MK2 cells using TPCK trypsin (trypsin, Catalog No. 3741, Worthington Biochemicals, Freehold, NJ) and was biologically cloned by four sequential plaque passages using an MEM-based agarose overlay as described [31] with the following changes: FBS was not included in the overlay and TPCK trypsin was used. Further in vitro and in vivo characterization of rPIV3-1.2HN was performed using the biologically cloned and amplified virus. To confirm the presence of the additional gene in rPIV3-1.2HN, viral RNA was extracted from concentrated virus, reverse transcribed, and amplified by PCR using primers specific to PIV1 F (5%-AGTGGCTAATTGCATTGCATCCACAT-3%) and PIV1 HN (5%-GCCGTCTGCATGGTGAATAGCAAT-3%) (see Fig. 1 for location of the primer pair) ORFs. Amplified DNA fragments were digested with restriction endonucleases and analyzed on a 1% tris–boric acid–EDTA (TBE) agarose gel. Control reactions were performed in parallel without the addition of RT.

Fig. 1. Structures of the antigenomic RNA of the parental virus rPIV3-1 and its derivative rPIV3-1.2HN. The cDNA encoding full-length antigenomic RNA for rPIV3-1 was modified by inserting an extra gene unit carrying the PIV2 HN glycoprotein gene as described in Section 2. The locations of restriction enzyme sites in the cDNAs from which the viruses were derived are indicated. Plasmid names are in parentheses. The location of the RT-PCR primers used to characterize the recombinant virus generated from these plasmids are indicated by arrows.

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2.5. Replication of PIVs in 6itro in LLC-MK2 cells Confluent LLC-MK2 monolayers in 6 well plates were infected in triplicate with rPIV3-1.2HN or rPIV3-1 at an MOI of 0.01 as earlier described [30]. After inoculation, infected monolayers were incubated at 32°C. A 0.5 ml aliquot of culture supernate was removed at 24 h intervals and replenished with 0.5 ml of serum free OptiMEM I. Specimens were flash frozen on dry ice and kept at − 80°C until assayed on LLC-MK2 monolayers, and the titers are expressed as log10TCID50 per ml [13].

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2.8. Replication of PIVs in the respiratory tract of hamsters Six to eight-week old Golden Syrian hamsters in groups of six were inoculated intranasally with 0.1 ml of L-15 medium containing 105.3 or 106.0 TCID50 of virus. Four days after inoculation, hamsters were sacrificed and their lungs and nasal turbinates were harvested and homogenized as earlier described [26]. Virus present in the tissue samples was titered on LLC-MK2 cell monolayers at 32°C as described above. The titers were expressed as mean log10TCID50 per g of tissue for each group of six hamsters.

2.6. PIV2 HN protein expression by rPIV3 -1.2HN 2.9. Immunization and protecti6e efficacy in hamsters The expression of the PIV2 HN glycoprotein by the recombinant chimeric virus was evaluated by radiolabeled immunoprecipitation assay (RIPA) as described earlier [20]. Briefly, confluent LLC-MK2 monolayers in T25 flasks were infected with virus at an MOI of 3 and were incubated at 32°C for 18 h. The infected cells were washed with methionine- and cysteine-free DMEM (BioWhittaker, Walkersville, MD) and were labeled with 120 mCi of 35S-ProMix (Pharmacia Amersham, Piscataway, NJ) in 3 ml of methionine- and cysteinefree DMEM at 32°C for 18 h. Labeled cells were scraped into PBS, washed, pelleted, and disrupted in 1 ml RIPA buffer. Clarified lysates were divided into two 0.5 ml aliquots, which were mixed with either 3 ml of PIV2 antibodies or PIV1 antibodies. After incubation at 4°C for 18 h, 0.2 ml RIPA buffer containing 10% Protein A Sepharose (Catalog No. 82506, Sigma) was added. The mixture was further incubated at 4°C for another 18 h. Beads coated with antibody– antigen complexes were pelleted, washed with RIPA buffer, and suspended in 100 ml of 1 × SDS sample buffer each. An 8 ml fraction was resolved on 4– 12% SDS polyacrylarnide gels using EXCEL II system (NOVEX, San Diego, CA). The gels were dried and autoradiographed. The expression of PIV2 HN glycoproteins by the recombinant chimeric on LLC-MK2 cells was also determined by immunostaining of viral plaques or monolayers as earlier described [32] using the PIV1 or PIV2 rabbit antibody.

