LIVE ATTENUATED RESPIRATORY SYNCYTIAL VIRUS

FIELD

The subject matter disclosed herein relates to respiratory syncytial virus (RSV) and attenuated, mutant strains thereof suitable for use as vaccines.

PARTIES TO A JOINT RESEARCH AGREEMENT

This invention was made under Public Health Service Cooperative Research and Development Agreement (PHS-CRADA) No. 2013-0810 between the National Institute of Allergy and Infectious Disease at the National Institutes of Health and Sanofi Pasteur, Inc.

BACKGROUND

Human respiratory syncytial virus (RSV) infects nearly everyone worldwide early in life and is responsible for considerable mortality and morbidity. In the United States alone, RSV is responsible for 75,000-125,000 hospitalizations yearly, and conservative estimates indicate that RSV is responsible worldwide for 64 million pediatric infections and 160,000 or more pediatric deaths each year. Another notable feature of RSV is that severe infection in infancy frequently is followed by lingering airway dysfunction, including a predisposition to airway reactivity, that in some individuals lasts for years and can extend into adolescence and beyond. RSV infection exacerbates asthma and may be involved in initiating asthma.

RSV is a member of the Pneumoviridae family and, as such, is an enveloped virus that replicates in the cytoplasm and matures by budding at the host cell plasma membrane. The genome of RSV is a single, negative-sense strand of RNA of 15.2 kilobases that is transcribed by the viral polymerase into 10 mRNAs by a sequential stop-start mechanism that initiates at a single viral promoter at the 3′ end of the genome. Each mRNA encodes a single major protein, with the exception of the M2 mRNA that has two overlapping open reading frames (ORFs) encoding two separate proteins M2-1 and M2-2. The 11 RSV proteins are: the RNA-binding nucleoprotein (N), the phosphoprotein (P), the large polymerase protein (L), the attachment glycoprotein (G), the fusion protein (F), the small hydrophobic (SH) surface glycoprotein, the internal matrix protein (M), the two nonstructural proteins NS1 and NS2, and the M2-1 and M2-2 proteins. The RSV gene order is: 3′-NS1-NS2-N-P-M-SH-G-F-M2-L. Each gene is flanked by short conserved transcription signals called the gene-start (GS) signal, present on the upstream end of each gene and involved in initiating transcription of the respective gene, and the gene-end (GE) signal, present at the downstream end of each gene and involved in directing synthesis of a polyA tail followed by release of the mRNA. Transcription initiates at a single promoter at the 3′ end and proceeds sequentially.

The development of RSV vaccines has been in progress since the 1960's but has been complicated by a number of factors. For example, immunization of RSV-naïve infants with inactivated RSV has been shown to prime for enhanced disease upon subsequent natural RSV infection, and studies in experimental animals indicate that disease enhancement also is associated with purified RSV subunit vaccines.

Another obstacle to immune protection is that RSV replicates and causes disease in the superficial cells of the respiratory airway lumen, where immune protection has reduced effectiveness. Thus, immune control of RSV infection is inefficient and often incomplete, and it is important for an RSV vaccine to be as immunogenic as possible. Another obstacle to RSV vaccines is that the magnitude of the protective immune response is roughly proportional to the extent of virus replication (and antigen production). Thus, the attenuation of RSV necessary to make a live vaccine typically is accompanied by a reduction in replication and antigen synthesis, and a concomitant reduction in immunogenicity, and therefore it is beneficial to identify a level of replication that is well tolerated yet satisfactorily immunogenic.

Another obstacle is that RSV grows only to moderate titers in cell culture and is often present in long filaments that are difficult to purify. RSV can readily lose infectivity during handling. Another obstacle is the difficulty in identifying and developing attenuating mutations. Appropriate mutations must be attenuating in vivo, but should be minimally restrictive to replication in vitro, since this is preferred for efficient vaccine manufacture. Another obstacle is genetic instability that is characteristic of RNA viruses, whereby attenuating mutations can revert to the wild-type (wt) assignment or to an alternative assignment that confers a non-attenuated phenotype. Instability and de-attenuation are particularly problematic for point mutations.

Taking these factors together, there is a need for live attenuated RSV strains that replicate efficiently in vitro, are maximally immunogenic, are satisfactorily attenuated, and are refractory to de-attenuation.

SUMMARY

Reported herein are embodiments of novel recombinant human RSVs having an attenuated phenotype that are suitable for use as live-attenuated RSV vaccines. The disclosed recombinant RSVs comprise one or more genetic mutations that lead to the attenuated phenotype.

In some embodiments, the recombinant human RSV comprises a genome comprising RSV F, G, NS2, N, P, M, SH, M2, and L genes located at gene positions 1-9, respectively, wherein the F and G genes located at gene positions 1 and 2 are shifted from the native gene positions 8 and 7, respectively, and wherein the genome comprises a deletion of the sequence encoding NS1 protein, and wherein the recombinant RSV is infectious, attenuated, and self-replicating. In several embodiments, the SH gene of the recombinant RSV comprises a deletion of positions 4499-4610 inclusive corresponding to the reference RSV sequence set forth as SEQ ID NO: 1, and C4489T, C4492T, A4495T, A4497G, and G4498A substitutions corresponding to the reference RSV sequence set forth as SEQ ID NO: 1,

In some embodiments, the deletion of the sequence encoding NS1 protein comprises a deletion of positions 99-627 inclusive corresponding to the reference RSV sequence set forth as SEQ ID NO: 1. In some embodiments, the L gene encodes an L protein comprising a S1313 residue encoded by an TCA codon and a Y1314K substitution encoded by a AAA codon, wherein the amino acid positions correspond to the reference L protein sequence set forth as SEQ ID NO: 13 (amino acid sequence of the L protein).

In some embodiments, the L gene encodes an L protein comprising a deletion of S1313 and an I1314L substitution, wherein the amino acid positions correspond to the reference L protein sequence set forth as SEQ ID NO: 13.

In some embodiments, the nucleic acid sequence encoding the F protein comprises SEQ ID NO: 14 (F WT) or SEQ ID NO: 15 (FBB). In some embodiments, the genome of the recombinant RSV does not comprise any heterologous genes. In some embodiments, the genome of the recombinant RSV further comprises a deletion of the NS2 gene.

In some embodiments, the RSV genome comprises the modifications as discussed above, and comprises a nucleotide sequence corresponding to a positive-sense sequence at least 99% identical to SEQ ID NO: 3 (LID/F1G2/ANS1), SEQ ID NO: 4 (LID/F1BBG2/ΔNS1), SEQ ID NO: 6 (LID/F1G2/ANS1/1030s), SEQ ID NO: 7 (LID/F1BBG2/ANS1/1030s), SEQ ID NO: 9 (LID/F1G2/ΔNS1/A1313/I1314L), or SEQ ID NO: 10 (LID/F1BBG2/ΔNS1/A1313/I1314L). For example, the RSV genome can comprise or consist of a nucleotide sequence corresponding to a positive-sense sequence denoted by SEQ ID NO: 3 (LID/F1G2/ΔNS1), SEQ ID NO: 4 (LID/F1BBG2/ΔNS1), SEQ ID NO: 6 (LID/F1G2/ΔNS1/1030s), SEQ ID NO: 7 (LID/F1BBG2/ΔNS1/1030s), SEQ ID NO: 9 (LID/F1G2/ΔNS1/Δ1313/I1314L), or SEQ ID NO: 10 (LID/F1BBG2/ΔNS1/Δ1313/I1314L).

The embodiments of recombinant RSV disclosed herein can be subtype A RSV or a subtype B RSV.

Also provided herein are methods and compositions related to the expression of the disclosed viruses. For example, isolated polynucleotide molecules that include a nucleic acid sequence encoding the genome or antigenome of the described viruses are provided. Additionally provided are methods of producing a recombinant RSV comprising transfecting a permissive cell culture with a vector comprising a nucleic acid molecule comprising the genome or antigenome of the disclosed recombinant RSV, incubating the cell culture for a sufficient period of time to allow for viral replication; and purifying the replicated recombinant RSV.

Pharmaceutical compositions including the recombinant RSV are also provided. The compositions can further include an adjuvant. Methods of eliciting an immune response in a subject by administering an effective amount of a disclosed recombinant RSV to the subject are also disclosed. In some embodiments, the subject is a human subject, for example, a human subject between 1 and 6 months of age, or between 1 and 12 months of age, or between 1 and 18 months of age, or older.

The foregoing and other features and advantages of this disclosure will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. Deletion of NS1 (ΔNS1), deletion of NS1 and NS2 together (ΔNS1+2), and deletion and modification of part of the SH gene of D46 (LID mutation). (FIG. 1A) Details of the ΔNS1 deletion. The 529-nt deletion (nucleotides 99-627 inclusive) begins with and includes nucleotide 99 that is the first nucleotide of the ATG of the NS1 ORF and extends to and includes nucleotide 627 that immediately precedes the ATG (nucleotides 628-630) of the NS2 ORF. This deletion joins the upstream non-translated region of the NS1 gene to the translational start codon of the NS2 ORF. Coded amino acids MDTTHNDN (SEQ ID NO: 21) are shown. (FIG. 1B) Details of the ΔNS1+2 mutation. The 1042-nt deletion (nucleotides 99-1140 inclusive) begins with and includes nucleotide 99 that is the first nucleotide of the ATG of the NS1 ORF and extends to and includes nucleotide 1140 that immediately precedes the ATG (1141-1143) of the N ORF. This joins the upstream non-translated region of the NS1 gene to the translational start codon of the N ORF. Coded amino acids MALSKVKL (SEQ ID NO: 22) are shown. (FIG. 1C) Details of the LID mutation. This consists of the combination of a deletion encompassing most of the downstream non-translated region of the SH gene of D46, together with five translationally-silent substitutions in the last three codons and termination codon of the SH ORF. The 112-nt deletion begins with and includes nucleotide 4499, which immediately follows the TAG termination codon of the SH ORF, and extends to and includes nucleotide 4610, which is 7 nucleotides upstream of the SH gene-end signal. This deletion is combined with synonymous nucleotide substitutions C4489T, C4492T, A4495T, A4497G, and G4498A (bold and underlined). Coded amino acids ARVNT (SEQ ID NO: 23) are shown. Note that RSV LID generally resembles a wild-type RSV and is used in some of the following experiments as a wild-type-like control.

FIGS. 2A and 2B. Shift of the G and F genes from their natural genome positions (gene positions 7 and 8, respectively) to be promoter proximal and to have F preceding G (rather than the natural order of G preceding F) in gene positions 1 and 2 for F and G, respectively. (FIG. 2A). Nucleotides 4630-7551 inclusive were deleted from the RSV antigenomic cDNA to remove the F and G genes. This has the effect of fusing the SH gene-end signal to the F/M2 intergenic region. This mutation is illustrated with an antigenomic D46 cDNA bearing the LID mutation (the LID mutation is indicated with an open triangle above or below the SH gene), although the modifications do not depend on the LID mutation. (FIG. 2B). The G and F ORFs (shaded sequences) were reconstructed into genes in the reverse of their natural order (i.e., F-G instead of G-F). Each ORF was flanked by short non-translated regions and gene-start and gene-end signals, and separated by a short intergenic region consisting of a single nucleotide. Nucleotide adapters with Xho I sites (boxed in FIG. 2B) were added upstream of the F ORF (CTCGAGTTCACC, XhoI site underlined, SEQ ID NO: 28) and downstream of the gene-end-intergenic-gene-start sequence (AGTTAATAAAAAATGGGGCAAATACTCGAG, XhoI site underlined, SEQ ID NO: 29) that follows the G ORF. Four nucleotide substitutions were used to introduce an Xho I site into the noncoding region preceding the NS1 ORF in the LID antigenomic cDNA shown at the bottom, and required mutation of nucleotides 88-91 in the sequence representing the upstream non-translated NS1 gene region, from CTTG (shown in large case) to tcga (shown in small case). Coded amino acids MELLILK (SEQ ID NO: 24), FSN, MSKN (SEQ ID NO: 25), TPRQ (SEQ ID NO: 26), and MDTT (SEQ ID NO: 27) are shown. As shown with the antigenome at the bottom, the F-G gene cassette (cross-hatched rectangles) was inserted into the genomic cDNA using the Xho I sites.

FIGS. 3A-3D. Genome maps of RSV strains bearing various combinations of mutations including the LID mutation (the LID mutation is indicated with an open triangle over the SH gene); deletion of G and F from their natural positions (ΔG+F) and shift to the 2^ndand 1^stgene positions, respectively (F1G2, with F and G in the reverse of their natural order G-F); F1G2 in which the F ORF has been codon optimized (F1BBG2); deletion of the NS1 gene (ΔNS1); addition of the “stabilized” 1030s mutation (i.e., refractory to de-attenuation), consisting of the L assignments 1321K(AAA) and 1313S(TCA); addition of the “stabilized” Δ1313 and I1314L(CTG) mutations (i.e., refractory to de-attenuation) in L; and deletion of both the NS1 and NS2 genes (ΔNS1+2). (FIG. 3A) RSV LID plus the ΔNS1 deletion, as is or with the F1G2 or F1BBG2 modifications. (FIG. 3B) RSV LID/ΔNS1 plus the 1030s mutation, as is or with the F1G2 or F1BBG2 modifications. (FIG. 3C) RSV LID/ΔNS1 plus the Δ1313 and I1314L mutations, as is or with the F1G2 or F1BBG2 modifications. (FIG. 3D) RSV LID plus the ΔNS1+2 deletion, as is or with the F1G2 or F1BBG2 modifications.

FIG. 4. Multi-cycle growth kinetics of the indicated recombinant RSVs in Vero cells. This shows that viruses bearing the LID, ΔNS1, F1G2, and F1BBG2 mutations in various combinations replicate to reasonable titers in Vero cells, the cell substrate for vaccine manufacture.

FIG. 5. Multi-cycle growth kinetics of the indicated recombinant RSVs in human airway epithelial (HAE) cell cultures. HAE cultures form a differentiated, polarized, pseudostratified, mucocilliary tissue that closely resembles authentic airway epithelium. Attenuated replication in these cultures is predictive of attenuated replication in non-human primates and humans. These data show that viruses bearing the ΔNS1, F1G2, and F1BBG2 modifications are strongly attenuated.

FIG. 6. Recombinant RSVs bearing the LID, F1BBG2, F1G2, and ΔNS1 mutations in various combinations readily form plaques on Vero cells.

FIG. 7. Recombinant RSVs bearing the F1G2 and F1BBG2 gene shifts direct the formation of large syncytia in Vero cells during multi-cycle replication, consistent with increased expression of the F protein. Examples of syncytia are indicated with arrows.

FIG. 8. Increased expression of the RSV F and G proteins in Vero cells infected with recombinant RSVs bearing the F1G2 and F1BBG2 modifications. Vero cells were infected at an MOI of 3 PFU/cell, and cells were harvested 24 h post-infection and analyzed by gel electrophoresis under denaturing and reducing conditions and Western blot analysis using antibodies of the indicated specificities. The levels of expression of the RSV F, G, N, P, and M proteins relative to RSV LID as 1.0 are shown at the right. This shows that the gene shifts of RSV G and F increase their expression with little effect on the other genes.

FIG. 9. Increased expression of type 1 (a, $) and type III (λ) interferons in human airway A549 cells infected with recombinant RSVs lacking the NS1 gene. A549 cells were infected with an MOI of 3 PFU/cell and tissue culture medium supernatants were collected 24 h post-infection. IFN concentrations were determined by ELISA. This shows that deletion of the RSV NS1 gene results in a substantial increase in the expression of host cell interferons.

FIGS. 10A and 10B. The presence of the F1G2 or F1BBG2 or ΔNS1 modifications results in reduced RSV replication in the respiratory tract of mice. Six-week-old BALB/c mice were infected with a dose of 10⁴or 10⁶PFU per animal of each indicated RSV. Five animals per virus and dose were sacrificed at 3 (FIG. 10A) and 5 (FIG. 10B) days post-infection, and nasal turbinates and lungs were harvested, homogenized, and viral titers determined by plaque assay.

FIG. 11. Despite their restricted replication, recombinant RSVs bearing the indicated combinations of the ΔNS1, F1G2, and F1BBG2 modifications induced titers of RSV-neutralizing serum antibodies that compared well with the LID virus. Animals were infected as described in FIG. 10, and serum samples were collected 28 days post-infection.

FIG. 12. AGM data from Tables 1 and 2.

FIG. 13. AGM data from Table 3.

FIGS. 14A-14F. Evaluation of a prime-boost regimen of selected immunogens in hamsters. (FIG. 14A) Study design. Ninety-six hamsters were confirmed to be RSV- and HPIV3-seronegative and were assigned into eight groups of 12 animals each. On day 0, Groups A-D were given a primary IN infection with 10⁶PFU of RSV D46 in 0.1 ml of L15 medium, and Groups E-H were left uninfected. Six weeks later, sera were collected for determination of pre-boost 60% RSV plaque reduction neutralization titers (RSV-PRNTs). Two days later, primed and unprimed groups in pairs were boosted IN (10⁵TCID₅₀for vectors and 10⁶PFU for RSV) with one of the four viruses: (i) the empty rB/HPIV3 vector (Groups A and E); (ii) DS-Cav1 vector (Groups B and F); (iii) DS-Cav1/B3TMCT vector (Groups C and G); and (iv) RSV D46 (Groups D and H). Five days after boosting, nasal turbinates (NT) and lungs of six hamsters per group were collected for virus titration. From the other six hamsters per group, sera were collected two weeks after boosting to measure RSV- and HPIV3-PRNT, and two days later the animals were challenged IN with 10⁶PFU RSV D46. Three days following the challenge, NT and lungs were collected for titration of RSV replication. (FIG. 14B, FIG. 14C) Titers of boosting viruses in (FIG. 14B) NT and (FIG. 14C) lung tissue homogenates five days post-boost. Dashed and dotted lines indicate the limit of detection (LOD) for the rB/HPIV3 vectors and RSV D46, respectively. (FIG. 14D) Serum HPIV3-PRNTs two weeks post-boost in primed and unprimed hamsters, assayed without complement. (FIG. 14E, FIG. 14F) Pre-boost and post-boost serum RSV-PRNTs in animals from Groups A-D, that were primed with RSV D46 and boosted with the indicated viruses, assayed with (FIG. 14E) and without (FIG. 14F) added complement. The significance of differences between the indicated comparisons was determined by Student's t-test: ns indicates not significant (P>0.05); * indicates 0.01<P<0.05; ** indicates 0.001<P<0.01; *** indicates 0.0001<P<0.001.