2.7. Stability of the PIV2 HN gene insert To test the genetic stability of the inserted PIV2 HN gene in the recombinant chimeric virus, rPIV3-1.2HN was serially passaged six times on LLC-MK2 cells at an MOI of 0.01 in serum free OptiMEM I. Virus present in the harvest from the sixth passage was plaque passaged once on LLC-MK2 cells and twelve plaques were of PIV2 HN protein expressed by picked and amplified twice. The presence the amplified viruses was analyzed using immunostaining and RIPA.

Hamsters in groups of twelve were immunized intranasally with 105.3 TCID50 of virus as described above. Serum was collected 2 days prior to and on day 28 after immunization. The PIV1- or PIV2- specific serum antibody titer was determined by neutralization of 100 TCID50 of PIV1 and PIV2 on LLC-MK2 monolayers using two-fold dilutions of serum and hemadsorption to identify infected cultures, as described earlier [33]. The antibody titers are presented as reciprocal mean log2. Six hamsters from each group were challenged with either PIV2 or PIV1 wild type virus 29 or 32 days, respectively, after immunization. They were sacrificed 4 days after challenge and their nasal turbinates and lungs were harvested for virus quantitation as earlier described [3].

3. Results

3.1. Reco6ery of chimeric rPIV3 -1 6irus carrying the PIV2 HN ORF as an additional transcriptional cassette The PIV2 HN gene was inserted as an additional transcriptional cassette into the PIV3-1 chimeric cDNA (Fig. 1). The final plasmid construct, pFLC31hc.2HN (Fig. 1), encodes a PIV3-1 chimeric antigenomic RNA plus the PIV2 HN ORF flanked by the required cis-acting transcriptional sequences from PIV3. The length of the encoded viral antigenome, 17382 nt for rPIV31.2HN, conforms to the rule of six [34,35]. HEp-2 cells were transfected with pFLC31hc.2HN in conjunction with MVA-T7 and the PIV3 support plasmids, and recombinant chimeric virus designated rPIV3-1.2HN was recovered. rPIV3-1.2HN was analyzed by RT-PCR to confirm the presence of the 2 kb PIV2 HN insert between the PIV1 F and HN genes. As shown in Fig. 2, a 1 kb fragment was amplified from the rPIV3-1 control virus, whereas a 3 kb cDNA fragment was amplified from rPIV3-1.2HN indicating the

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Fig. 2. RT-PCR amplification of the F-HN gene junction from vRNA of rPIV3-1.2HN or rPIV3-l, and restriction enzyme analysis. (A-C) The RT-PCR products generated from rPIV3-l.2HN and rPIV3-1 using the RT-PCR primer pair indicated in Fig. 1 are diagrammed showing the positions of the (A) PpuMI, (B) NdeI, and (C) NcoI sites. The nucleotide length of each predicted restriction fragment is indicated. Panel D shows the fragments obtained by digestion with the indicated restriction enzyme followed by electrophoresis on a 1% tris – boric acid – EDTA agarose gel. Viral RNA extracted from concentrated rPIV3-1.2HN or rPIV3-1 virus was reverse transcribed and then amplified by PCR. The absence of an amplified product in the two RT negative control lanes (lanes A and C in Panel D for rPIV3-1.2HN or rPIV3-1, respectively) demonstrates that the template was a viral RNA and not a contaminating DNA. rPIV3-1.2HN (lane B) yields a 3 kb fragment, 2 kb larger than its rPIV3-1 parent (lane D) indicating the presence of an appropriately-sized PIV2 HN insert. Restriction enzyme digestion of this 3 kb fragment (lane 1, 3, and 5) produced patterns different from that of rPIV3-l (lane 2, 4, and 6) for each restriction endonuclease tested. The patterns obtained are consistent with the design of PIV2 HN gene insert as indicated by panels A – C. Representative insert bands unique to rPIV3-1.2HN virus are indicated by white arrows. Lane M contains 1 kb DNA size markers (Life Technologies).

presence of a 2 kb insert. The RT step was required for generation of the RT-PCR products for both rPIV3-1 and rPIV3-1.2HN confirming that the template was RNA rather than contaminating DNA (Fig. 2, Panel D, Lanes A-D). Restriction enzyme digestion of the RT-PCR product for rPIV3-1.2HN indicated that its restriction patterns conformed to the cDNA design (Fig. 2, panels A–C) with the PIV2 HN gene present

between the PIV1 F and HN genes (Fig. 2, panel D, lanes 1, 3, and 5).