FIGS. 15A-15E. AGM experiment #1: Viral replication and serum RSV-PRNTs in AGMs when the interval between priming and boosting was ˜2 months (two months minus nine days). (FIG. 15A) Study design. Twelve AGMs were previously administered a primary infection with one of three RSVs (Table 8) by the combined IN/IT routes. Sera were collected on Day 37 (two weeks before boosting), and RSV-PRNTs were determined in the presence of complement. The AGMs were organized into two groups of six animals each that were balanced with regard to the Day 37 RSV-PRNTs, identity of the priming virus, and sex ratio (Table 8). On day 51 (two months minus nine days) following priming, the groups were boosted with RSV 276 (which is a AM2-2 virus; SEQ ID NO: 12) or DS-Cav1/B3TMCT vector by the combined IN/IT routes. Virus shedding was monitored on Days 1-10 post-boost by NP and TL and virus titration. Sera were collected on days 7, 14, 21, and 28 post-boosting. (FIG. 15B, FIG. 15C) Viral titers in the (FIG. 15B) NP and (FIG. 15C) TL, shown as means with brackets indicating SEMs and limits of detection shown as dashed lines (vectors) and dotted lines (RSV). (FIG. 15D) Serum RSV-PRNTs at Day 37 post-priming and Days 0, 7, 14, 21, and 28 post-boosting, assayed in the presence of complement. (FIG. 15E) Serum RSV-PRNT at Days 0, 7, 14, 21, and 28 post-boosting, assayed without complement. D and E are annotated to show the mean serum RSV-PRNT for the combined two groups at the time of boosting (black dashed lines, with mean arithmetic values shown); in addition, dashed colored lines indicate the highest mean serum RSV-PRNT for each group, with the arithmetic values shown. Mean serum RSV-PRNTs are shown with brackets indicating SEMs. Peak mean titers of two groups were compared by Student's t-test: ** indicates 0.001<P<0.01; *** indicates 0.0001<P<0.001.

FIGS. 16A-16G. AGM experiment #2: Viral replication and serum RSV-PRNTs in AGMs when the interval between priming and boosting was ˜6 months (six months plus nine days). (FIG. 16A) Study design. Twenty AGMs were previously administered a primary infection with one of five attenuated RSVs (Table 9) by the combined IN/IT routes. Sera were collected on Days 28 and 154 (the latter being 35 days before boosting), and RSV-PRNTs were determined in the presence of complement. The AGMs were organized into three groups (n=6, 7, and 7) that were balanced with regard to Day-154 serum RSV-PRNTs, identity of the priming virus, and sex ratio (Table 9). On Day 189 (six months plus nine days) post-priming, the groups were boosted with one of three viruses: (i) RSV 276 (n=6), (ii) DS-Cav1 vector (n=7), or (iii) DS-Cav1/B3TMCT vector (n=7). Viral shedding and serum RSV-PRNTs were monitored as described in the legend to FIG. 15. Nasal mucosal lining fluid was collected for IgA analysis using SAM strips on Days 0, 14, 21, 28 post-boosting (see Methods and Materials for details). (FIG. 16B, FIG. 16C) Viral titers in the (FIG. 16B) NP and (FIG. 16C) TL were determined, shown as means with brackets indicating standard error of means (SEMs) and limits of detection shown as dashed lines (vectors) and dotted lines (RSV). (FIG. 16D) Serum RSV-PRNTs at 28, 154, and 189 days post-priming, and 7, 14, 21, and 28 days post-boosting, assayed with complement. (FIG. 16E) RSV-PRNTs at 0, 7, 14, 21, 28 days post-boosting, assayed without complement shown as means with brackets indicating the SEMs. D and E are annotated to show the mean serum RSV-PRNT for the combined three groups at the time of boost (black dashed lines, with mean arithmetic values given); in addition, dashed colored lines indicate the highest mean RSV-PRNT for each group, with arithmetic values given. (FIG. 16F, FIG. 16G) Serum and nasal mucosal IgA responses in AGMs. IgA antibody titers were determined in (FIG. 16F) serum samples collected Days 0, 7, 14, 21, and 28 post-boosting, and (FIG. 16G) nasal mucosal lining fluid collected on Days 0, 14, 21, and 28 post-boosting (note that in the DS-Cav1/BTMCT group, one of the Day 28 SAM specimens did not provide sufficient volume; thus, the Day 28 mean titer of the DS-Cav1/BTMCT vector was calculated for 6 animals only). IgA titers were measured by binding to purified RSV DS-Cav1 F protein in a DELFIA TRF assay. The antibody titers are given as the log₂dilution of serum or mucosal specimen yielding 400 fluorescence units, shown as means with brackets indicating the SEMs. Mean antibody titers for the combined three groups at the time of boost (Day 0) are indicated by the larger black dotted lines; the titers of all SAM samples on Day 0 were below the detection level (5.3 log₂). The limits of detection (LOD) are indicated by the smaller black dotted line. The dashed colored lines indicate the highest mean antibody titer for each group. Peak mean titers of three groups were compared pairwise by Student's t-test (GraphPad Prism): * indicates 0.01<P<0.05; ** indicates 0.001<P<0.01; *** indicates 0.0001<P<0.001; ns indicates P>0.05, not significant.

FIGS. 17A-17E. AGM experiment #3: Viral replication and serum RSV-PRNTs in AGMs when the interval between priming and boosting was ˜15 months (15 months minus seven days). (FIG. 17A) Study design. Four AGMs were previously administered a primary infection with the live-attenuated vaccine candidate RSV 276 (Table 10). Sera were collected on Day 429 (two weeks before boosting) and RSV-PRNTs were determined with complement. The AGMs were distributed into two groups of two animals each, such that the groups had similar individual and mean RSV-PRNTs based on the Day 429 sera (Table 10). On Day 443 (15 months minus seven days) post-priming, sera were collected and the two groups were boosted with DS-Cav1/B3TMCT vector (n=2) or RSV 276 (n=2) administered IN and IT at dose of 10⁶TCID₅₀(DS-Cav1/B3TMCT vector) or PFU (RSV 276) per site. Viral replication and serological responses were monitored as described in the legends to FIGS. 15 and 16. (FIG. 17B, FIG. 17C) Viral titers in the (FIG. 17B) NP and (FIG. 17C) TL, shown as means with limits of detection shown as dashed lines. Symbols indicate titers for individual animals, and the lines indicates mean values. (FIG. 17D) Serum RSV-PRNTs at Day 429 post-priming and Days 0, 7, 14, 21, and 28 post-boosting, assayed with complement. (FIG. 17E) Serum RSV-PRNTs at Days 0, 7, 14, 21, 28 post-boosting, assayed without complement. D is annotated to show the mean serum RSV-PRNT for the combined two groups at the time of boosting (black dashed line in D, with the mean arithmetic value 1:26 shown); in addition, D and E are annotated with dashed colored lines that indicate the highest mean serum RSV-PRNT for each group, with the arithmetic values shown.

FIGS. 18A-18C. Multicycle replication of rB/HPIV3 vectors in vitro in the presence or absence of RSV-neutralizing antibodies. LLC-MK2 cells were infected by empty rB/HPIV3 vector (FIG. 18A), DS-Cav1 vector (FIG. 18B), or DS-Cav1/B3TMCT vector (FIG. 18C) at an MOI of 0.01 TCID₅₀per cell. After adsorption for one h, cells were washed three times with cell culture medium and then incubated with culture medium containing 10% of one of the following sera (which had been previously heated at 56° C. for 30 min): pre-immune hamster serum, or pooled sera from hamsters infected with RSV D46 or with empty rB/HPIV3 vector, in the absence of added complement. The hamster sera were from the experiment in FIG. 14. The different treatments were performed in triplicate. An aliquot of medium was taken daily for three consecutive days after infection, flash-frozen, and viral titers were determined. The significance of difference between the replication in the presence of the pre-immune serum and RSV immune serum were determined by student-t tests: *, P<0.05; **, P<0.01.

FIG. 19. Replication of RSV 276 and wt rRSV in RSV-seronegative AGMs. RSV-seronegative AGMs were infected by the combined IN/IT routes with 10⁶PFU of RSV 276 (n=8) or wt rRSV (n=4) in a 1-mL inoculum per site. NP and TL were collected daily and every second day, respectively, for 10 days plus on Day 12. Viral titers were determined by immunoplaque assay and are shown as group means for each time point. Brackets indicate the SEM, and the limits of detection shown as dotted lines. The eight animals infected with RSV 276 here are ones shown in Tables 8 and 10 that subsequently were boosted in AGM experiments #1 and #3.

FIGS. 20A-20B. Replication of boosting RSV 276 in the tracheal lavage (TL) from AGMs primed with attenuated RSV ˜6 months earlier. From the experiment in FIG. 16, the titers of RSV 276 in TL on Days 2, 4, 6, 8 and 10 post-boosting were quantified by immunoplaque assay (FIG. 20A) and RT-qPCR specific for both positive- and negative-sense RSV M gene sequence (FIG. 20B). Each color represents an individual monkey. The limit of detection is indicated with a dotted line.

FIG. 21. Serum HPIV3-PRNTs induced by DS-Cav1 and DS-Cav1/B3TMCT vectors in AGMs primed with live-attenuated RSV 6 months earlier. From the experiment in FIG. 16, sera collected 2 weeks after boosting by DS-Cav1 and DS-Cav1/B3TMCT vectors were analyzed by HPIV3 PRNT assays without complement. The dots indicate individual animals; group means are indicated with short horizontal lines. The dotted line indicates the limit of detection. Mean serum HPIV3-PRNTs of two groups were compared by Student's t-test: ns indicates P>0.05, not significant.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file in the form of the file named “Sequence.txt” (˜356 kb), which was created on Jun. 4, 2021 which is incorporated by reference herein. In the accompanying sequence listing:

SEQ ID NO: 1 is the antigenomic nucleic acid sequence of RSV D46.

SEQ ID NO: 2 is the antigenomic nucleic acid sequence of RSV LID/ΔNS1.

SEQ ID NO: 3 is the antigenomic nucleic acid sequence of RSV LID/F1G2/ΔNS1.

SEQ ID NO: 4 is the antigenomic nucleic acid sequence of RSV LID/F1BBG2/ΔNS1.

SEQ ID NO: 5 is the antigenomic nucleic acid sequence of RSV LID/ΔNS1/1030s.

SEQ ID NO: 6 is the antigenomic nucleic acid sequence of RSV LID/F1G2/ΔNS1/1030s.

SEQ ID NO: 7 is the antigenomic nucleic acid sequence of RSV LID/F1BBG2/ΔNS1/1030s.

SEQ ID NO: 8 is the antigenomic nucleic acid sequence of RSV LID/ΔNS1/Δ1313/I1314L.

SEQ ID NO: 9 is the antigenomic nucleic acid sequence of RSV LID/F1G2/ΔNS1/Δ1313/I1314L.

SEQ ID NO: 10 is the antigenomic nucleic acid sequence of RSV LID/F1BBG2/ΔNS1/Δ1313/I1314L.

SEQ ID NO: 11 is the antigenomic nucleic acid sequence of RSV D46/NS2/N/ΔM2-2-HindIII.

SEQ ID NO: 12 is the antigenomic nucleic acid sequence of RSV 276 genome.

SEQ ID NO: 13 is the amin acid sequence of the L protein from D46.

SEQ ID NO: 14 is the amino acid sequence of the RSV F protein from D46.

SEQ ID NO: 15 is the amino acid sequence of RSV FBB protein.

SEQ ID NO: 16 is the antigenomic sequence of B/HPIV3 DS-Cav1.

SEQ ID NO: 17 is the antigenomic sequence of B/HPIV3 DS-Cav1/B3TMCT.

SEQ ID NO: 18 is the antigenomic sequence of RSV LID/ANS2/1030s.

SEQ ID NO: 19 is the antigenomic sequence of RSV LID/ANS2/Δ1313/I1314L.

SEQ ID NOs: 20-29 are fragments of DNA and protein sequences.

DETAILED DESCRIPTION

Major challenges to developing pediatric vaccines against RSV include the immaturity of the immune system during infancy, immune-suppression by maternal antibodies, inefficient immune protection at the superficial epithelium of the respiratory tract, viral suppression of host responses such as the suppression of host interferon and apoptosis responses by NS1 and NS2, and vaccine-induced enhanced disease that has been observed in studies with inactivated or subunit RSV vaccines in virus-naïve recipients (Kim et al., Amer. J. Epidemiol. 89:422-434, 1969; Ottolini et al., Viral Immunol. 13:231-236, 2000; Schneider-Ohrum et al., J. Virol. 91:e02180-16, 2017). Thus, despite substantial effort, a need remains for an effective immunogen that induces a protective immune response to RSV.

The present disclosure provides recombinant RSV that is attenuated, infectious, and self-replicating and that meets the above-discussed need.

I. SUMMARY OF TERMS

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes, 2008; and other similar references.

As used herein, the term “comprises” means “includes.” Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various embodiments, the following explanations of terms are provided:

About: Plus or minus 5% from a reference amount. For example, “about 5” refers to 4.75 to 5.25. A ratio of “about 5:1” refers to a ratio of from 4.75:1 to 5.25:1.

Adjuvant: A vehicle used to enhance antigenicity. Adjuvants include a suspension of minerals (alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; or water-in-oil emulsion, for example, in which antigen solution is emulsified in mineral oil (Freund incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or causes influx of macrophages). Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants. Adjuvants include biological molecules (a “biological adjuvant”), such as costimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL, immune stimulating complex (ISCOM) matrix, and toll-like receptor (TLR) agonists, such as TLR-9 agonists, Poly J:C, or PolyICLC. Adjuvants are described, for example, in Singh (ed.) Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007.

Administration: The introduction of a composition into a subject by a chosen route. Administration can be local or systemic. For example, if the chosen route is intranasal, the composition (such as a composition including a disclosed attenuated RSV) is administered by introducing the composition into the nasal passages of the subject. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes.

Amino acid substitution: The replacement of one amino acid in a polypeptide with a different amino acid.

Attenuated: A virus that is “attenuated” or has an “attenuated phenotype” refers to a virus that has decreased virulence compared to a reference virus under similar conditions of infection. Attenuation usually is associated with decreased virus replication as compared to replication of a reference wild-type virus under similar conditions of infection, and thus “attenuation” and “restricted replication” often are used synonymously. In some hosts (typically non-natural hosts, including experimental animals), disease is not evident during infection with a reference virus in question, and restriction of virus replication can be used as a surrogate marker for attenuation. In some embodiments, a disclosed attenuated RSV exhibits at least about 10-fold or greater decrease, such as at least about 100-fold or greater decrease in virus titer in the upper or lower respiratory tract of a mammal compared to non-attenuated, wild type virus titer in the upper or lower respiratory tract, respectively, of a mammal of the same species under the same conditions of infection. Examples of mammals include, but are not limited to, humans, mice, rabbits, rats, hamsters, such as for example Mesocricetus auratus, and non-human primates, such as for example Ceropithiecus aethiops. An attenuated RSV may display different phenotypes including without limitation altered growth, temperature sensitive growth, host range restricted growth, or plaque size alteration.

Control: A reference standard. In some embodiments, the control is a negative control sample obtained from a healthy patient. In other embodiments, the control is a positive control sample obtained from a patient diagnosed with RSV infection. In still other embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of RSV patients with known prognosis or outcome, or group of samples that represent baseline or normal values).

A difference between a test sample and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference. In some examples, a difference is an increase or decrease, relative to a control, of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or greater than 500%.

Effective amount: An amount of agent that is sufficient to elicit a desired response, such as an immune response in a subject. It is understood that to obtain a protective immune response against a virus of interest can require multiple administrations of a disclosed immunogen, and/or administration of a disclosed immunogen as the “prime” in a prime boost protocol wherein the boost immunogen can be different from the prime immunogen. Accordingly, an effective amount of a disclosed immunogen can be the amount of the immunogen sufficient to elicit a priming immune response in a subject that can be subsequently boosted with the same or a different immunogen to elicit a protective immune response.

In one example, a desired response is to inhibit or reduce or prevent RSV infection. The RSV infection does not need to be completely eliminated or reduced or prevented for the method to be effective. For example, administration of an effective amount of the agent can decrease the RSV infection (for example, as measured by infection of cells, or by number or percentage of subjects infected by RSV) by a desired amount, for example by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable RSV infection), as compared to a suitable control.

Gene: A nucleic acid sequence that comprises control and coding sequences necessary for the transcription of an RNA, whether an mRNA or otherwise. For instance, a gene may comprise a promoter, one or more enhancers or silencers, a nucleic acid sequence that encodes a RNA and/or a polypeptide, downstream regulatory sequences and, possibly, other nucleic acid sequences involved in regulation of the expression of an mRNA.

A “gene” of a recombinant RSV as described herein refers to a portion of the recombinant RSV encoding an mRNA and typically begins at the upstream (3′) end with a gene-start (GS) signal and ends at the downstream (5′) end with the gene-end (GE) signal. In this context, the term gene also embraces what is referred to as a “translational open reading frame”, or ORF, particularly in the case where a protein is expressed from an additional ORF rather than from a unique mRNA. To construct a disclosed recombinant RSV, one or more genes or genome segments may be deleted, inserted or substituted in whole or in part.

Heterologous: Originating from a different genetic source. A heterologous gene included in a recombinant genome is a gene that does not originate from that genome.

Host cells: Cells in which a vector can be propagated and its nucleic acid expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term “host cell” is used.

Infectious and Self-Replicating Virus: A virus that is capable of entering and replicating in a cultured cell or cell of an animal or human host to produce progeny virus capable of the same activity.

Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an “antigen-specific response”). In one embodiment, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response, and results in the production of specific antibodies.

Immunogenic composition: A preparation of immunogenic material capable of stimulating an immune response, which in some examples can be administered for the prevention, amelioration, or treatment of infectious or other types of disease. The immunogenic material may include attenuated or killed microorganisms (such as bacteria or viruses), or antigenic proteins, peptides or DNA derived from them. Immunogenic compositions comprise an antigen (such as a virus) that induces a measurable T cell response against the antigen, or induces a measurable B cell response (such as production of antibodies) against the antigen. In one example, an immunogenic composition comprises a disclosed recombinant RSV that induces a measurable CTL response and/or a measurable B cell response (such as production of antibodies) against RSV when administered to a subject. For in vivo use, the immunogenic composition will typically include a recombinant virus in a pharmaceutically acceptable carrier and may also include other agents, such as an adjuvant.