3.2. Multicycle growth characteristics of rPIV3 -1.2HN The multicycle replication of rPIV3-1.2HN and rPIV3-1 was analyzed by infecting cell cultures in triplicate at an MOI of 0.01 and harvesting samples over a

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6-day period (Fig. 3). The presence of the PIV2 HN gene insert in rPIV3-1.2HN was found to slightly restrict its replication in vitro, with the peak titer being about ten-fold lower than that of rPIV3-1. Nonetheless, the peak titer of rPlV3-1.2HN was almost 108 TCID50 per ml.

As described earlier [14,26], replication of rPIV3-l was moderately restricted at 40°C, and rPIV3-1.2HN also manifested this level of temperature sensitivity (Table 1). These findings indicate that the presence of PIV2 HN insert did not alter the level of temperature sensitivity of replication of rPIV3-1.

3.3. The le6el of temperature sensiti6ity of replication in 6itro for rPIV3 -1.2HN is similar to that of its parent 6irus

3.4. In 6itro expression of PIV2 HN by rPIV3 -1.2HN During early passages of rPIV3-1.2HN, expression of the PIV2 HN protein from the gene insert was confirmed by RIPA (data not shown), and we next sought to determine if expression of PIV2 HN by rPIV3-1.2HN remained stable following multiple passages in cell culture. Following transfection, rPIV3-l.2HN was passaged twice, biologically cloned 4 times by plaque passage, amplified twice, serially passaged at a MOI of

Since it has earlier been demonstrated that the insertion of an additional transcriptional unit into rPIV3 can specify a temperature sensitive phenotype [23], we determined the level of temperature sensitivity of rPIV3-l.2HN, its rPIV3-1 parental virus, PIV3cp45, and the PIV1, -2, and -3 wild type control viruses (Table 1).

Fig. 3. Multicycle growth characterization of rPIV3-1.2HN and rPIV3-1 in vitro. LLC-MK2 monolayers were infected in triplicate with rPIV3-1.2HN or its parental virus rPIV3-1 at an MOI of 0.01. Infected cells were incubated at 32°C. Tissue culture supernatant aliquots were taken at 24 h intervals, and the quantity of virus present was determined by titration on LLC-MK2 monolayer cultures. The mean titers of virus are expressed as log10TCID50/ml. Table 1 The rPIV3-1 virus carrying the PIV2 HN insertion exhibits a level of temperature sensitivity similar to that of its parental virus Virus

Titera at 32°C (log10TCID50 per ml)

Titer reduction (log10TCID50 per ml) at indicated temperatures (°C)a 36

PIV2/V9412 b PIV1/Wash64 b rPIV3 PlV3cp45b rPIV3-1 rPIV3-1.2HN a

7.8 8.5 7.9 7.8 8.0 8.3

(0.1)c 1.1 0.1 0.3 0.5 (0.3)

37 0.0 1.4 0.1 1.3 0.6 0.3

38

39

40

(0.4) 0.6 (0.3) 3.4d 0.9 0.6

(0.4) 0.5 (0.4) 6.8 1.1 1.5

0.0 0.9 0.4 ]6.9 2.6d 2.6d

Data presented are the mean of two experiments. Biologically derived virus. The other viruses are recombinant. c Numbers in parentheses signify a titer increase. d Value indicates shut-off temperature at which the virus titer showed a titer reduction of 100-fold or more compared to titer at 32°C. b

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Fig. 4. Analysis of the expression of PIV2 HN protein by twelve plaque populations of serially-passaged rPIV3-1.2HN using RIPA. LLC-MK2 monolayers in T25 flasks were each infected with one of 12 plaque populations of serially passaged rPIV3-1.2HN or with wt PIV1 or PIV2 and were labeled with 35S-methionine and 35S-cysteine. The labeled viral proteins were immunoprecipitated with PIV2-specific antibodies (Panel A) or PIV1-specific antibodies (Panel B). The arrows indicate the precipitated HN and F proteins of PIV2 or PIV1. Molecular weight standards are marked with lines and number (in kDa).

0.01 six times, and plaque passaged once to yield 12 plaque populations. These plaque populations were amplified twice and examined for their expression of the PIV2 HN protein. Thus, each plaque population examined had been passaged 17 times, and the majority of these passages had been at low MOI. Cells infected with the serially-passaged rPIV3-1.2HN biological clones were examined by RIPA using PIV2 or PIV1 antibodies in panel A and B of Fig. 4, respectively. As

indicated in panel A using PlV2-specific antibodies, the PIV2 HN protein was precipitated from cells infected with each of the plaque populations of multiply-passaged rPlV3-1.2HN virus (labeled 8-1 through 8-12) and with wild type PIV2. This PIV2 HN protein band was absent from cells infected with PIV1. As a control, aliquots of the same lysates were immunoprecipitated with PIV1-specific antibody (Fig. 4, panel B). Two major bands containing the PIV1 HN and F proteins

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were present in cells infected with the rPIV3-1.2HN plaque populations, and these bands were similar in size to those from cells infected with wild type PIV1 with one difference: the F protein band migrated at a slower rate than that of PIV1. This suggested that the majority of PIV1 F glycoprotein in rPIV3-1.2HN infected cells was in the uncleaved F0 form whereas that in PIV1 infected cells was largely cleaved. The sequence of the F genes of PIV1 and rPIV3-1.2HN were determined and found to be identical suggesting that the PIV1 F cleavage is delayed or inhibited in the PIV3-1 chimeric background. Similar PIV1 HN or F bands were not present in cells infected with PIV2. These findings were confirmed by immunostaining of the 12 plaque populations, in which plaques of each of the 12 clones tested were stained with either PIV1 or PIV2 antibodies (data not shown).

3.5. rPIV3 -1.2HN is attenuated in the respiratory tract of hamsters To determine if the insertion of an extra transcriptional cassette modifies the level of replication of rPIV3-1 in vivo, rPIV3-1.2HN was used to infect hamsters. HPIV3 causes an inapparent infection in hamsters, and the reduction in the level of replication of a recombinant HPIV3 virus versus that of its parent is used to assess the level of attenuation of the vaccine candidate [11–14,20,30,36,37]. As shown in Table 2, rPIV3-1.2HN replicated 30-fold lower than its rPIV3-1 parent in both the upper and lower respiratory tract of hamsters, indicating that insertion of an extra transcriptional cassette of this size and at this position attenuates the virus for hamsters. However, rPIV3-1.2HN was not as attenuated as rPlV3-1cp45 in the lower respiratory tract.

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3.6. Immunization of hamsters with rPIV3 -1.2HN elicits serum neutralizing antibodies against PIV1 and PIV2 and induces a high le6el of resistance against challenge with PIV1 and PIV2 Hamsters were immunized with rPIV3-1.2HN or control viruses and were challenged with wild type PIV1 or PIV2 4 weeks later. As shown in Table 3, immunization with rPIV3-1.2HN induced a strong immune response to both PIV1 and PIV2, and the hamsters immunized with rPIV3-1.2HN were highly resistant to challenge with wild type PIV1 or PIV2 virus. rPIV3-1.2HN elicited a 4-fold lower titer of neutralizing antibodies against PIV1 when compared with its rPIV3-1 parent. This decreased immunogenicity of rPIV3-1.2HN could be a consequence of (i) its decreased replication [20]; (ii) the more distal location of the PIV1 HN from the 3% promoter in rPIV3-1.2HN than in rPIV3-1, which could have decreased its level of expression [20], and consequently, its immunogenicity; or (iii) antigenic competition. There is a low level of neutralizing activity in the hamster sera which might be due to the presence of sialic acid binding glycoproteins or glycolipids in the sera that are analogous to the gamma inhibitors of influenza viruses [38]. rPIV31.2HN induced slightly less protection against PIV1 challenge than rPIV3-1 in the upper respiratory tract.

4. Discussion The similar age distribution of illness caused by PIV1 and PIV2 suggested the strategy to develop a bivalent live-attenuated virus vaccine that can be used to prevent the serious disease caused by these two viruses. We have been pursuing two approaches using reverse genetics to develop this bivalent vaccine.

Table 2 Insertion of the PIV2 HN transcriptional unit into rPIV3-1 attenuates virus growth in hamsters Experiment No.

1c 2d

Virus

rPIV3-1 rPIV3-1.2HN rPIV3-1 rPIV3-1.2HN rPIV3-1cp45 rPIV3

Virus titer grouping]b

a

in indicated tissue (log10TCID50 per g9 S.E.) [Duncan

NT

Lung

6.9 90.1 5.4 90.2 6.7 90.1 5.1 9 0.1 4.6 90.3 6.5 90.2

6.0 90.3 4.4 90.4 6.6 9 0.2 5.2 9 0.2 1.8 9 0.4 6.7 9 0.1

[A] [B] [B] [A]

[A] [B] [C] [A]

a Lungs and nasal turbinates of the hamsters were harvested on day 4. Virus titers in tissue were determined, and the mean titer was expressed as log10TCID50 per g 9 standard error (SE). NT, nasal turbinates. b Grouping as analyzed by Duncan multirange test. Values in the same column that are indicated with the same letter are not significantly different from each other whereas those with different letters are significantly different. c Groups of six animals were inoculated intranasally with 106 pfu of indicated virus in 0.1 ml medium on day 0. d Groups of six hamsters were inoculated intranasally as in experiment 1 with 105.3 TCID50 of indicated virus on day 0.