Isolated: An “isolated” biological component has been substantially separated or purified away from other biological components, such as other biological components in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA, RNA, and proteins. Proteins, peptides, nucleic acids, and viruses that have been “isolated” include those purified by standard purification methods. Isolated does not require absolute purity, and can include protein, peptide, nucleic acid, or virus molecules that are at least 50% pure, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% pure.

Nucleic acid molecule: A polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. The term “nucleic acid molecule” as used herein is synonymous with “polynucleotide.” A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. The term includes single- and double-stranded forms of DNA. A nucleic acid molecule may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked nucleic acid sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed immunogens.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In particular embodiments, suitable for administration to a subject the carrier may be sterile, and/or suspended or otherwise contained in a unit dosage form containing one or more measured doses of the composition suitable to induce the desired immune response. It may also be accompanied by medications for its use for treatment purposes. The unit dosage form may be, for example, in a sealed vial that contains sterile contents or a syringe for injection into a subject, or lyophilized for subsequent solubilization and administration or in a solid or controlled release dosage.

Polypeptide: Any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). “Polypeptide” applies to amino acid polymers including naturally occurring amino acid polymers and non-naturally occurring amino acid polymer as well as in which one or more amino acid residue is a non-natural amino acid, for example an artificial chemical mimetic of a corresponding naturally occurring amino acid. A “residue” refers to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic. A polypeptide has an amino terminal (N-terminal) end and a carboxy terminal (C-terminal) end. “Polypeptide” is used interchangeably with peptide or protein, and is used herein to refer to a polymer of amino acid residues.

Prime-boost immunization: An immunization protocol including administration of a first immunogenic composition (the prime immunization) followed by administration of a second immunogenic composition (the boost immunization) to a subject to induce a desired immune response. A suitable time interval between administration of the prime and the boost, and examples of such timeframes are disclosed herein. In some embodiments, the prime, the boost, or both the prime and the boost additionally include an adjuvant.

Recombinant: A recombinant nucleic acid molecule or protein or virus is one that has been produced by recombinant DNA methods, typically from cloned cDNA(s). The cDNA sequence(s) may be identical to that of a biologically-derived molecule(s), or may contain a sequence(s) that is not naturally-occurring: for example, includes one or more nucleic acid substitutions, deletions or insertions, and/or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished, for example, by chemical synthesis, targeted mutation of a naturally occurring nucleic acid molecule or protein, or, artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.

Respiratory Syncytial Virus (RSV): An enveloped non-segmented negative-sense single-stranded RNA virus of the family Pneumoviridae, genus Orthopneumovirus. The RSV genome is ˜15,000 nucleotides in length (15,223 for KT 992094) and includes 10 genes encoding 11 proteins, including the glycoproteins SH, G and F. The F protein mediates fusion, allowing entry of the virus into the cell cytoplasm and also promoting the formation of syncytia. Two antigenic subgroups of human RSV strains have been described, the A and B subgroups, based primarily on differences in the antigenicity of the G glycoprotein. RSV strains for other species are also known, including bovine RSV. Several animal models of infection by human RSV and closely-related animal counterparts are available, including model organisms infected with human RSV, as well as model organisms infected with species-specific RSV, such as use of bRSV infection in cattle (see, e.g., Bern et al., Am J, Physiol. Lung Cell Mol. Physiol., 301: L148-L156, 2011; and Nam and Kun (Eds.). Respiratory Syncytial Virus: Prevention, Diagnosis and Treatment. Nova Biomedical Nova Science Publisher, 2011; and Cane (Ed.) Respiratory Syncytial Virus. Elsevier Science, 2007.)

Unless context indicates otherwise, the positioning of RSV nucleic acid residues is according to the reference RSV antigenomic sequence provided herein as SEQ ID NO: 1, which is an RSV strain A2 antigenomic sequence, also provided as Genbank accession number KT 992094.1 (incorporated by reference herein), and is described in Collins, et al., Proc Natl Acad Sci USA, 92:11563-11567 1995. This sequence is also known as the “D46” antigenomic RSV sequence.

Sequence identity: The percentage of nucleotide or amino acid sequence assignments that are identical between two or more compared nucleotide or amino acid sequences, with gaps permitted to maximize the percent identity. The higher the percentage, the more similar the two sequences are. Homologs, orthologs, or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. In the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

Variants of a polypeptide are typically characterized by possession of at least about 75%, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full length alignment with the amino acid sequence of interest. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet.

As used herein, reference to “at least 90% identity” (or similar language) refers to “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence.

Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals. In an example, a subject is a human. In a particular example, the subject is a newborn infant. In an additional example, a subject is selected that is in need of inhibiting an RSV infection. For example, the subject is either uninfected and at risk of RSV infection or is infected in need of treatment.

Under conditions sufficient for: A phrase that is used to describe any environment that permits a desired activity.

II. RECOMBINANT RSV

Disclosed herein are mutations that are useful in producing recombinant strains of human RSV exhibiting a range of attenuation phenotypes and are suitable for use as attenuated, live vaccines in humans. As reported herein, particular combinations of mutations to wt RSV result in a live, attenuated virus that elicits a superior immune response.

Further disclosed herein are methods and compositions related to the expression of the disclosed viruses. For example, isolated polynucleotide molecules that include a nucleic acid sequence encoding the genome or antigenome of the described viruses are disclosed.

The recombinant RSV provided herein comprises a genome or antigenome containing modifications or mutations as described in detail herein relative to wild-type RSV that attenuate the recombinant RSV. The wild-type RSV genome or antigenome encodes the following 11 proteins: the RNA-binding nucleoprotein (N), the phosphoprotein (P), the large polymerase protein (L), the attachment surface glycoprotein (G), the fusion surface glycoprotein (F), the small hydrophobic surface glycoprotein (SH), the internal matrix protein (M), the two nonstructural proteins NS1 and NS2, and the M2-1 and M2-2 proteins. The genome of RSV is a single strand of negative sense RNA of about 15.2 kb comprising 10 genes encoding 10 mRNAs. Each mRNA encodes a single protein, except for the M2 mRNA which encodes two separate proteins M2-1 and M2-2. The RSV gene order is: 3′-NS1-NS2-N-P-M-SH-G-F-M2-L with a single viral promoter located at the 3′end. Thus, in the native RSV genome NS1 is at position 1, NS2 at position 2, N at position 3, P at position 4, M at position 5, SH at position 6, G at position 7, F at position 8, M2 at position 9 and L at position 10. This organization is shown schematically in FIG. 1C.

In several embodiments, the recombinant RSV provided herein comprises a genome with RSV F, G, NS2, N, P, M, SH, M2, and L genes located at gene positions 1-9, respectively, wherein the F and G genes located at gene positions 1 and 2 are shifted from the native gene positions 8 and 7, respectively. The genome of the recombinant RSV further comprises a modification that deletes the sequence encoding the NS1 protein. In some embodiments, the deletion comprises a deletion of positions 99-626 corresponding to the reference RSV sequence set forth as SEQ ID NO: 1 (wt recombinant RSV strain A2, Genbank KT 992094). In several embodiments, the genome of the recombinant RSV provided herein further comprises a deletion of 112 nucleotides corresponding to positions 4499-4610 inclusive corresponding to the reference RSV sequence set forth as SEQ ID NO: 1, and C4489T, C4492T, A4495T, A4497G, and G4498A of SEQ ID NO: 1. This deletion and the five nucleotide substitution are collectively called the “LID” mutation or modification. As discussed in the examples below, the novel combination of these modifications to the native RSV genome results in a recombinant RSV that elicits a superior immune response.

As used herein, and unless contect indicates otherwise, virus names are descriptive rather than limiting. The full set of modifications or mutations in each virus is not necessarily listed fully in the name. Additionally, unless context indicates otherwise, the order of appearance of modifications/mutations and forward slashes in a virus name can vary: for example, “LID/F1G2/ΔNS1” is the same as “LID/ΔNS1/F1G1.”

In several embodiments, the recombinant RSV provided herein comprises a genome comprising a deletion of the sequence encoding NS1 protein, wherein the deletion is a deletion of of positions 99-627 corresponding to the reference RSV sequence set forth as SEQ ID NO: 1, and wherein the recombinant RSV is infectious, attenuated, and self-replicating. In several such embodiments, the genome of the recombinant RSV provided herein further comprises a deletion of 112 nucleotides corresponding to positions 4499-4610 inclusive corresponding to the reference RSV sequence set forth as SEQ ID NO: 1, and C4489T, C4492T, A4495T, A4497G, and G4498A of SEQ ID NO: 1. As discussed in the examples below, the novel combination of these modifications to the native RSV genome results in a recombinant RSV that elicits a superior immune response.

In some embodiments, the recombinant RSV further comprises an L protein comprising a S1313 residue encoded by a TCA codon and a Y1314K substitution encoded by a AAA codon, wherein the amino acid positions correspond to the reference L protein sequence set forth as SEQ ID NO: 13 (amino acid sequence of the L protein). This pair of mutations is called “1030s” and was developed and optimized by reverse genetics to be highly refractory to de-attenuation (Luongo, et al. 2012. J Virol 86:10792-10804).

In some embodiments, the recombinant RSV further comprises an L protein comprising a deletion of S1313 and an I1314L substitution, wherein the amino acid positions correspond to the reference L protein sequence set forth as SEQ ID NO: 13. This pair of mutations is called “Δ1313/I1314L” and was developed and optimized by reverse genetics to be highly refractory to de-attenuation (Luongo, et al. 2013. J Virol 87:1985-1996).

In some embodiments, the F protein of the recombinant RSV is encoded by a wild-type sequence, such as SEQ ID NO: 14. In other embodiments, the recombinant RSV may comprise one or more changes in the F protein sequence. For example, in some embodiments, a native or naturally occurring nucleotide sequence encoding a protein of the RSV may be replaced with a codon optimized sequence designed for increased expression in a selected host, in particular the human. For example, in some embodiments, the F protein of the recombinant RSV is encoded by the codon optimized sequence FBB (“FBB”) (SEQ ID NO: 15). Different versions of codon optimization can be obtained.

In several embodiments, the genome of the recombinant RSV does not comprise any heterologous genes.

In some embodiments, the genome of the recombinant RSV comprises the one or more mutations as discussed above, and a nucleic acid sequence complementary to an antigenomic sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 1 (D46 sequence). In some embodiments, the genome of the recombinant RSV is a D46 genome modified with the one or more mutations as discussed above. In several embodiments, the genome of the recombinant RSV comprises the one or more mutations as discussed herein, and any remaining sequence difference of the genome of the recombinant RSV compared to the genomic sequence of D46 RSV (SEQ ID NO: 1) is biologically insignificant (for example, the remaining sequence differences do not include changes to the wild-type genomic sequence that modify a known cis-acting signal or change amino acid coding, or measurably affect in vitro replication or plaque size of the virus).

The embodiments of recombinant RSV disclosed herein can be subtype A RSV or a subtype B RSV. The embodiments of recombinant RSV disclosed herein are infectious, attenuated, and self-replicating.

In some embodiments, the recombinant RSV comprises a RSV genome comprising RSV NS2, N, P, M, SH, G, F M2, and L genes located at gene positions 1-9, respectively, wherein the genome comprises a deletion of the sequence encoding NS1 protein. In several such embodiments, the SH gene of the recombinant RSV comprises a deletion of positions 4499-4610 corresponding to the reference RSV sequence set forth as SEQ ID NO: 1, and C4489T, C4492T, A4495T, A4497G, and G4498A substitutions corresponding to the reference RSV sequence set forth as SEQ ID NO: 1. In some such embodiments, the genome of the recombinant RSV comprises a nucleic acid sequence complementary to a positive-sense anitgenomic sequence at least 90%, at least 95%, at least 98%, and/or at least 99% identical to SEQ ID NO: 2 (RSV LID/ΔNS1). In some such embodiments, the genome of the recombinant RSV comprises a nucleic acid sequence complementary to the positive-sense anitgenomic sequence set forth as SEQ ID NO: 2 (RSV LID/ΔNS1).

In some embodiments, the recombinant RSV comprises a RSV genome comprising RSV F, G, NS2, N, P, M, SH, M2, and L genes located at gene positions 1-9, respectively, wherein the F and G genes located at gene positions 1 and 2 are shifted from the native gene positions 8 and 7, respectively, and the genome comprises a deletion of the sequence encoding NS1 protein. In several such embodiments, the SH gene of the recombinant RSV comprises a deletion of positions 4499-4610 corresponding to the reference RSV sequence set forth as SEQ ID NO: 1, and C4489T, C4492T, A4495T, A4497G, and G4498A substitutions corresponding to the reference RSV sequence set forth as SEQ ID NO: 1. In some such embodiments, the genome of the recombinant RSV comprises a nucleic acid sequence complementary to a positive-sense anitgenomic sequence at least 90%, at least 95%, at least 98%, and/or at least 99% identical to SEQ ID NO: 3 (RSV LID/F1G2/ΔNS1). In some such embodiments, the genome of the recombinant RSV comprises a nucleic acid sequence complementary to the positive-sense anitgenomic sequence set forth as SEQ ID NO: 3 (RSV LID/F1G2/ΔNS1).

In some embodiments, the recombinant RSV comprises a RSV genome comprising RSV F, G, NS2, N, P, M, SH, M2, and L genes located at gene positions 1-9, respectively, wherein the F and G genes located at gene positions 1 and 2 are shifted from the native gene positions 8 and 7, respectively, and the L gene encodes an L protein comprising a S1313 residue encoded by an TCA codon and a Y1314K substitution encoded by a AAA codon, wherein the amino acid positions correspond to the reference L protein sequence set forth as SEQ ID NO: 13. In several such embodiments, the SH gene of the recombinant RSV comprises a deletion of positions 4499-4610 corresponding to the reference RSV sequence set forth as SEQ ID NO: 1, and C4489T, C4492T, A4495T, A4497G, and G4498A substitutions corresponding to the reference RSV sequence set forth as SEQ ID NO: 1. In some such embodiments, the genome of the recombinant RSV comprises a nucleic acid sequence complementary to a positive-sense anitgenomic sequence at least 90%, at least 95%, at least 98%, and/or at least 99% identical to SEQ ID NO: 6 (RSV LID/F1G2/ΔNS1/1030s). In some such embodiments, the genome of the recombinant RSV comprises a nucleic acid sequence complementary to the positive-sense anitgenomic sequence set forth as SEQ ID NO: 6 (RSV LID/F1G2/ΔNS1/1030s).

In several such embodiments, the SH gene of the recombinant RSV comprises a deletion of positions 4499-4610 corresponding to the reference RSV sequence set forth as SEQ ID NO: 1, and C4489T, C4492T, A4495T, A4497G, and G4498A substitutions corresponding to the reference RSV sequence set forth as SEQ ID NO: 1. In some such embodiments, the genome of the recombinant RSV comprises a nucleic acid sequence complementary to a positive-sense anitgenomic sequence at least 90%, at least 95%, at least 98%, and/or at least 99% identical to SEQ ID NO: 9 (RSV LID/F1G2/ΔNS1/Δ1313/I1314L). In some such embodiments, the genome of the recombinant RSV comprises a nucleic acid sequence complementary to the positive-sense anitgenomic sequence set forth as SEQ ID NO: 9 (RSV LID/F1G2/ΔNS1/Δ1313/I1314L).

In some embodiments, the recombinant RSV comprises a RSV genome comprising RSV F, G, NS2, N, P, M, SH, M2, and L genes located at gene positions 1-9, respectively, wherein the F and G genes located at gene positions 1 and 2 are shifted from the native gene positions 8 and 7, respectively, the F protein is encoded by the sequence set forth as SEQ ID NO: 15 (FBB), and the genome comprises a deletion of the sequence encoding NS1 protein. In several such embodiments, the SH gene of the recombinant RSV comprises a deletion of positions 4499-4610 corresponding to the reference RSV sequence set forth as SEQ ID NO: 1, and C4489T, C4492T, A4495T, A4497G, and G4498A substitutions corresponding to the reference RSV sequence set forth as SEQ ID NO: 1. In some such embodiments, the genome of the recombinant RSV comprises a nucleic acid sequence complementary to a positive-sense anitgenomic sequence at least 90%, at least 95%, at least 98%, and/or at least 99% identical to SEQ ID NO: 4 (RSV LID/F1BBG2/ΔNS1). In some such embodiments, the genome of the recombinant RSV comprises a nucleic acid sequence complementary to the positive-sense anitgenomic sequence set forth as SEQ ID NO: 4 (RSV LID/F1BBG2/ΔNS1).

In some embodiments, the recombinant RSV comprises a RSV genome comprising RSV F, G, NS2, N, P, M, SH, M2, and L genes located at gene positions 1-9, respectively, wherein the F and G genes located at gene positions 1 and 2 are shifted from the native gene positions 8 and 7, respectively, the F protein is encoded by the sequence set forth as SEQ ID NO: 15 (FBB), the genome comprises a deletion of the sequence encoding NS1 protein, and the L gene encodes an L protein comprising a S1313 residue encoded by an TCA codon and a Y1314K substitution encoded by a AAA codon, wherein the amino acid positions correspond to the reference L protein sequence set forth as SEQ ID NO: 13. In several such embodiments, the SH gene of the recombinant RSV comprises a deletion of positions 4499-4610 corresponding to the reference RSV sequence set forth as SEQ ID NO: 1, and C4489T, C4492T, A4495T, A4497G, and G4498A substitutions corresponding to the reference RSV sequence set forth as SEQ ID NO: 1. In some such embodiments, the genome of the recombinant RSV comprises a nucleic acid sequence complementary to a positive-sense anitgenomic sequence at least 90%, at least 95%, at least 98%, and/or at least 99% identical to SEQ ID NO: 7 (RSV LID/F1BBG2/ΔNS1/1030s). In some such embodiments, the genome of the recombinant RSV comprises a nucleic acid sequence complementary to the positive-sense anitgenomic sequence set forth as SEQ ID NO: 7 (RSV LID/F1BBG2/ΔNS1/1030s).

In some embodiments, the recombinant RSV comprises a RSV genome comprising RSV F, G, NS2, N, P, M, SH, M2, and L genes located at gene positions 1-9, respectively, wherein the F and G genes located at gene positions 1 and 2 are shifted from the native gene positions 8 and 7, respectively, and wherein the genome comprises a deletion of the sequence encoding NS1 protein, and wherein the L gene encodes an L protein comprising a deletion of S1313 and an I1314L substitution, wherein the amino acid positions correspond to the reference L protein sequence set forth as SEQ ID NO: 13. In several such embodiments, the SH gene of the recombinant RSV comprises a deletion of positions 4499-4610 corresponding to the reference RSV sequence set forth as SEQ ID NO: 1, and C4489T, C4492T, A4495T, A4497G, and G4498A substitutions corresponding to the reference RSV sequence set forth as SEQ ID NO: 1. In some such embodiments, the genome of the recombinant RSV comprises a nucleic acid sequence complementary to a positive-sense anitgenomic sequence at least 90%, at least 95%, at least 98%, and/or at least 99% identical to SEQ ID NO: 10 (RSV LID/F1BBG2/ΔNS1/Δ1313/I1314L). In some such embodiments, the genome of the recombinant RSV comprises a nucleic acid sequence complementary to the positive-sense anitgenomic sequence set forth as SEQ ID NO: 10 (RSV LID/F1BBG2/ΔNS1/Δ1313/I1314L).

In some embodiments, the recombinant RSV comprises a deletion of the non-translated sequences in genes, in the intergenic regions, and in the trailer region, in addition to the modifications noted above.

Viruses are named herein by listing the combination of mutations present in them and are descriptive rather than limiting. The use of the symbol “/” in a virus name (as in RSV LID/ΔNS1/1030s which denotes RSV D46 comprising the mutations ΔNS1, LID, and 1030s) has no significance apart from being present to make the name easier to read, particularly when present in text. Hence, RSV LID/ΔNS1/1030s is the same as RSV LID/ΔNS1/1030s and RSV LID/ΔNS1/1030s. RSV is not always used in a name. The names of antigenomic cDNAs and their encoded RSVs typically are interchangeable.

Unless context indicates otherwise, the numbering used in this disclosure is based on the sequence of the RSV A2 strain D46, provided herein as SEQ ID NO: 1 and viral genomic sequences described are in positive-sense. With regard to sequence numbering of nucleotide and amino acid sequence positions for the described viruses, a convention was used whereby each nucleotide or amino acid residue in a given viral sequence retained the sequence position number that it has in the RSV D46 strain provided as SEQ ID NO: 1, irrespective of any modifications. Thus, although a number of genomes contain deletions and/or insertions that cause changes in nucleotide length, and in some cases amino acid length, the numbering of all of the other residues (nucleotide or amino acid) in the genome and encoded proteins remains unchanged. It also is recognized that, even without the expedient of this convention, one skilled in the art can readily identify corresponding sequence positions between viral genomes or proteins that might differ in length, guided by sequence alignments as well as the positions of open reading frames, well-known RNA features such as gene-start and gene-end signals, and amino acid sequence features.

The Examples utilize RSV strain A2 of antigenic subgroup A, which is the most widely used experimental strain and also is the parent of numerous live attenuated RSV vaccine candidates that have been evaluated in clinical studies. Given that a variety of additional RSV strains exist (e.g., RSV B1, RSV Long, RSV Line 19), those skilled in the art will appreciate that certain strains of RSV may have nucleotide or amino acid insertions or deletions that alter the position of a given residue. For example, if a protein of another RSV strain had, in comparison with strain A2, two additional amino acids in the upstream end of the protein, this would cause the amino acid numbering of downstream residues relative to strain A2 to increase by an increment of two. However, because these strains share a large degree of sequence identity, those skilled in the art would be able to determine the location of corresponding sequences by simply aligning the nucleotide or amino acid sequence of the A2 reference strain with that of the strain in question. Therefore, it should be understood that the amino acid and nucleotide positions described herein, though specifically enumerated in the context of this disclosure, can correspond to other positions when a sequence shift has occurred or due to sequence variation between virus strains. In the comparison of a protein, or protein segment, or gene, or genome, or genome segment between two or more related viruses, a “corresponding” amino acid or nucleotide residue is one that is thought to be exactly or approximately equivalent in function in the different species.

In addition to the above described modifications to recombinant RSV, different or additional modifications in RSV clones can be made to facilitate manipulations, such as the insertion of unique restriction sites in various intergenic regions or elsewhere. Nontranslated gene sequences can be removed to increase capacity for inserting foreign sequences.

Introduction of the foregoing, defined mutations into an infectious RSV clone can be achieved by a variety of well-known methods. By “infectious clone” is meant cDNA or its product, synthetic or otherwise, which can be transcribed into genomic or antigenomic RNA capable of producing an infectious virus. The term “infectious” refers to a virus or viral structure that is capable of replicating in a cultured cell or animal or human host to produce progeny virus or viral structures capable of the same activity. Thus, defined mutations can be introduced by conventional techniques (e.g., site-directed mutagenesis) into a cDNA copy of the genome or antigenome. The use of antigenome or genome cDNA subfragments to assemble a complete antigenome or genome cDNA is well-known by those of ordinary skill in the art and has the advantage that each region can be manipulated separately (smaller cDNAs are easier to manipulate than large ones) and then readily assembled into a complete cDNA. Thus, the complete antigenome or genome cDNA, or any subfragment thereof, can be used as template for oligonucleotide-directed mutagenesis. A mutated subfragment can then be assembled into the complete antigenome or genome cDNA. Mutations can vary from single nucleotide changes to replacement of large cDNA pieces containing one or more genes or genome regions.

In some embodiments, the disclosed recombinant RSV can be produced using a recombinant DNA-based technique called reverse genetics (Collins, et al. 1995. Proc Natl Acad Sci USA 92:11563-11567). This system allows de novo recovery of infectious virus entirely from cDNA in a qualified cell substrate under defined conditions. Reverse genetics provides a means to introduce predetermined mutations into the RSV genome via the cDNA intermediate. Specific attenuating mutations were characterized in preclinical studies and combined to achieve the desired level of attenuation. Derivation of vaccine viruses from cDNA minimizes the risk of contamination with adventitious agents and helps to keep the passage history brief and well documented. Once recovered, the engineered virus strains propagate in the same manner as a biologically derived virus. As a result of passage and amplification, the vaccine viruses do not contain recombinant DNA from the original recovery.

Recombinant RSV may be produced by the intracellular coexpression of a cDNA that encodes the RSV genomic RNA, together with those viral proteins necessary to generate a transcribing, replicating nucleocapsid. Plasmids encoding other RSV proteins may also be included with these essential proteins. Alternatively, RNA may be synthesized in in vitro transcription reactions and transfected into cultured cells.

To propagate a RSV virus for vaccine use and other purposes, a number of cell lines which allow for RSV growth may be used. RSV grows in a variety of human and animal cells. Preferred cell lines for propagating attenuated RS virus for vaccine use include DBSFRhL-2, MRC-5, and Vero cells. Highest virus yields are usually achieved with epithelial cell lines such as Vero cells. Cells are typically inoculated with virus at a multiplicity of infection ranging from about 0.001 to 1.0, or more, and are cultivated under conditions permissive for replication of the virus, e.g., at about 30-37° C. and for about 3-10 days, or as long as necessary for virus to reach an adequate titer. Temperature-sensitive viruses often are grown using 32° C. as the “permissive temperature.” Virus is removed from cell culture and separated from cellular components, typically by well-known clarification procedures, e.g., centrifugation, and may be further purified as desired using procedures well known to those skilled in the art.

RSV which has been attenuated as described herein can be tested in various well known and generally accepted in vitro and in vivo models to confirm adequate attenuation, resistance to phenotypic reversion, and immunogenicity for vaccine use. In in vitro assays, the modified virus, which can be a multiply attenuated, biologically derived or recombinant RSV, is tested for temperature sensitivity of virus replication or “ts phenotype,” and for the small plaque phenotype. Modified virus also may be evaluated in an in vitro human airway epithelium (HAE) model, which appears to provide a means of ranking viruses in the order of their relative attenuation in non-human primates and humans (Zhang et al 2002 J Virol 76:5654-5666; Schaap-Nutt et al 2010 Vaccine 28:2788-2798; Ilyushina et al 2012 J Virol 86:11725-11734). Modified viruses are further tested in animal models of RSV infection. A variety of animal models (e.g., murine, cotton rat, and primate) have been described and are known to those skilled in the art.

Recombinant viruses may be evaluated in cell culture, rodents and non-human primates for infectivity, replication kinetics, yield, efficiency of protein expression, and genetic stability. While these semi-permissive systems may not reliably detect every difference in replication, substantial differences in particular may be detected. Also recombinant strains may be evaluated successively in adults, seropositive children, and seronegative children. In some cases, where a previous similar strain has already been shown to be well-tolerated in seronegative children, a new strain may be evaluated directly in seronegative children. Evaluation may be done, for example, in groups of 10 vaccine recipients and 5 placebo recipients, which is a small number that allows simultaneous evaluation of multiple candidates. Candidates may be evaluated in the period immediately post-immunization for vaccine virus infectivity, replication kinetics, shedding, tolerability, immunogenicity, and genetic stability, and the vaccinees may be subjected to surveillance during the following RSV season for safety, RSV disease, and changes in RSV-specific serum antibodies, as described in Karron, et al. 2015, Science Transl Med 2015 7(312):312ra175, which is incorporated herein in its entirety. Thus, analysis of selected representative viruses may provide for relatively rapid triage to narrow down candidates to identify the most optimal.

Also provided herein are isolated polynucleotides that encode the described mutated viruses, make up the described genomes or antigenomes, express the described genomes or antigenomes, or encode various proteins useful for making recombinant RSV in vitro. The nucleic acid sequences of a number of exemplary polynucleotides are also provided. Included within the embodiments provided herein are polynucleotides comprising sequences that consist or consist essentially of any of the aforementioned nucleic acid sequences. Further included are polynucleotides that possess at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent or more identity, or any number in between, to any of the aforementioned sequences or SEQ ID NOs provided herein, as well as polynucleotides that hybridize to, or are the complements of the aforementioned molecules.

These polynucleotides can be included within or expressed by vectors in order to produce a recombinant RSV. Accordingly, cells transfected with the isolated polynucleotides or vectors are also included.

In additional embodiments, compositions (e.g., isolated polynucleotides and vectors incorporating an RSV-encoding cDNA) and methods are provided for producing a recombinant RSV. Also provided are novel, isolated polynucleotide molecules and vectors incorporating such molecules that comprise a RSV genome or antigenome which is modified as described herein. Also provided is the same or different expression vector comprising one or more isolated polynucleotide molecules encoding the RSV proteins. These proteins also can be expressed directly from the genome or antigenome cDNA. The vector(s) are preferably expressed or coexpressed in a cell or cell-free lysate, thereby producing an infectious mutant RSV particle or subviral particle.

Also provided is a method for producing one or more purified RSV protein(s) which involves infecting a host cell permissive of RSV infection with a recombinant RSV strain under conditions that allow for RSV propagation in the infected cell. After a period of replication in culture, the cells are lysed and recombinant RSV is isolated therefrom. One or more desired RSV protein(s) is purified after isolation of the virus, yielding one or more RSV protein(s) for vaccine, diagnostic and other uses.

The above methods and compositions yield infectious viral or subviral particles, or derivatives thereof. An infectious virus is comparable to the authentic RSV virus particle and is infectious as is. It can directly infect fresh cells. An infectious subviral particle typically is a subcomponent of the virus particle which can initiate an infection under appropriate conditions. For example, a nucleocapsid containing the genomic or antigenomic RNA and the N, P, L and M2-1 proteins is an example of a subviral particle which can initiate an infection if introduced into the cytoplasm of cells. Subviral particles include viral particles which lack one or more protein(s), protein segment(s), or other viral component(s) not essential for infectivity.

In other embodiments the invention provides a cell or cell free lysate containing an expression vector which comprises an isolated polynucleotide molecule encoding mutant RSV genome or antigenome as described above, and an expression vector (the same or different vector) which comprises one or more isolated polynucleotide molecules encoding the N, P, L and RNA polymerase elongation factor proteins of RSV. One or more of these proteins also can be expressed from the genome or antigenome cDNA. Upon expression the genome or antigenome and N, P, L, and RNA polymerase elongation factor proteins combine to produce an infectious RSV viral or sub-viral particle.

III. IMMUNOGENIC COMPOSITIONS

Immunogenic compositions comprising a disclosed recombinant RSV and a pharmaceutically acceptable carrier are also provided. Such compositions can be administered to a subject by a variety of modes, for example, by an intranasal route. Standard methods for preparing administrable immunogenic compositions are described, for example, in such publications as Remingtons Pharmaceutical Sciences, 19^thEd., Mack Publishing Company, Easton, Pa., 1995.

Potential carriers include, but are not limited to, physiologically balanced culture medium, phosphate buffer saline solution, water, emulsions (e.g., oil/water or water/oil emulsions), various types of wetting agents, cryoprotective additives or stabilizers such as proteins, peptides or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., sodium glutamate), or other protective agents. The resulting aqueous solutions may be packaged for use as is or lyophilized. Lyophilized preparations are combined with a sterile solution prior to administration for either single or multiple dosing.

The immunogenic composition can contain a bacteriostat to prevent or minimize degradation during storage, including but not limited to effective concentrations (usually ≤1% w/v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and/or propylparaben. A bacteriostat may be contraindicated for some patients; therefore, a lyophilized formulation may be reconstituted in a solution either containing or not containing such a component.

The immunogenic composition can contain as pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.

The immunogenic composition may optionally include an adjuvant to enhance the immune response of the host. Suitable adjuvants are, for example, toll-like receptor agonists, alum, AlPO4, alhydrogel, Lipid-A and derivatives or variants thereof, oil-emulsions, saponins, neutral liposomes, liposomes containing the recombinant virus, and cytokines, non-ionic block copolymers, and chemokines. Non-ionic block polymers containing polyoxyethylene (POE) and polyxylpropylene (POP), such as POE-POP-POE block copolymers, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.), among many other suitable adjuvants well known in the art, may be used as an adjuvant (Newman et al., 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142). These adjuvants have the advantage in that they help to stimulate the immune system in a non-specific way, thus enhancing the immune response to a pharmaceutical product.

In some instances it may be desirable to combine the immunogenic composition including the recombinant RSV, with other pharmaceutical products (e.g., vaccines) which induce protective responses to other viral agents, particularly those causing other childhood illnesses. For example, a composition including a recombinant RSV as described herein can also include other vaccines recommended by the Advisory Committee on Immunization Practices (ACIP; cdc.gov/vaccines/acip/index.html) for the targeted age group (e.g., infants from approximately one to six months of age). These additional vaccines include, but are not limited to, IN-administered vaccines. As such, a recombinant RSV as described herein may be administered simultaneously with vaccines against, for example, hepatitis B (HepB), diphtheria, tetanus and pertussis (DTaP), pneumococcal bacteria (PCV), Haemophilus influenzae type b (Hib), polio, influenza and rotavirus.

In some embodiments, the immunogenic composition can be provided in unit dosage form for use to induce an immune response in a subject, for example, to prevent RSV infection in the subject. A unit dosage form contains a suitable single preselected dosage for administration to a subject, or suitable marked or measured multiples of two or more preselected unit dosages, and/or a metering mechanism for administering the unit dose or multiples thereof.

IV. METHODS OF ELICITING AN IMMUNE RESPONSE

Provided herein are methods of eliciting an immune response in a subject by administering an immunogenic composition containing a disclosed recombinant RSV to the subject. Upon immunization, the subject responds by producing antibodies specific for RSV. In addition, innate and cell-mediated immune responses are induced, which can provide antiviral effectors as well as regulating the immune response. As a result of the immunization the host becomes at least partially or completely immune to RSV infection, or resistant to developing moderate or severe RSV disease, particularly of the lower respiratory tract.

In additional embodiments, a method of eliciting an immune response in a subject by administering to the subject an immunogenic composition containing a recombinant RSV comprising a genome comprising a deletion of the sequence encoding NS1 protein (such as a deletion of of positions 99-627 corresponding to the reference RSV sequence set forth as SEQ ID NO: 1), and wherein the recombinant RSV is infectious, attenuated, and self-replicating. In some such embodiments, the genome of the recombinant RSV comprises a SH gene comprising a deletion of positions 4499-4610 corresponding to the reference RSV sequence set forth as SEQ ID NO: 1, and C4489T, C4492T, A4495T, A4497G, and G4498A substitutions corresponding to the reference RSV sequence set forth as SEQ ID NO: 1. In some such embodiments, the genome of the recombinant RSV comprises a nucleic acid seuqnece complementary to an antigenomic sequence set forth as SEQ ID NO: 2 (LID/ΔNS1), or at least 99% identical thereto. Upon immunization, the subject responds by producing antibodies specific for RSV. In addition, innate and cell-mediated immune responses are induced, which can provide antiviral effectors as well as regulating the immune response. As a result of the immunization the host becomes at least partially or completely immune to RSV infection, or resistant to developing moderate or severe RSV disease, particularly of the lower respiratory tract.

Because nearly all humans are infected with RSV by the age of 5, the entire birth cohort is included as a relevant population for immunization. This could be done, for example, by beginning an immunization regimen anytime from birth to 6 months of age, from 6 months of age to 5 years of age, in pregnant women (or women of child-bearing age) to protect their infants by passive transfer of antibody, family members of newborn infants or those still in utero, and subjects greater than 50 years of age. The scope of this disclosure is meant to include maternal immunization. In several embodiments, the subject is a human subject that is seronegative for RSV specific antibodies. In additional embodiments, the subject is no more than one year old, such as no more than 6 months old, no more than 3 months, or no more than 1 month old.

Subjects at greatest risk of RSV infection with severe symptoms (e.g. requiring hospitalization) include children with prematurity, bronchopulmonary dysplasia, and congenital heart disease are most susceptible to severe disease. During childhood and adulthood, disease is milder but can be associated with lower airway disease and is commonly complicated by sinusitis. Disease severity increases in the institutionalized elderly (e.g., humans over 65 years old). Severe disease also occurs in persons with severe combined immunodeficiency disease or following bone marrow or lung transplantation. In some embodiments, these subjects can be selected for administration of a disclosed recombinant RSV.