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Table 3 rPIV3-1.2HN expressing the HN glycoprotein of PlV2 protects hamsters against challenge with both PIV1 and PlV2 Immunizing virusa

Serum neutralizing antibody titerb against indicated virus (mean reciprocal log2 9 S.E.) [Duncan grouping]

Titer of challenge virusd in indicated tissues (log10TClD50/g+S.E.) [Duncan grouping]c

PIV1

PIV1

Pre rPIV3 PIV2 rPIV3-1 rPIV3-1.2HN rPIV3-1cp45

PIV2 Post

Pre

Post

NTe

PIV2 Lung

54.090.0 54.09 0.0[C] 4.5 9 0.1 4.69 0.2[C] 5.4 9 0.2 [A] 5.1 90.1 54.090.0 54.090.0 [C] 4.3 90.2 9.69 0.2 [A] 5.7 90.2 [A] 5.7 90.2 4.2 9 0.1 8.5 9 0.3[A] 4.0 9 0.0 4.29 0.1 [D] 51.290.0 [D] 51.29 0.0 54.09 0.0 6.2 90.2 [B] 4.1 90.1 8.3 90.2 [B] 2.3+0.5 [C] 51.290.0 54.090.0 6.29 0.4 [B] 54.09 0.0 54.09 0.0 [D] 4.1 9 0.3 [B] 1.8 90.5

NTe [A] 6.8 90.2 [A] 51.29 0.0 [B] 6.3 90.1 [B] 51.290.0 [B] 6.0 9 0.1

Lung [A] 6.0 90.3 [AB] [C] 51.290.0 [C] [B] 6.5 90.2 [A] [C] 51.290.0 [C] [B] 5.7 9 0.4 [B]

a

Hamsters in groups of 12 were immunized with 105.3 TCID50 of indicated virus intranasally on day 0. Serum was diluted 1:10 with OptiMEM and incubated at 56° for 30 min before being assayed for neutralization titer. c Grouping as analyzed by Duncan multirange test. Values in the same column indicated with the same letter are not significantly different from each other, whereas values with different letters are statistically different. d Half of the hamsters from each group were challenged with 106 TCID50 PIV2 on day 29, and the remaining half were challenged with 106 TCID50 PIV1 on day 32. Tissue samples were harvested 4 days after challenge. e Nasal turbinates. b

The first approach, not addressed in this study, involves the development of separate live-attenuated PIV1 and PIV2 vaccines that each contains the two protective antigens of the parainfluenza viruses, i.e. the HN and F glycoproteins. The PIV1 component was generated by modification of the extensively studied cold-passaged PIV3cp45 vaccine candidate [14]. The HN and F glycoproteins of the PIV3cp45 candidate vaccine virus were replaced with those of PIV1. This created a live-attenuated PIV1 vaccine candidate, designated rPIV3-lcp45, which contained the attenuation backbone of the PIV3cp45 vaccine virus together with the protective HN and F antigens of PIV1. This virus was satisfactorily attenuated for hamsters and induced a high level of resistance to replication of PIV1 challenge virus, even in animals immune to PIV3 [3]. The PIV2 component of this bivalent vaccine consisted of a PlV2-PlV3 chimeric virus in which the HN and F ORFs of a wild type PIV3 virus were replaced by chimeric HN and F ORFs in which the PIV2 ectodomain and transmembrane domain were fused to the PIV3 cytoplasmic domain [30]. This antigenic chimeric virus, termed rPIV3-2CT, replicated efficiently in vitro and was attenuated in both the upper and the lower respiratory tract of hamsters and African green monkeys. This observation indicated that the chimerization of the HN and F proteins of PIV2 and PIV3 itself specified an attenuation phenotype in vivo. Despite this attenuation, rPIV3-2CT was highly immunogenic and protective against challenge with PIV2 wild type virus in both species. The compatibility of rPIV3-1cp45 and rPIV32CT as components of a bivalent PIV1 and PIV2 vaccine is currently being evaluated. The second approach, the subject of the present study, uses a PIV3/PIV1 antigenic chimeric virus as a