The immunogenic compositions containing the recombinant RSV are administered to a subject susceptible to or otherwise at risk of RSV infection in an “effective amount” which is sufficient to induce or enhance the individual's immune response capabilities against RSV. The immunogenic composition may be administered by any suitable method, including but not limited to, via injection, aerosol delivery, nasal spray, nasal droplets, oral inoculation, or topical application. In some embodiments, the vaccine may be administered intranasally or subcutaneously or intramuscularly. In some embodiments, it may be administered to the upper respiratory tract. This may be performed by any suitable method, including but not limited to, by spray, droplet or aerosol delivery. Often, the composition will be administered to an individual seronegative for antibodies to RSV or possessing transplacentally acquired maternal antibodies to RSV.

Upon immunization with an effective amount of a disclosed recombinant RSV, the subject responds by producing antibodies specific for RSV virus proteins, e.g., F and G glycoproteins. In addition, innate and cell-mediated immune responses are induced, which can provide antiviral effectors as well as regulating the immune response. As a result of the immunization the host becomes at least partially or completely immune to RSV infection, or resistant to developing moderate or severe RSV disease, particularly of the lower respiratory tract.

In a preferred embodiment, the attenuated virus is administered according to established human intranasal administration protocols (e.g., as discussed in Karron et al. JID 191:1093-104, 2005). Briefly, adults or children are inoculated intranasally via droplet with an effective amount of the recombinant RSV, typically in a volume of 0.5 ml of a physiologically acceptable diluent or carrier. This has the advantage of simplicity and safety compared to parenteral immunization with a non-replicating virus. It also provides direct stimulation of local respiratory tract immunity, which plays a major role in resistance to RSV. Further, this mode of vaccination effectively bypasses the immunosuppressive effects of RSV-specific maternally-derived serum antibodies, which typically are found in the very young. Also, while the parenteral administration of RSV antigens can sometimes be associated with immunopathologic complications, this has not been observed with a live virus.

The precise amount of immunogen administered and the timing and repetition of administration will be determined by various factors, including the patient's state of health and weight, the mode of administration, the nature of the formulation, etc. Dosages will generally range from about 3.0 log₁₀to about 6.0 log₁₀plaque forming units (“PFU”) or more of virus per patient, more commonly from about 4.0 log₁₀to 5.0 log₁₀PFU virus per patient. In one embodiment, about 5.0 log₁₀to 6.0 log₁₀PFU per patient may be administered during infancy, such as between 1 and 6 months of age, and one or more additional booster doses could be given 2-6 months or more later. In another embodiment, young infants could be given a dose of about 5.0 log₁₀to 6.0 log₁₀PFU per patient at approximately 2, 4, and 6 months of age, which is the recommended time of administration of a number of other childhood vaccines. In yet another embodiment, an additional booster dose could be administered at approximately 10-15 months of age.

The embodiments of recombinant RSV described herein, and immunogenic compositions thereof, are administered to a subject in an amount effective to induce or enhance an immune response against the RSV antigens in the recombinant RSV in the subject. An effective amount will allow some growth and proliferation of the virus, in order to produce the desired immune response, but will not produce viral-associated symptoms or illnesses. Based on the guidance provided herein and knowledge in the art, the proper amount of recombinant RSV to use for immunization cane determined.

The resulting immune response can be characterized by a variety of methods. These include taking samples of nasal washes or sera for analysis of RSV-specific antibodies, which can be detected by tests including, but not limited to, complement fixation, plaque neutralization, enzyme-linked immunosorbent assay, luciferase-immunoprecipitation assay, and flow cytometry. In addition, immune responses can be detected by assay of cytokines in nasal washes or sera, ELISPOT of immune cells from either source, quantitative RT-PCR or microarray analysis of nasal wash or serum samples, and restimulation of immune cells from nasal washes or serum by re-exposure to viral antigen in vitro and analysis for the production or display of cytokines, surface markers, or other immune correlates measures by flow cytometry or for cytotoxic activity against indicator target cells displaying RSV antigens. In this regard, individuals are also monitored for signs and symptoms of upper respiratory illness.

A desired immune response is to inhibit subsequent infection with RSV. The RSV infection does not need to be completely inhibited for the method to be effective. For example, administration of an effective amount of a disclosed recombinant RSV can decrease subsequent RSV infection (for example, as measured by infection of cells, or by number or percentage of subjects infected by RSV) by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (prevention of detectable RSV infection), as compared to a suitable control.

Determination of effective dosages is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in the subject, or that induce a desired response in the subject (such as a neutralizing immune response). Suitable models in this regard include, for example, murine, rat, hamster, cotton rat, bovine, ovine, porcine, feline, ferret, non-human primate, and other accepted animal model subjects known in the art. Alternatively, effective dosages can be determined using in vitro models (for example, immunologic and histopathologic assays). Using such models, only ordinary calculations and adjustments are required to determine an appropriate concentration and dose to administer a effective amount of the composition (for example, amounts that are effective to elicit a desired immune response or alleviate one or more symptoms of a targeted disease).

Administration of the recombinant RSV to a subject can elicit the production of an immune response that is protective against serious lower respiratory tract disease, such as pneumonia and bronchiolitis, or croup, when the subject is subsequently infected or re-infected with a wild-type RSV. While the naturally circulating virus is still capable of causing infection, particularly in the upper respiratory tract, there is a reduced possibility of rhinitis as a result of the immunization and a possible boosting of resistance by subsequent infection by wild-type virus. Following immunization, there are detectable levels of host engendered serum and secretory antibodies which are capable of neutralizing homologous (of the same subgroup) wild-type virus in vitro and in vivo. In many instances the host antibodies will also neutralize wild-type virus of a different, non-vaccine subgroup.

An immunogenic composition including one or more of the disclosed recombinant RSV viruses can be used in coordinate (or prime-boost) immunization protocols or combinatorial formulations. It is contemplated that there can be several boosts, and that each boost can be a different disclosed immunogen. It is also contemplated in some examples that the boost may be the same immunogen as another boost, or the prime. In certain embodiments, novel combinatorial immunogenic compositions and coordinate immunization protocols employ separate immunogens or formulations, each directed toward eliciting an anti-viral immune response, such as an immune response to RSV proteins. Separate immunogenic compositions that elicit the anti-viral immune response can be combined in a polyvalent immunogenic composition administered to a subject in a single immunization step, or they can be administered separately (in monovalent immunogenic compositions) in a coordinate (or prime-boost) immunization protocol.

In some embodiments, a disclosed recombinant RSV (such as any one of RSV LID/ΔNS1 (SEQ ID NO: 2), RSV LID/F1G2/ΔNS1 (SEQ ID NO: 3), RSV LID/F1BBG2/ΔNS1 (SEQ ID NO: 4), RSV LID/F1G2/ΔNS1/1030s (SEQ ID NO: 6), RSV LID/F1BBG2/ΔNS1/1030s (SEQ ID NO: 7), RSV LID/F1G2/ΔNS1/Δ1313/I1314L (SEQ ID NO: 9), or RSV LID/F1BBG2/ΔNS1/Δ1313/I1314L (SEQ ID NO: 10)) is used for the prime immunization and the boost comprises immunization with a RSV 276 virus (SEQ ID NO: 12), a B/HPIV3 DS-Cav1 virus (SEQ ID NO: 16), or a B/HPIV3 DS-Cav1/B3TMCT virus (SEQ ID NO: 17).

In some embodiments, the prime comprises administration of a RSV LID/ANS2/Δ1313/I1314Lvirus (SEQ ID NO: 19), a RSV LID/F1BBG2/ΔNS1 virus (SEQ ID NO: 4), a RSV LID/ANS virus (SEQ ID NO: 2), a RSV LID/ANS2/1030s virus (SEQ ID NO: 18), or a RSV D46/NS2/N/ΔM2-2-HindIII virus (SEQ ID NO: 11), and the boost comprises administration of a RSV 276 virus (SEQ ID NO: 12).

In some embodiments, the prime comprises administration of a RSV LID/ANS2/Δ1313/I1314Lvirus (SEQ ID NO: 19), a RSV LID/F1BBG2/ΔNS1 virus (SEQ ID NO: 4), a RSV LID/ANS virus (SEQ ID NO: 2), a RSV LID/ANS2/1030s virus (SEQ ID NO: 18), a RSV D46/NS2/N/ΔM2-2-HindIII virus (SEQ ID NO: 11), or a RSV 276 virus (SEQ ID NO: 12), and the boost comprises administration of a B/HPIV3 DS-Cav1/B3TMCT virus (SEQ ID NO: 17).

In some embodiments, the prime comprises administration of a RSV LID/ANS2/Δ1313/I1314Lvirus (SEQ ID NO: 19), a RSV LID/F1BBG2/ΔNS1 virus (SEQ ID NO: 4), a RSV LID/ANS virus (SEQ ID NO: 2), a RSV LID/ANS2/1030s virus (SEQ ID NO: 18), a RSV D46/NS2/N/ΔM2-2-HindIII virus (SEQ ID NO: 11), or a RSV 276 virus (SEQ ID NO: 12), and the boost comprises administration of a B/HPIV3 DS-Cav1 virus (SEQ ID NO: 16).

In some embodiments, the prime comprises administration of a RSV LID/ANS2/Δ1313/I1314L virus (SEQ ID NO: 19) and the boost comprises administration of a B/HPIV3 DS-Cav1 virus (SEQ ID NO: 16). In some embodiments, the prime comprises administration of a RSV LID/ANS2/Δ1313/I1314Lvirus (SEQ ID NO: 19) and the boost comprises administration of a B/HPIV3 DS-Cav1/B3TMCT virus (SEQ ID NO: 17). In some embodiments, the prime comprises administration of a RSV LID/ΔNS2/Δ1313/I1314Lvirus (SEQ ID NO: 19) and the boost comprises administration of a RSV 276 virus (SEQ ID NO: 12).

In some embodiments, the prime comprises administration of a RSV LID/ΔNS2/1030s virus (SEQ ID NO: 18) and the boost comprises administration of a B/HPIV3 DS-Cav1 virus (SEQ ID NO: 16). In some embodiments, the prime comprises administration of a RSV LID/ΔNS2/1030s virus (SEQ ID NO: 18) and the boost comprises administration of a B/HPIV3 DS-Cav1/B3TMCT virus (SEQ ID NO: 17). In some embodiments, the prime comprises administration of a RSV LID/ΔNS2/1030s virus (SEQ ID NO: 18) and the boost comprises administration of a RSV 276 virus (SEQ ID NO: 12).

In some embodiments, the prime comprises administration of a RSV 276 virus (SEQ ID NO: 12) and the boost comprises administration of a B/HPIV3 DS-Cav1 virus (SEQ ID NO: 16). In some embodiments, the prime comprises administration of a RSV 276 virus (SEQ ID NO: 12) and the boost comprises administration of a B/HPIV3 DS-Cav1/B3TMCT virus (SEQ ID NO: 17).

In some embodiments, the prime comprises administration of a RSV LID/F1BBG2/ΔNS1 virus (SEQ ID NO: 4) and the boost comprises administration of a B/HPIV3 DS-Cav1 virus (SEQ ID NO: 16). In some embodiments, the prime comprises administration of a RSV LID/F1BBG2/ΔNS1 virus (SEQ ID NO: 4) and the boost comprises administration of a B/HPIV3 DS-Cav1/B3TMCT virus (SEQ ID NO: 17). In some embodiments, the prime comprises administration of a RSV LID/F1BBG2/ΔNS1 virus (SEQ ID NO: 4) and the boost comprises administration of a RSV 276 virus (SEQ ID NO: 12).

In some embodiments, the prime comprises administration of a RSV LID/ΔNS1 virus (SEQ ID NO: 2) and the boost comprises administration of a B/HPIV3 DS-Cav1 virus (SEQ ID NO: 16). In some embodiments, the prime comprises administration of a RSV LID/ΔNS1 virus (SEQ ID NO: 2) and the boost comprises administration of a B/HPIV3 DS-Cav1/B3TMCT virus (SEQ ID NO: 17). In some embodiments, the prime comprises administration of a RSV LID/ΔNS1 virus (SEQ ID NO: 2) and the boost comprises administration of a RSV 276 virus (SEQ ID NO: 12).

In some embodiments, the prime comprises administration of a RSV D46/NS2/N/ΔM2-2-HindIII virus (SEQ ID NO: 11) and the boost comprises administration of a B/HPIV3 DS-Cav1 virus (SEQ ID NO: 16). In some embodiments, the prime comprises administration of a RSV D46/NS2/N/ΔM2-2-HindIII virus (SEQ ID NO: 11) and the boost comprises administration of a B/HPIV3 DS-Cav1/B3TMCT virus (SEQ ID NO: 17). In some embodiments, the prime comprises administration of a RSV D46/NS2/N/ΔM2-2-HindIII virus (SEQ ID NO: 11) and the boost comprises administration of a RSV 276 virus (SEQ ID NO: 12).

In any of the prime-boost immunization methods provided herein, the prime and boost may be administered intranasally to the subject at a dose of about 5.0 log₁₀PFU to about 6.0 log₁₀PFU. In any of the prime-boost immunization methods provided herein, a suitable subject may be selected for immunization, such as a human subject of five years old or younger. In the prime-boost immunization methods provided herein, the virus is administered to the subject by a suitable method, such as intranasal administration.

In some embodiments, the recombinant RSV as disclosed herein is administered intranasally to human subject 5 years of age or younger (such as about 2 years of age or younger) at a dose of 3.0 log₁₀to 7.0 log₁₀PFU, for example, a dose of about 5.0 log₁₀to about 6.0 log₁₀PFU (such as a dose of about 6.0 log₁₀PFU).

In some embodiments, the recombinant RSV as disclosed herein is administered intranasally to a RSV-seropositive human subject 5 years of age or younger (such as about 2 years of age or younger) at a dose of 3.0 log₁₀to 7.0 log₁₀PFU (such as a dose of about 6.0 log₁₀PFU), and wherein a titer of the recombinant RSV in nasal wash taken during the first 2 weeks post administration is 3.0 log₁₀PFU/ml of nasal wash or less, and the recombinant RSV administered to the subject is attenuated such that within the first month following intranasal administration to a subject, the subject exhibits no or mild (Grade 1) respiratory/febrile illness that is comparable in kind and severity to that of comparable subjects who did not receive the virus.

EXAMPLES

The materials, information, and methods described in this disclosure provide an array of attenuated strains with graded attenuation phenotypes, and provide guidance in selecting suitable vaccine candidate strains based on clinical benchmarks. The following examples are provided to illustrate particular features of certain embodiments, but the scope of the claims should not be limited to those features exemplified.

Example 1
Recombinant Respiratory Syncytial Virus Bearing NS1 Deletion and F and G Gene Shifts

This example describes recombinant attenuated RSV including a deletion of the NS1 gene in combination with one or more additional mutations.

The NS1 and NS2 proteins independently—and to some extent cooperatively—mediate strong suppression of host innate immune responses and apoptosis, and also have been found to suppress and alter adaptive immune responses. NS1 appears to play the greater role in many of these activities. The mechanisms by which NS1 and NS2 suppress and alter host responses are numerous and are incompletely identified and incompletely characterized. Both proteins, and especially NS1, inhibit the induction of type I and type III interferons (IFNs) as well as pro-inflammatory cytokines. This inhibition involves multiple mechanisms. For example, NS1 and NS2 inhibit intracellular signaling pathways, such as by binding to RIG-I and MAVS to block their interaction, and by promoting the degradation of RIG-I, TRAF3, TBK1, and IKKε (Boyapalle, et al. 2012. PLoS One 7:e29386; Sweden, et al. 2009. J Virol 83:9682-9693). NS1 can block IRF3 activation, its nuclear translocation, and its interaction with transcription co-activators on the IFN-β promoter (Spann, et al. 2005. J Virol 79:5353-5362; Ren, et al. 2011. J Gen Virol 92:2153-2159). Both proteins have been reported to inhibit signaling from the type I IFN receptor (IFNAR), which otherwise amplifies the INF response and induces dozens of proteins that contribute to an antiviral state. For example, both proteins, and especially NS2, have been described to promote degradation of STAT2 (Lo, et al. 2005, J Virol 79:9315-9319; Xu, et al. 2014. Intervirology 57:65-73). NS1 also has been shown to directly interfere with a component of the IFN-induced antiviral state, namely the 2′-5′-Oligoadenylate Synthetase-Like (OASL) protein that otherwise inhibits RSV replication (Dhar, et al. 2015. J Virol 89:10115-10119). Both proteins, and especially NS1, suppress the induction of the cellular apoptosis in response to RSV infection, with the effect of prolonging the survival of the infected cells and increasing the production of progeny RSV (Bitko, et al. 2007. J Virol 81:1786-1795). Both proteins, and especially NS1, suppress the development of adaptive immunity by suppressing the maturation of dendritic cells (DC), by skewing the activation of T cell subsets—partially through antagonizing the IFN response, and by altering the response of regulatory T cells (Munir, et al. 2008. J Virol 82:8780-8796; Munir, et al. 2011. PLoS Pathogen 7:e1001336; Yang, et al. 2015. Virology 485:223-232). The NS1 protein also has been shown to facilitate RSV infection by modifying the expression of specific miRNAs (Bakre, et al. 2015. J Gen Virol 96:3179-3191; Zhang, et al. 2016. Biochem Biophys Res Comm 478:1436-1441). The NS1 protein, and to a lesser extent the NS2 protein, also have been shown to downregulate RNA synthesis in a mini-genome system, suggesting that they have a direct effect on viral gene expression and/or genome replication that remains to be defined (Atreya, et al. 1998. J Virol 72:1452-1461). This list of activities and mechanisms attributed to NS1 and NS2 is by no means complete or fully characterized.