vector to express the major protective antigen of PIV2, the HN protein, thereby generating a single virus that can protect against both PIV1 and PIV2. This approach borrows from the recent literature that indicates that mononegaviruses can be modified to express foreign antigens, some of which can function as vaccine candidates [17–23,39–54]. When placed under the control of viral transcription gene-start and gene-end signals and inserted into the gene order, the foreign gene is expressed as a separate mRNA, and a high level of protein expression occurs. The mononegaviruses are potentially useful as vectors for several reasons. First, the inserts in most cases remain stably expressed even after many passages in vitro [40,45,49]. This differs from findings with single-stranded, positive-sense RNA viruses in which the inserts are subject to deletion [55,56]. Insert stability is important for a vaccine virus because extensive replication is required for vaccine production. The PIV2 HN insert in the rPIV3-1.2HN virus of the present study was stably expressed after 17 passages confirming the high degree of stability of such constructs. Second, in most cases mononegaviruses have accepted relatively large inserts without drastic reduction in replication [16,23,48,52]. This makes it possible to express one or more viral protective antigens while maintaining efficient replication in vitro. Third, attenuated derivatives of certain mononegaviruses are available and could serve as safe vectors [14,18,41,52,57–59]. Two findings of importance to the development of a bivalent PIV1 and PIV2 vaccine were derived from the present study. First, it was possible to produce a single virus, rPIV3-1.2HN, that was able to induce immunity against two human respiratory tract pathogens, PIV1 and PIV2. The PIV1 HN and F glycoproteins of the

T. Tao et al. / Vaccine 19 (2001) 3620–3631

vector backbone induced immunity to PIV1 and the inserted PIV2 HN gene induced resistance to PIV2. Second, the insertion of the PIV2 HN gene into rPIV3-1 attenuated the virus for hamsters. We have earlier observed that the insertion of the HA gene of measles virus attenuated wild type rPIV3 for hamsters [20]. In addition, insertion of the measles virus HA gene also further attenuated rPlV3cp45L, which contains the three attenuating cp45 mutations in the L polymerase protein, for hamsters [20]. Thus, insertion of either the PIV2 HN or the measles virus HA leads to attenuation of a parainfluenza virus for the respiratory tract of hamsters for reasons that remain undefined. It is known that insertion of a transcriptional cassette of the same overall length of the HA or HN inserts that had been modified so that it did not encode a protein does not attenuate wild type PIV3 [23]. However, insertion of this non-coding transcriptional cassette does further attenuate an attenuated virus bearing the cp45 mutations in the L polymerase [23]. This indicates that the expressed protein rather than the insertion of the transcriptional cassette attenuates wild type virus. If the rPIV3-1.2HN vaccine candidate is found to be insufficiently attenuated in humans, the cp45 attenuating mutations should be added incrementally to achieve the proper balance between attenuation and immunogenicity needed for a live-attenuated PIV2 vaccine for use in humans. We have earlier found that a rPIV3 virus expressing the measles virus HA and bearing only the three cp45 attenuating mutations in L was satisfactorily attenuated for the upper and lower respiratory tract of hamsters, and hamsters immunized with the rPIV3-HA chimeric virus developed a high level of antibody to both measles virus and to PIV3 [20]. These observations considered in the context of findings from the present study indicate that the development of a vaccine against PIV1 and PIV2 using the PIV vector approach should be possible. A final vaccine might include a second virus expressing the second protective antigen of PIV2, the F glycoprotein, or might be a single virus expressing both the PIV2 HN and F glycoproteins. Both PIV2 HN and F expressing PIV3-1 recombinants might be further modified to place the inserted transcriptional cassette closer to 3%-promoter to optimize expression and immunogenicity of the PIV2 antigen as has been recently found for rPIV3cp45L expressing the measles virus HA [20]. Taken together, these findings demonstrate the flexibility of the PIV3 reverse genetics system to develop vaccines for the three major human parainfluenza viruses that are associated with serious lower respiratory disease.

Acknowledgements We thank Dr Robert Chanock and Dr Stephen Whitehead for their suggestions and comments on this

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manuscript. We also thank Ernest Williams, Chris Cho, and Sandra Cooper for their technical assistance. This work is part of a continuing program of research and development with Wyeth–Lederle Vaccines and Pediatrics through CRADA contract AI-000087 and AI000099.

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