Deletion or silencing of either or both genes attenuates RSV replication in vivo, with deletion of NS1 having a greater attenuating effect than NS2 in seronegative chimpanzees (Whitehead, et al. 1999. J Virol 73:3438-3442; Teng, et al. 2000. J Virol 74:9317-9321), which are considered the most predictive surrogate for RSV-naïve humans. It is presumed that the increased IFN and apoptosis responses in the absence of the NS1 and/or NS2 proteins are major factors in this attenuation. However, the significance and impact of the many NS1-mediated and NS2-mediated activities on RSV replication in its native human host, and on the attenuation and the immunogenicity of live-attenuated RSV vaccine candidates in humans, remain under investigation and are incompletely understood. Several experimental vaccines bearing the deletion of NS2 in combination with various point mutations have been evaluated in humans. One of these vaccine viruses, rA2cpΔNS2, which combined a ΔNS2 deletion with five point mutations in the N, F, and L proteins from a cold-passaged (cp) RSV strain, was under-attenuated in seropositive children (Wright, et al. 2006. J Infect Dis 193:573-581). Two other vaccine viruses, rA2cp248/404ΔNS2 and rA2cp530/1009ΔNS2, which each had the further addition of a different pair of attenuating temperature-sensitivity (ts) mutations (248/404 or 530/1009), were over-attenuated in seronegative children (Wright, et al. 2006. J Infect Dis 193:573-581). These studies confirmed that deletion of NS2 was attenuating in the human host, but none of the viruses was considered suitably attenuated for further evaluation as a pediatric vaccine. Previously, no RSV bearing the deletion of the NS1 gene had been evaluated in humans; in the present disclosure, we present data from a Phase I clinical trial in RSV-seropositive children 12-59 months of age and RSV-seronegative children 6-24 months of age.

Design of Recombinant RSV with ΔNS1 and Additional Mutations

The RSV antigenome that was used for constructing the recombinant RSV was the wild-type RSV strain A2 antigenomic cDNA called D46. This antigenomic cDNA also is sometimes referred to in the literature as D53. D46 is the basis for the present reverse genetics system (Collins, et al. 1995. Proc Natl Acad Sci USA 92:11563-11567), and its complete sequence is shown in U.S. Pat. No. 6,790,449 and in GenBank KT992094, and provided herein as SEQ ID NO: 1.

FIG. 1A illustrates the deletion of the NS1 gene. The 529-nt deletion (nucleotides 99-627 inclusive) begins with and includes nucleotide 99 that is the first nucleotide of the ATG of the NS1 ORF and extends to and includes nucleotide 627 that immediately precedes the ATG (nucleotides 628-630) of the NS2 ORF. This deletion joins the upstream non-translated region of the NS1 gene to the translational start codon of the NS2 ORF.

FIG. 1B illustrates the deletion of the NS1 and NS2 ORFs along with most of their flanking gene sequences from their native positions 1 and 2 in the genome. The 1042-nt deletion (nucleotides 99-1140 inclusive) begins with and includes nucleotide 99 that is the first nucleotide of the ATG of the NS1 ORF and extends to and includes nucleotide 1140, which immediately precedes the ATG (1141-1143) of the N ORF. This joins the upstream non-translated region of the NS1 gene to the translational start codon of the N ORF.

FIG. 1C illustrates the “LID” mutation, which includes a 112-nucleotide deletion of the downstream non-translated region of the SH gene of D46 together with 5 nucleotide substitutions (C4489T, C4492T, A4495T, A4497G, and G4498A) that involve the last three codons and stop codon of the SH ORF and do not change amino acid coding (Bukreyev, et al. 2001. J Virol 75:12128-12140). Note that RSV bearing the LID mutation generally appeared to have a wild-type-like phenotype in pre-clinical studies (Bukreyev, et al. 2001. J Virol 75:12128-12140), although this does not preclude possible subtle differences such as modestly increased replication in cell culture.

FIG. 2 illustrates the shift of the G and F genes from their natural genome positions to be promoter proximal and to have F preceding G (rather than the natural order of G preceding F). FIG. 2A illustrates deletion of the F and G genes from RSV LID. Nucleotides 4630-7551 inclusive were deleted from the RSV antigenomic cDNA to remove the F and G genes. This has the effect of fusing the SH gene-end signal to the F/M2 intergenic region. This mutation is illustrated with an antigenomic cDNA bearing the LID mutation (indicated with an open triangle above or below the SH gene), although the modifications do not depend on the LID mutation. FIG. 2B illustrates insertion of the F and G genes in gene positions 1 and 2, respectively, of the recombinant RSV antigenome. The G and F ORFs (shaded sequences) were reconstructed into genes in the reverse of their natural order (i.e., F-G instead of G-F). Each ORF was designed so that in the final construct (the antigenomic cDNA) it would be flanked by short non-translated regions and gene-start and gene-end signals, with the F and G genes separated by a short intergenic region consisting of a single nucleotide. Nucleotide adapters with Xho I sites were added upstream and downstream of the F and G ORFs: creation of the upstream Xho I site required mutation of nucleotides 88-91, in the sequence representing the upstream non-translated NS1 gene region, from CTTG (shown in large case) to tcga (shown in small case) in the LID antigenomic cDNA. As shown with the antigenome at the bottom, the F-G gene cassette (cross-hatched rectangles) was inserted into the genomic cDNA using the Xho I sites.

FIG. 3 provides genome maps of RSV strains bearing various combinations of mutations including the LID mutation (the LID mutation is indicated with an open triangle above the SH gene); deletion of G and F from their natural positions (ΔG+F) and their shift to the 2^ndand 1^stgene positions, respectively (F1G2, with F and G in the reverse of their natural order G-F); F1G2 in which the F ORF has been codon optimized (F1BBG2); deletion of the NS1 gene (ΔNS1); addition of the 1030s mutation, consisting of the L assignments 1321K(AAA) and 1313S(TCA); addition of the Δ1313 and I1314L(CTG) mutations in L; and deletion of both the NS1 and NS2 genes (ΔNS1+2). The LID/F1BBG2/ΔNS1, LID/F1BBG2/ΔNS1/1030s, LID/F1BBG2/ΔNS1/Δ1313/I1314L, and LID/ΔNS1+2/F1BBG2 constructs shown contain the codon optimized F1BB2 ORF: parallel constructs called LID/F1G2/ΔNS1, LID/F1G2/ΔNS1/1030s, LID/F1G2/ΔNS1/Δ1313/I1314L, and LID/F1G2/ΔNS1+2 contained the non-optimized wild-type version (not shown).

The recombinant RSVs were recovered by reverse genetics (see Collins et al. 1995. Proc Natl Acad Sci USA 92:11563-7). RSVs were grown at 32° C. in Vero cells. The complete genome sequences of all viruses were confirmed by automated Sanger sequencing analysis of un-cloned RT-PCR products to be free of adventitious mutations detectable above background.

Assessment of the Recombinant RSVs In Vitro

The kinetics and yield of multi-cycle replication of various recombinant RSV was assessed in African green monkey Vero cells (FIG. 4), which are unable to produce type I interferons in response to virus infection. This shows that viruses bearing the LID, ΔNS1, F1G2, and F1BBG2 mutations in various combinations replicate to reasonable titers in Vero cells, the cell substrate for vaccine manufacture. Note that the RSV LID virus is a wild-type-like virus and thus serves as a wild-type-like control in this and subsequent experiments.

The multicycle growth kinetics of the various recombinant RSV were also assessed in human airway epithelial (HAE) cell cultures (FIG. 5). HAE cultures form a differentiated, polarized, pseudostratified, mucocilliary tissue that closely resembles authentic airway epithelium. Attenuated replication in these cultures is predictive of attenuated replication in non-human primates and humans. These data show that viruses bearing the ΔNS1 modification, alone or combined with the F1G2 and F1BBG2 modifications, was strongly attenuated compared to the RSV LID parent.

The plaque- and syncytia forming properties of the recombinant RSVs were assessed on Vero cells (FIGS. 6 and 7). Recombinant RSVs bearing the LID, F1BBG2, F1G2, and ΔNS1 mutations in various combinations readily form plaques on Vero cells, and recombinant RSVs bearing the F1G2 and F1BBG2 gene shifts direct the formation of large syncytia in Vero cells during multi-cycle replication, consistent with increased expression of the F protein.

Additionally, Vero cells infected with recombinant RSVs bearing the F1G2 and F1BBG2 modifications showed increased expression of the RSV F and G proteins in Vero cells (FIG. 8), as determined by gel electrophoresis under denaturing and reducing conditions and Western blot analysis using antibodies of the indicated specificities, with the levels of RSV F, G, N, P, and M protein expression shown to the right relative to RSV LID as 1.0.

The recombinant RSV were also assessed in human airway A549 cells, which are competent for interferon responses to viral infection. Cells infected with recombinant RSVs lacking the NS1 gene showed increased expression of type 1 (α, β) and type III (λ) interferons (FIG. 9). The A549 cells were infected with an MOI of 3 PFU/cell and tissue culture medium supernatants were collected 24 h post-infection. IFN concentrations in the medium supernatants were determined by ELISA. This showed that deletion of the NS1 gene resulted in a substantial increase in the expression of type I and type III interferons, which may stimulate immune responses and attenuate virus replication.

Assessment of the Recombinant RSVs in Experimental Animals

The recombinant RSVs were assessed in mice (FIG. 10). The presence of the F1G2 or F1BBG2 modifications with or without deletion of NS1 and the LID mutations led to reduced RSV replication in the upper and lower respiratory tract of mice compared to the RSV LID virus, which is a wild-type-like virus. For these assays, six-week-old BALB/c mice were infected with a dose of 10⁴or 10⁶PFU per animal of each indicated RSV. Five animals per virus and dose were sacrificed at 3 and 5 days post-infection, and nasal turbinates and lungs were harvested, homogenized, and viral titers determined by plaque assay.

Despite their restricted replication in the upper and lower respiratory tract, the recombinant RSVs bearing the indicated combinations of the LID, ΔNS1, F1G2, and F1BBG2 modifications induced titers of RSV-neutralizing serum antibodies that compared well with the LID virus, which is a wild-type-like virus (FIG. 11). For these assays, six-week-old BALB/c mice were infected with a dose of 10⁴or 10⁶PFU per animal of each indicated RSV, and serum samples were collected 28 days post-infection.

African green monkeys (AGMs) were used to assess RSV LID/ΔNS1 and LID/F1BBG2/ΔNS1, in comparison with the ΔNS2-bearing RSV ΔNS2/Δ1313/I1314L, the ΔM2-2-bearing RSV 276, and wild type virus. Four animals were infected for each virus, and virus titers were determined from nasopharyngeal and tracheal lavage samples from each animal (Tables 1 and 2; FIG. 12). Serum samples were also obtained from each animal, and serum PRNT₆₀titers determined (Table 3; FIG. 13). The results show that LID/F1BBG2/ΔNS1 has a promising attenuation phenotype and elicits a superior immune response.

TABLE 1

Viral Titers of Nasopharyngeal Swab Samples from AGMs Inoculated with RSV ΔNS1

Mutants, Compared with ΔNS2 and ΔM2-2 Mutants and Recombinant wt RSV rA2.

Peak
Sum of

Virus Test
AGM
NP virus titer (log₁₀PFU/mL) on indicated days^b
virus
daily

Article^a
ID
1
2
3
4
5
6
7
8
9
10
12/14
titer
titers^c

Wt RSV A2
7209
—
—
3.1
4.4
4.4

5.2

3.6
4.1
3.4
2.0
—
5.2
31.3

7467
—
0.7
2.8
3.8
2.1

3.9

3.4
3.2
1.9
2.0
—
3.9
24.5

7468
—
1.7
2.6

4.0

3.9
3.7
3.6
2.7
3.6
2.6
—
4.0
29.1

7492
0.7
1.2
2.3

3.9

2.9

3.9

3.9

2.4
1.7
1.0
—
3.9
24.3

Mean

4.2

27.2

LID/ΔNS1
8960
—
0.7
—
1.7
2.2
2.7

2.8

1.8
—
—
—
2.8
13.7

8992
—
0.7
—
0.7
2.6
2.7

2.9

2.6
1.9
0.7
—
2.9
15.8

8904
—
—
—
1.7
1.2
2.4

2.8

1.3
—
1.2
—
2.8
12.3

8951
—
—
—
2.3
2.3
3.1

3.5

2.6
1.5
—
—
3.5
17.0

Mean

3.0

14.7

LID/ΔNS1/
8922
—
0.7
—
—
2.3
2.6

2.7

—
—
—
—
2.7
10.7

F1BBG2
8974
—
—
—
1.3
1.8

2.7

2.5
0.7
—
—
—
2.7
11.1

8966
—
—
0.7
—
1.8

2.2

1.7
—
—
—
—
2.2
8.9

8994
—
—
—
—
1.0
1.0

1.5

1.0
1.0
—
—
1.5
7.6

Mean

2.2

9.2

RSV ΔNS2/
7648
—
—
1.7
1.2
1.8
2.9
2.2

4.0

3.7
3.4
—
4.0
22.0

Δ1313/I1314L
7692
—
—
—
—
1.9
—
—

3.3

1.0
1.5
—
3.3
10.2

7714
—
—
0.7
—
1.3
—
—
1.4
2.4

2.8

—
2.8
10.7

7764
—
—
—
0.7
0.7
1.0
—
2.3
1.2

2.9

—
2.9
10.5

Mean

3.2

13.3

RSV 276^d
8918
—
—
—
—
—
—
—
—
—
—
—
—
3.9

8902
—
1.5
2.0
2.9
2.5
3.0

3.1

2.5
1.4
1.3
—
3.1
21.0

8913
—
—
—
—
—
0.7

1.4

1.0
—
—
—
1.4
6.0

8952
—
—
—
—
0.7
0.7

1.5

—
—
1.2
—
1.5
6.5

Mean

1.6

9.3

^aMonkeys were inoculated i.n. and i.t. with 10⁶PFU of the indicated virus in a 1 mL inoculum per site (total dose = 2 × 10⁶PFU/AGM).

^bVirus titrations were performed on Vero cells. The lower limit of detection was 1.0 log₁₀PFU/mL. Samples with no detectable virus are represented as “—”.

Peak titers for each animal are underlined. Mean peak titers of ^cThe sum of daily titers is used as an estimate for the magnitude of shedding (area under the curve). A value of 0.35 was used for samples with no detectable virus.

^dA ΔM2-2 virus.

TABLE 2

Viral Titers of Tracheal Lavage Samples from AGMs

Inoculated with RSV ΔNS1 Mutants, Compared with

ΔNS2 and ΔM2-2 Mutants and Recombinant wt RSV rA2.

TL virus titer (log₁₀
Peak
Sum of

Virus Test

PFU/mL) on indicated day^b
virus
daily

Article^a
AGM ID
2
4
6
8
10
12/14
titer
titers ^c

Wt RSV A2
7209
3.0
3.8
4.3

4.5

2.6
—
4.5
18.9

7467
3.0
3.0

4.3

3.4
2.8
—
4.3
17.2

7468
2.5
1.7
2.9

3.0

2.9
—
3.0
13.7

7492
1.9
3.5

4.7

3.3
—
—
4.7
14.8

Mean:

4.1

16.2

LID/ΔNS1
8960
—
—
1.5

2.7

—
—
2.7
7.0

8992
—
2.9

3.6

2.7
1.0
—
3.6
11.6

8904
2.3
2.7

3.8

3.1
—
—
3.8
13.3

8951
—
3.0
1.5

3.3

—
—
3.3
9.9

Mean:

3.3

10.5

LID/ΔNS1/
8922

3.0

2.5
1.3
1.3
—
—
3.0
9.5

F1BBG2
8974
2.3
1.3

2.4

1.7
—
—
2.4
9.1

8966
3.1
—

3.2

—
—
—
3.2
10.4

8994
1.0
—

2.0

—
—
—
2.0
5.8

Mean:

2.7

8.7

RSV ΔNS2/
7648
—
—
—

1.5

—
—
1.5
5.0

Δ1313/I1314L
7692
—
—
—
1.0

1.7

—
1.7
5.5

7714
—
—
—
—
—
—
—
4.2

7764
—
—
—

1.3

—
—
1.3
4.8

Mean:

1.4

4.9

RSV 276^d
8918
—
1.7

2.3

1.6
1.0
—
2.3
8.0

8902
2.2
1.6

2.9

2.1
—
—
2.9
10.3

8913
—
—

3.2

1.3
—
—
3.2
7.3

8952
1.8

2.1

1.6
1.8
—
—
2.1
8.8

Mean:

2.6

8.6

^aMonkeys were inoculated i.n. and i.t. with 10⁶PFU of the indicated virus in a 1 mL inoculum per site (total dose = 2 × 10⁶PFU/AGM).

^bVirus titrations were performed on Vero cells. The lower limit of detection was 1.0 log₁₀PFU/mL. Samples with no detectable virus are represented as “—”. Underlined value indicates maximum titer for each animal.

^cThe sum of daily titers is used as an estimate for the magnitude of shedding (area under the curve). Values of 0.7 are used for samples with no detectable virus.

^dA ΔM2-2 virus.

TABLE 3

Serum PRNT₆₀Titers in AGMs Inoculated with RSV ΔNS1

Mutants, Compared with ΔNS2 and ΔM2-2 Mutants

and Recombinant wt RSV rA2.

RSV Neutralization Titer

(Log2 of reciprocal) on days

Virus Test Article
AGM ID
0
21
28

Wt RSV rA2
7209
<3.3 ^a
7.1
6.7

7467
<3.3
8.1
8.7

7468
<3.3
5.9
5.9

7492
<3.3
7.7
8.8

Mean:

7.2
7.5

LID/ΔNS1
8960
<3.3
8.4
8.9

8992
<3.3
9.5
9.5

8904
<3.3
8.9
9.8

8951
<3.3
9.4
9.0

Mean:

9.1
9.3

LID/F1BBG2/ΔNS1
8922
<3.3
10.9
11.6

8974
<3.3
9.2
9.6

8966
<3.3
10.3
10.5

8994
<3.3
8.2
8.8

Mean:

9.7
10.1

RSV ΔNS2/
7648
<3.3
6.4
6.8

Δ1313/I1314L
7692
<3.3
6.6
6.4

7714
<3.3
4.4
6.1

7764
<3.3
5.5
6.0

Mean:

5.8
6.3

RSV 276 ^b
8918
<3.3
6.1
6.2

8902
<3.3
8.4
8.8

8913
<3.3
8.8
8.9

8952
<3.3
9.7
9.9

Mean:

8.4
8.5

^aThe lower limit of detection of the 60% Plaque Reduction assay is 3.3 (Log₂of the dilution reciprocal). Samples below the lower limit of detection are recorded as “—“.

^bA ΔM2-2 virus.

Example 2
Assessment of Recombinant RSVs in Human Subjects

Information from studies in experimental animals often is not fully accurate for the prediction of the magnitude of attenuation, tolerability, and immunogenicity in humans, and in particular in infants and young children who are most at risk for RSV infection and are the primary targets of vaccine development. Further, experimental animals typically are adult animals with mature immune systems, compared to the immature and less-effective immune systems of infants and young children. Relevant to a respiratory virus, the size, structure, and composition of the upper and lower respiratory tracts of experimental animals can have differences compared to humans. In addition, the effects of deleting RSV accessory proteins—such as the NS1 and NS2 proteins described in the present disclosure—cannot be assumed to be identical in experimental animals and humans, since these viral proteins have evolved to interact with human host cell proteins and may not have the same interactions and effects with corresponding proteins in experimental animals. Thus, clinical trials in infants and young children are important for evaluation of candidate pediatric RSV vaccines.

Notably, most live-attenuated RSV strains that appeared to be promising as pediatric vaccine candidates based on pre-clinical studies including in experimental animals, and that were advanced to Phase I clinical studies, were found to be insufficiently promising to be advanced to further clinical studies. Karron et al recently reviewed clinical studies of 19 live-attenuated RSV strains as pediatric vaccine candidates evaluated over more than 25 years (Karron et al 2013 In Challenges and Opportunities for Respiratory Syncytial Virus Vaccines, Current Topics in Microbiology and Immunology 372, pp 259-284). Of ten biologically-derived live-attenuated RSV strains in that review, none had been found suitable for further study. Of nine additional recombinantly-derived live-attenuated candidates in that review, only one, RSV Medi/ΔM2-2 (Karron et al Sci Transl Med 312:ra175; ClinicalTrials.gov Identifier: NCT01459198), appeared possibly suitable for further clinical study but was not advanced. Subsequently, five additional recombinantly-derived live-attenuated RSV strains have been evaluated in Phase I clinical studies for which sufficient data were obtained to determine their suitability for further clinical evaluation. These additional strains are (i) RSV LID/ΔM2-2 (McFarland et al. 2018. Live-attenuated respiratory syncytial virus vaccine candidate with deletion of RSV synthesis regulatory protein M2-2 is immunogenic in children. J. Infect. Dis. 217:1347-55; ClinicalTrials.gov Identifier: NCT02040831 and NCT02237209); (ii) RSV D46/cp/M2-2 (ClinicalTrials.gov Identifier NCT02601612); (iii) RSV LID/cp/ΔM2-2 (Cunningham et al. 2019. Live-attenuated respiratory syncytial virus vaccine with deletion of RNA synthesis regulatory protein M2-2 and cold-passage mutations is over-attenuated. Open Forum Infect Dis. 2019 May 6; 6(6):ofz212. doi: 10.1093/ofid/ofz212. eCollection 2019 June; ClinicalTrials.gov Identifier: NCT02890381 and NCT02948127); (iv) RSV LID/ΔM2-2/1030s (McFarland et al. 2020. Live respiratory syncytial attenuated by M2-2 deletion and stabilized temperature sensitivity mutation 1030s is a promising vaccine candidate in children. J. Infect. Dis. 221:534-43; ClinicalTrials.gov Identifier: NCT02952339 and NCT02794870), and (v) RSV D46/NS2/N/ΔM2-2-HindIII (McFarland et al. 2020. Live-attenuated respiratory syncytial virus vaccine with M2-2 deletion and with SH non-coding region is highly immunogenic in children. J. Infect. Dis. 2020 Feb. 1. pii: jiaa049. doi: 10.1093/infdis/jiaa049. [Epub ahead of print]; ClinicalTrials.gov Identifier: NCT03102034). Of these five additional viruses, only RSV LID/ΔM2-2/1030s appears suitable to advance to further clinical study. Therefore, out of a total of 24 live-attenuated RSV strains evaluated over more than 25 years that looked promising in pre-clinical studies and were advanced to pediatric clinical studies, only two (Medi/ΔM2-2 and LID/ΔM2-2/1030s) were considered candidates for further clnical study. Thus, clinical evaluation of live-attenuated RSV vaccine candidates in infants and young children is unpredictable and historically has not had a high success rate.

Two viruses in this disclosure were advanced to a Phase I clinical study in children as candidate live-attenuated pediatric RSV vaccines, namely RSV LID/ΔNS1 and RSV LID/F1G2/ΔNS1 (see FIG. 3A for genome maps). This study provides the first clinical evaluation of two independent modifications/mutations in a live RSV strain: (i) deletion of the NS1 gene and (ii) shifting the F and G genes from their usual positions 8 and 7, respectively, to positions 1 and 2, respectively. To prepare CTM acceptable for human use, seed virus for each mutant was generated de novo by reverse genetics from cDNAs electroporated into cGMP-qualified Vero cells that had been generated from World Health Organization Vero cells. cGMP-qualified CTM for each mutant was prepared from seed virus in the same cell substrate at Charles River Laboratories. Full-genome sequence analysis using RT-PCR amplification confirmed that the two preparations of seed virus and the two preparations of CTM were free of detectable adventitious mutations. Pre-clinical safety testing confirmed that the two preparations of CTM were suitable for human administration.

Single doses of LID/ΔNS1 and LID/FlG2/ΔNS1 were evaluated side-by-side with placebo recipients in sequential, randomized, double-blind, placebo-controlled studies in RSV-seropositive children aged 12-59 months at a dose of 6.0 log₁₀PFU (LID/ΔNS1) or 5.8 log₁₀PFU (LID/F1G2/ΔNS1) and in RSV-seronegative children aged 6-24 months at a dose of 5.0 log₁₀PFU. Children were randomized 2:2:1 to receive LID/ΔNS1, LID/F1G2/ΔNS1, or placebo, respectively, administered as nose drops (0.5 ml per subject, given as approximately 0.25 ml per nostril). The clinical protocol, consent forms, and Investigator's Brochure were developed by CIR and NIAID investigators; Investigational New Drug application, FDA revew, informed consent, Institutional Review Board review, randomization, blinding, unblinding, study conduct, and review by the NIAID Data Safety Monitoring Board were performed essentially as described previously (Karron et al. 2015. A gene deletion that up-regulates viral gene expression yields an attenuated RSV vaccine with improved antibody responses in children. Sci Transl Med7:312ra175.).

Children were enrolled between April 1 and October 31 each year, outside of RSV season. Clinical assessments were performed and nasal wash (NW) samples were obtained on the following days following vaccine administration: RSV-seropositive children, study days 0, 3-7 and 10; RSV-seronegative children, study days 0, 3, 5, 7, 10, 12, 14, 17, 28±1 day at each time point. Adverse events were collected through day 28 for RSV-seropositive and RSV-seronegative children; serious adverse events and LRIs were collected through day 56 for RSV-seronegative children, with additional physical examinations performed and NW samples obtained in the event of LRI. Fever, upper respiratory illness (URI; including rhinorrhea, pharyngitis, and hoarseness), cough, LRI, and otitis media were defined as described elsewhere (Karron et al. 1997. Evaluation of two live, cold-passaged, temperature-sensitive respiratory syncytial virus vaccines in chimpanzees and in adult humans. J Infect Dis 176:1428-36). When illnesses occurred, NW samples were tested for other viruses and mycoplasma by means of real-time reverse-transcription polymerase chain reaction (RT-PCR) (Respiratory Pathogens 21 kit; Fast Track Diagnostics, Luxembourg). Vaccine virus in NW fluid was quantified by immunoplaque assay using a mixture of 3 monoclonal antibodies (mAbs) to RSV F (mAbs 1129, 1243, and 1269 and by quantitative RT-PCR (RT-qPCR) (Karron 2015 ibid). Serum samples were obtained before inoculation and approximately 1 month after inoculation of RSV-seropositive participants and 2 months after inoculation of RSV-seronegative participants. Serum samples were tested for RSV-neutralizing antibodies using a complement-enhanced 60% RSV plaque-reduction neutralization assay, and for immunoglobulin G (IgG) antibodies to the RSV F glycoprotein using an enzyme-linked immunosorbent assay (Karron et al. 2015, ibid). The plaque reduction neutralization titer (PRNT) and RSV F IgG titer are expressed as reciprocal log₂values. Antibody responses were defined as ≥4-fold increases in titer compared to pre-vaccination titer. Infection with vaccine was defined as detection of vaccine virus by culture or RT-qPCR and/or a ≥4-fold rise in serum RSV PRNT or in RSV F IgG. The mean peak titer of vaccine virus shedding (in log₁₀PFU/mL) was calculated for infected vaccinees only. PRNT and RSV F IgG titers were transformed to log₂values for calculation of means, and the Student t test was used to compare means between groups. Serum antibody titers were calculated for all subjects: those with titers below the limit of detection were assigned values of 2.3 log₂(PRNT) and 4.6 log 2 (ELISA). Rates of illness and antibody responses were compared using the 2-tailed Fisher exact test.

The RSV LID/ΔNS1 and LID/F1G2/ΔNS1 vaccine candidates were evaluated first in RSV-seropositive children 12-59 months of age in order to confirm that they were sufficiently attenuated to evaluate in RSV-seronegative children (Tables 4 and 5). The RSV-seropositive children were enrolled in years 2018 and 2019. Each vaccine was administered to 10 subjects each, and the placebo (consisting of L-15 tissue culture medium) was administered to 5 subjects. Mild respiratory or febrile illness occurring between study days 0 and 28 was observed in 30%, 50%, and 40% of participants who received RSV LID/ΔNS1, LID/F1G2/ΔNS1, and placebo, respectively (Table 4). No instances of lower respiratory tract illness (LRI) were observed during this period. In the vaccine recipients, each of these incidents of mild respiratory/febrile illness was contemporaneous with detection of adventitious respiratory pathogens (data not shown). None of the subjects in any group shed detectable vaccine virus, indicative of attenuation (Table 4). A single vaccine recipient (LID/F1G2/ΔNS1 group) had a ≥4-fold rise in serum RSV-neutralizing and RSV-F-binding antibodies, but without detected vaccine virus shedding or adventitious RSV (Tables 4 and 5). This low level of immunogenicity also was indicative of attenuation. Thus, both of these new vaccine candidates were sufficiently attenuated to be advanced to evaluation in RSV-seronegative, younger children.

Therefore, the RSV LID/ΔNS1 and LID/F1G2/ΔNS1 vaccine candidates were evaluated in RSV-seronegative children 6-24 months of age, the age group that likely will be the major target for a pediatric RSV vaccine (Tables 5 and 6). Enrollment initiated in 2019 and is still in progress. Data are available for 4 subjects in each vaccine group and 2 placebo recipients (complete enrollment will be 14-20 subjects per vaccine group and 7-10 for the placebo recipients, for a total of 35-50 subjects). Mild respiratory or febrile illness between study days 0 and 28 was observed in 50%, 75%, and 50% of participants who received RSV LID/ΔNS1, LID/F1G2/ΔNS1, and placebo, respectively (Table 6). None of these incidents appeared to be linked to vaccine virus shedding (data not shown). There were no incidents of LRI. Two of the vaccine recipients in the LID/ΔNS1 group shed vaccine virus, with mean peak titers of 2.1 log 10 PFU (culture) and 5.4 log₁₀copy number (RT-PCR). In the LID/F1G2/ΔNS1 group, all four of the vaccine recipients shed vaccine virus detectable by culture and/or RT-PCR, with mean peak titers of 1.3 log 10 PFU (culture) and 3.9 log₁₀copy number (RT-PCR) (Table 6). A ≥4-fold rise in serum RSV-neutralizing and/or RSV-F-binding antibodies was not observed for any recipients of the RSV LID/ΔNS1 virus, but was detected for 75% of the recipients of the RSV LID/F1G2/ΔNS1 virus, with mean titers of 5.5 log₂(PRNT) and 10.8 log₂(ELISA)(Table 7).

Based on these initial data, the two new viruses (LID/ΔNS1 and LID/F1G2/ΔNS1) appeared to be highly attenuated and well-tolerated (attenuated). In both RSV-seropositive and RSV-seronegative subjects, the frequency of mild respiratory/febrile disease was approximately the same as for placebo recipients, and usually appeared to be contemporaneous with adventitious agents.

Both viruses appeared to be highly restricted. In RSV-seropositive subjects, there was no observed shedding of either virus, whether assayed by culture or RT-PCR, and only one vaccine recipient had a serum RSV-specific antibody response. In RSV-seronegative subjects, 50% of recipients of LID/ΔNS1 shed virus detected by culture and RT-PCR, but only at low titers. In RSV-seronegative subjects, 50% and 100% of recipients of LID/F1G2/ΔNS1 shed virus detected by culture and RT-PCR, respectively, and only at low titers.

In RSV-seronegative recipients, the LID/ΔNS1 virus did not induce ≥4-fold increases in serum RSV-PRNT and ELISA antibody responses, whereas the LID/F1G2/ΔNS1 virus induced ≥4-fold increases in serum RSV-PRNT and ELISA antibodiesin 75% of recipients.

In summary, this study provided the first evaluation in humans of two modifications to a live-attenuated RSV vaccine candidate: (i) deletion of the NS1 gene, and (ii) shifting the positions of the F and G genes from 8 and 7 to 1 and 2, respectively. Deletion of the NS1 gene resulted in viruses that were highly attenuated, well-tolerated highly-restricted and, in the case of LID/F1G2/ΔNS1, moderately immunogenic. The F1G2 gene shift appeared to increase immunogenicity, increasing the percentage of recipients with a ≥4-fold serum antibody response from 0% (LID/ΔNS1) to 75% (LID/F1G2/ΔNS1). This study remains in progress and will enroll additional RSV-seronegative subjects 6-24 months of age.

The available data raised the possibility that these viruses may be over-attenuated at the dose of 5.0 log₁₀PFU, since we generally consider it preferable to have a frequency of infectivity detected by culture of approximately 85% or greater, and to have mean peak titers of shed virus detected by culture of 3.0 to 4.0 log₁₀PFU/ml in order to have sufficient antigenic load. Of course, further data from the ongoing clinical study should have clarify the properties of these viruses. If either or both viruses are deemed to be over-attenuated, this likely can be compensated for by increasing the dose from 5.0 log₁₀PFU to 6.0 log₁₀PFU, as was done previously with another live-attenuated RSV candidate described below.

We previously evaluated, in RSV-seropositive and RSV-seronegative children, a virus called RSV ΔNS2/Δ1313/I1314L (with deletion of the NS2 gene and the presence of the Δ1313 codon deletion in L and stabilizing I1314L mutation in L) (Karron 2019. Safety and Immunogenicity of the Respiratory Syncytial Virus Vaccine RSV/ΔNS2/Δ1313/I1314L in RSV-Seronegative Children. J Infect Dis. pii: jiz408. doi: 10.1093/infdis/jiz408. [Epub ahead of print]; ClinicalTrials.gov Identifier: NCT01893554). When administed to RSV-seronegative children at a dose of 5.0 log₁₀PFU, the RSV ΔNS2/Δ1313/I1314L virus was well-tolerated. Shedding was detectable in 1 of 15 recipients by culture (peak titer 1.4 log₁₀PFU/ml) and in 11 of 15 recipients by RT-PCR (mean peak titer 3.0 log₁₀copies/ml). When administered at the 10-fold higher dose of 6.0 log₁₀PFU, infectivity was substantially improved, with shedding detected in 16 of 20 recipients by culture (mean peak titer of 1.8 log₁₀PFU/ml) and 18 of 20 recipients by RT-PCR (mean peak titer of 3.5 log₁₀copies/ml). Importantly, the percentage of recipients with a ≥4-fold rise in serum PRNT increased from 53% to 80%. The increase in dose had no effect on tolerability: the incidence of mild respiratory/febrile illness at the 5.0 and 6.0 log₁₀doses was 73% and 55%, respectively, compared to 14% and 70% for each respective placebo recipient group.

In conclusion, a Phase I clinical study in RSV-seronegative children 6-24 months of age showed that, at a dose of 5.0 log₁₀PFU, RSV LID/ΔNS1 and LID/F1G2/ΔNS1 are highly-attenuated, highly restricted, well tolerated, and that LID/F1G2/ΔNS1 is moderately immunogenic. Shifting the F and G genes to positions 1 and 2 was associated with increased immunogenicity.

TABLE 4

Clinical and virologic responses of RSV-seropositive children 12 to 59 months

of age to 10⁶PFU of recombinant live-attenuated respiratory syncytial virus vaccine,

RSV LID/ΔNS1, or 10^5.8PFU of RSV LID/F1G2/ΔNS1, or placebo.

Viral Detection
Viral Detection

(Immunoplaque assay)
(PCR)

Group,
Age at

Peak

Peak Copy
Respiratory
Any

Subject
Inoculation

Titer

Number
or Febrile
Antibody

number
(months)
Duration
Days
(log10)
Duration
Days
(log10)
Illness
Response
Infected

RSV LID/ΔNS1

1
59
0
0
0.5
0
0
1.7
0
0
0

2
59
0
0
0.5
0
0
1.7
1
0
0

3
38
0
0
0.5
0
0
1.7
0
0
0

4
22
0
0
0.5
0
0
1.7
1
0
0

5
25
0
0
0.5
0
0
1.7
0
0
0

6
24
0
0
0.5
0
0
1.7
1
0
0

7
38
0
0
0.5
0
0
1.7
0
0
0

8
39
0
0
0.5
0
0
1.7
0
0
0

9
29
0
0
0.5
0
0
1.7
0
0
0

10
51
0
0
0.5
0
0
1.7
0
0
0

Mean
38.4
0
0
0.5
0
0
1.7
3/10
0/10
0/10

SD
14.0
0
0
0.0
0
0
0.0

RSV LID/F1G2/ΔNS1

11
59
0
0
0.5
0
0
1.7
1
0
0

12
54
0
0
0.5
0
0
1.7
1
0
0

13
37
0
0
0.5
0
0
1.7
0
0
0

14
53
0
0
0.5
0
0
1.7
1
0
0

15
25
0
0
0.5
0
0
1.7
1
0
0

16
26
0
0
0.5
0
0
1.7
0
0
0

17
44
0
0
0.5
0
0
1.7
0
1
1

18
33
0
0
0.5
0
0
1.7
1
0
0

19
17
0
0
0.5
0
0
1.7
0
0
0

20
51
0
0
0.5
0
0
1.7
0
0
0

Mean
39.9
0
0
0.5
0
0
1.7
5/10
1/10
1/10

SD
14.4
0
0
0.0
0
0
0.0

Placebo

21
28
0
0
0.5
0
0
1.7
1
0
0

22
34
0
0
0.5
0
0
1.7
0
0
0

23
49
0
0
0.5
0
0
1.7
0
0
0

24
36
0
0
0.5
0
0
1.7
1
0
0

25
18
0
0
0.5
0
0
1.7
0
0
0

Mean
33
0
0
0.5
0
0
1.7
2/5
0/5
0/5

SD
11.4
0
0
0.0
0
0
0.0

TABLE 5

Immunologic responses of RSV-seropositive children 12 to 59 months of age

to 10⁶PFU of recombinant live-attenuated respiratory syncytial virus vaccine,

RSV LID/ΔNS1, or 10^5.8PFU of RSV LID/F1G2/ΔNS1, or placebo.

Serum RSV

neutralizing antibody
Serum IgG ELISA RSV F

(reciprocal log2)
antibody (reciprocal log2)

Group,
Age at

≥4

≥4

Subject
Inoculation
Pre-
Post-
fold
Pre-
Post-
fold

number
(months)
inoculation
inoculation
rise
inoculation
inoculation
rise

RSV LID/ΔNS1

1
59
7.8
7.3
0
13.4
13.1
0

2
59
10.5
10.1
0
17.2
15.8
0

3
38
6.9
7.2
0
14.2
14.1
0

4
22
10.9
9.1
0
16.7
16.4
0

5
25
12.5
10.7
0
17.7
17.6
0

6
24
6.7
5.6
0
12.8
11.8
0

7
38
6.5
5.5
0
14.2
13.9
0

8
39
6.2
5.8
0
14.2
14.2
0

9
29
7.1
6.1
0
14.4
14.0
0

10
51
9.7
9.4
0
14.6
14.7
0

Mean
38.4
8.5
7.7
0/10
14.9
14.6
0/10

SD
14.0
2.2
2.0

1.7
1.7

RSV LID/F1G2/ΔNS1

11
59
8.0
7.4
0
14.3
14.2
0

12
54
7.6
7.6
0
14.2
13.9
0

13
37
7.1
7.0
0
14.9
14.7
0

14
53
8.0
8.0
0
14.8
14.4
0

15
25
8.2
7.5
0
15.4
15.3
0

16
26
6.8
6.1
0
12.4
12.9
0

17
44
6.3
9.1
1
13.3
16.3
1

18
33
5.1
4.9
0
12.0
12.1
0

19
17
5.6
6.5
0
12.7
13.3
0

20
51
5.6
6.6
0
12.2
12.2
0

Mean
39.9
6.8
7.1
1/10
13.6
13.9
1/10

SD
14.4
1.1
1.1

1.3
1.3

Placebo

21
28
8.5
7.2
0
15.3
14.7
0

22
34
7.5
7.1
0
13.9
14.1
0

23
49
6.7
6.7
0
13.7
13.5
0

24
36
9.8
8.9
0
15.1
15.2
0

25
18
5.9
5.4
0
12.3
13.1
0

Mean
33
7.7
7.1
0/5
14.1
14.1
0/5

SD
11.4
1.5
1.3

1.2
0.9

TABLE 6

Clinical and virologic responses of RSV-seronegative children 6 to 24 months of age to 10⁵PFU of recombinant

live-attenuated respiratory syncytial virus vaccine, RSV LID/ΔNS1, or RSV LID/F1G2/ΔNS1, or placebo.

Viral Detection

(Immunoplaque assay)
Viral Detection (PCR)

Group,
Age at

Peak

Peak Copy
Respiratory
Any

Subject
Inoculation

Titer

Number
or Febrile
Antibody

number
(months)
Duration
Days
(log10)
Duration
Days
(log10)
Illness
Response
Infected

RSV LID/ΔNS1

1
11
0
0
0.5
0
0
1.7
0
0
0

2
7
7
5, 7
2.7
10
5, 7, 10
5.6
0
0
1

3
8
14
9, 12, 14
1.5
16
5, 7, 9,
5.1
1
0
1

12, 14, 16

4
14
0
0
0.5
0
0
1.7
1
0
0

Mean
10.0
10.5

2.1
13.0

5.4
2/4
0/4
2/4

SD
3.2
4.9

0.8
4.2

0.4

RSV LID/F1G2/ΔANS1

5
15
0
0
0.5
14
5, 9, 12, 14
3.7
1
1
1

6
8
0
0
0.5
12
7, 12
3.6
1
0
1

7
22
7
7
1.3
14
7, 10, 12, 14
4.0
1
1
1

8
16
11
9, 11
1.2
16
9, 11, 14, 16
4.3
0
1
1

Mean
15.3
9.0

1.3
14.0

3.9
3/4
3/4
4/4

SD
5.7
2.8

0.1
1.6

0.3

Placebo

9
14
0
0
0.5
0
0
1.7
1
0
0

10
16
0
0
0.5
0
0
1.7
0
0
0

Mean
15
0.0

0.5
0.0

1.7
1/2
0/2
0/2

SD
1.4
0.0

0.0
0.0

0.0

TABLE 7

Immunologic responses of of RSV-seronegative children 6 to 24 months

of age to 10⁵PFU of recombinant live-attenuated respiratory syncytial

virus vaccine, RSV LID/ΔNS1, or RSV LID/F1G2/ΔNS1, or placebo.

Serum RSV

neutralizing antibody
Serum IgG ELISA RSV F

(reciprocal log2)
antibody (reciprocal log2)

Group,
Age at

≥4

≥4

Subject
Inoculation
Pre-
Post-
fold
Pre-
Post-
fold

number
(months)
inoculation
inoculation
rise
inoculation
inoculation
rise

RSV LID/ΔNS1

1
11
2.3
3.6
0
7.2
7.7
0

2
7
2.3
4.2
0
8.1
9.9
0

3
8
2.3
2.3
0
7.1
6.1
0

4
14
3.6
2.3
0
11.2
11.3
0

Mean
10.0
2.6
3.1
0/4
8.4
8.8
0/4

SD
3.2
0.6
0.9

1.9
2.3

RSV LID/F1G2/ΔNS1

5
15
2.3
4.6
1
4.6
10.5
1

6
8
2.3
3.5
0
7.6
7.8
0

7
22
2.3
5.8
1
4.6
11.9
1

8
16
2.3
8.2
1
4.6
12.8
1

Mean
15.3
2.3
5.5
3/4
5.4
10.8
3/4

SD
5.7
0.0
2.0

1.5
2.2

Placebo

9
14
2.3
2.3
0
4.6
4.6
0

10
16
2.3
2.3
0
4.6
4.6
0

Mean
15
2.3
2.3
0/2
4.6
4.6
0/2

SD
1.4
0.0
0.0

0.0
0.0

Example 3
Prime-Boost Immunization with Live-Attenuated RSV Vaccines

In the present study, the boosting effect of rB/HPIV3 vectors expressing DS-Cav1-stabilized RSV pre-F protein, with or without the B3TMCT mutation, were evaluated in comparison with RSV. Boosting was evaluated in hamsters and African Green monkeys (AGMs) that previously had a primary RSV infection. In addition, the effect of different time intervals (˜2, ˜6, and ˜15 months) between the prime and boost was evaluated in AGMs. The results show that, in either experiment animal and for any of the time intervals, a boost by a rB/HPIV3 vector expressing RSV F engineered for enhanced immunogenicity induced significantly higher titers of serum RSV-neutralizing antibodies compared to a boost by RSV, particularly antibodies that are capable of neutralizing RSV in vitro without added complement and thus are particularly potent.

The RSV F and G proteins are the two RSV neutralization antigens and the major protective antigens. F is generally considered to be a more potent neutralization and protective antigen than G, and its amino acid sequence is much more conserved among RSV strains. RSV F is produced in a pre-fusion (pre-F) conformation that is metastable and can readily be triggered to undergo a major irreversible conformational rearrangement that drives membrane fusion and leaves F in a highly stable post-fusion (post-F) conformation (See McLellan et al. 2010. Structure of a major antigenic site on the respiratory syncytial virus fusion glycoprotein in complex with neutralizing antibody 101F. J Virol 84:12236-44; Swanson et al. 2011. Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers. Proc Natl Acad Sci USA 108:9619-24; McLellan et al. 2013. Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science 340:1113-7). Pre-F and post-F share some neutralizing epitopes, but most of the neutralizing activity in convalescent human sera recognizes epitopes specific to pre-F (Graham. 2017. Vaccine development for respiratory syncytial virus. Curr Opin Virol 23:107-112). RSV F can be substantially stabilized in the pre-F conformation by structure-based engineering, such as by the introduction of a disulfide bond called “DS” and two hydrophobic cavity-filling amino acid substitutions called “Cav1” (McLellan et al. 2013. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 342:592-8). DS-Cav-stabilized pre-F is substantially more immunogenic in rodents and non-human primates than post-F either as a subunit vaccine or expressed by a parainfluenza virus (PIV) vector (Liang et al. 2015. Enhanced Neutralizing Antibody Response Induced by Respiratory Syncytial Virus Prefusion F Protein Expressed by a Vaccine Candidate. J Virol 89:9499-510).

A number of attenuated derivatives of RSV strain A2 have been developed as candidate live-attenuated IN RSV vaccines (See Karron et al. 2013. Live-attenuated respiratory syncytial virus vaccines. Curr Top Microbiol Immunol 372:259-84). Some of these attenuated viruses have been shown to be well-tolerated and immunogenic in phase I studies in infants and young children.

Additionally, a PIV vector strategy to express RSV antigen, principally the F protein, from an added gene has been pursued. HPIV serotypes 1, 2, and 3 are important pediatric respiratory viruses, and HPIV3 is second only to RSV as a major cause of severe pediatric respiratory infection. The PIVs are enveloped non-segmented negative-sense RNA viruses that are related to RSV and belong to the family Paramyxoviridae of the order Mononegavirales. rB/HPIV3 consists of the bovine PIV3 (BPIV3) genome (which confers attenuation in humans and non-human primates by host range restriction) in which the BPIV3 F and hemagglutinin-neuraminidase (HN) glycoprotein genes (encoding the two PIV3 neutralization and major protective antigens) were replaced by their counterparts from human PIV3 (HPIV3).

Results

Comparison of booster immunization with RSV versus rB/HPIV3-RSV-pre-F vectors in hamsters previously infected with RSV. Two rB/HPIV3 vectors were previously described (Liang et al. 2017. Improved Prefusion Stability, Optimized Codon Usage, and Augmented Virion Packaging Enhance the Immunogenicity of Respiratory Syncytial Virus Fusion Protein in a Vectored-Vaccine Candidate. J Virol 91): rB/HPIV3/DS-Cav1 (abbreviated DS-Cav1 vector) that expresses RSV F with greatly increased stability in the pre-F conformation by the DS-Cav1 mutations; and rB/HPIV3/DS-Cav1/B3TMCT (abbreviated DS-Cav1/B3TMCT vector) that expresses RSV F with the same DS-Cav1 mutations plus replacement of its TMCT domains with those of the BPIV3 F protein to achieve efficient incorporation into the vector virion. These two vectors were compared to RSV D46 for the ability to infect and induce a secondary or booster response of serum RSV-neutralizing antibodies in hamsters that previously had received a single primary IN infection with RSV D46 (FIG. 14A). RSV D46 was used for the infections because attenuated RSV strains are poorly infectious in hamsters due to a strong host range restriction.

Forty-eight hamsters were confirmed to be seronegative for RSV and HPIV3, placed into four groups (Groups A-D, n=12 each), and primed by IN inoculation with 10⁶PFU RSV D46 per animal (FIG. 14A). Forty-eight additional RSV- and HPIV3-seronegative hamsters in four groups (Groups E-H, n=12 each) were left unprimed for comparison. Six weeks post-priming, sera were collected to measure RSV- and HPIV3-PRNTs. Two days later, pairs of primed and unprimed groups were boosted IN with: (i) empty rB/HPIV3 vector (Groups A and E); (ii) DS-Cav1 vector (Groups B and F); (iii) DS-Cav1/B3TMCT vector (Groups C and G); or (iv) RSV D46 (Groups D and H). The vectors were given at a dose of 10⁵TCID₅₀per animal, and RSV D46 at a dose of 10⁶PFU. Five days after boosting, six hamsters per group were sacrificed and nasal turbinates (NT) and lungs were harvested and assayed by immunoplaque assay (RSV) or limiting dilution assay (rB/HPIV3 vectors) to measure infectious virus titers. For the other six hamsters per group, sera were collected two weeks post-boost to measure RSV- and HPIV3-PRNT. Two days later, the animals were challenged IN with RSV D46 as described below.

Analysis of virus titers in unprimed animals five days following the boost showed that the DS-Cav1 and DS-Cav1/B3TMCT vectors replicated to lower titers than the empty vector in the NT and lungs (FIGS. 14B and 14C, lanes 3 and 5 versus 1, respectively), indicating attenuation due to the presence of the RSV F insert. The DS-Cav1/B3TMCT vector was somewhat more attenuated than the DS-Cav1 vector and consistent with the idea that incorporation of RSV F into the vector envelope might interfere with vector replication.

Comparison of RSV-primed versus unprimed animals (FIGS. 14B and 14C: even lanes versus odd lanes) showed that the priming immunization completely restricted the replication of the RSV D46 boost (FIGS. 14B, 14C, lane 8 versus 7) but had no effect on replication of the empty rB/HPIV3 vector (lane 2 versus 1), consistent with the expectation that RSV-specific immunity would restrict the replication of RSV but not rB/HPIV3. Priming with RSV resulted in a modest restriction (2-fold in the NT, 4-fold in the lungs) of the replication the DS-Cav1 vector (FIGS. 14B, 14C, lane 4 versus 3), and a somewhat greater restriction of the DS-Cav1/B3TMCT vector (32-fold in the NT, 10-fold in the lungs; FIGS. 14B and 14C, lane 6 versus 5), although the difference between primed and unprimed groups was statistically significant only for the DS-Cav1/B3TMCT vector in the NT (FIG. 14B, lane 6 versus 5). These observations suggested that the expression of RSV F by rB/HPIV3 conferred a low level of sensitivity to restriction by RSV-specific immunity that was increased somewhat when RSV F was packaged into the vector virion.

Analysis of the titers of HPIV3-neutralizing antibodies in sera collected from unprimed animals two weeks following the boost showed that the presence of either RSV insert in the rB/HPIV3 vector was associated in a decrease in HPIV3-neutralizing antibodies compared to the empty vector (FIG. 14D, lanes 3 and 5 versus 1), consistent with the decreased replication associated with the insert as noted above. The presence of the DS-Cav1 insert did not detectably reduce HPIV3 immunogenicity in primed versus unprimed animals (FIG. 14D, lane 4 versus 3), but the DS-Cav1/B3TMCT insert resulted in a significant decrease in HPIV3 immunogenicity in primed versus unprimed animals (FIG. 14D, lane 6 versus 5), consistent with its restriction as noted above.

Serum RSV-PRNTs induced by priming and boosting were determined by assays with or without added guinea pig complement (FIGS. 14E and 14F). The addition of complement can confer steric hindrance and viral lysis activity to antibodies, resulting in enhanced detection of RSV-specific antibodies that might otherwise have poor neutralizing activity. The neutralization assay in the absence of added complement is a more stringent assay for “high-quality” antibodies that directly neutralize RSV (see Liang et al. 2016. Packaging and Prefusion Stabilization Separately and Additively Increase the Quantity and Quality of Respiratory Syncytial Virus (RSV)-Neutralizing Antibodies Induced by an RSV Fusion Protein Expressed by a Parainfluenza Virus Vector. J Virol 90:10022-10038; Liang et al. 2017. Improved Prefusion Stability, Optimized Codon Usage, and Augmented Virion Packaging Enhance the Immunogenicity of Respiratory Syncytial Virus Fusion Protein in a Vectored-Vaccine Candidate. J Virol 91).

The initial priming infection with RSV D46 induced high serum RSV-PRNTs measured in the presence of complement, and lower RSV-PRNTs measured in the absence of complement. Specifically, the sera collected from Groups A-D six weeks following priming had mean serum RSV-PRNTs within the range 10.6 log₂(1:1,552) to 11.2 log₂(1:2,353) determined with complement (FIG. 14E), and 4.5 log₂(1:23) to 5.2 log₂(1:37) determined without complement (FIG. 14F).

Boosting with RSV D46 increased the mean serum RSV-PRNT measured with complement (FIG. 14E, lane 7 versus 8) by 3-fold, to a post-boost mean titer of 12.3 log₂(1:5,043), and increased the mean serum RSV-PRNT measured without complement (FIG. 14F, lane 7 versus 8) also by 3-fold, to a post-boost mean titer of 6.6 log₂PRNT (1:97).

Boosting with the DS-Cav1 and DS-Cav1/B3TMCT vectors increased the mean serum RSV-PRNT measured with complement by 8- and 6-fold, respectively, to remarkably high post-boost mean titers of 13.8 log 2 (1:14,263) and 13.7 log 2 (1:13,308) (FIG. 14E, lane 3 versus 4 for DS-Cav1, and lane 5 versus 6 for DS-Cav1/B3TMCT). Pre- and post-boost mean serum RSV-PRNTs measured without complement increased by 9- and 18-fold, respectively, to post-boost mean titers of 8.3 log 2 (1:315) and 9.3 log 2 (1:630) (FIG. 14F, lanes 3 versus 4 for DS-Cav1 vector and 5 versus 6 for DS-Cav1/B3TMCT vector).

The six animals per group used for post-boost serology also were challenged two days following serum collection by IN infection with 10⁶PFU of RSV D46 per animal, and NT and lungs were collected 3 days post-challenge and challenge RSV titers were determined by immunoplaque assay. Animals that were not primed with RSV and were boosted with empty rB/HPIV3 vector, and thus had no RSV-specific immunity, had approximately 5.0 log₁₀PFU/g tissue of challenge RSV in the NT and lungs. All of the hamsters in the other groups, which had been primed and/or boosted with RSV D46 and/or rB/HPIV3 expressing RSV F, had no detectable infectious challenge RSV in the NT and lungs (data not shown). Thus, all of the combinations of priming and/or boosting were highly protective in this semi-permissive model for RSV replication and could not be distinguished based on protective efficacy.

In summary, when rB/HPIV3 vectors were used for the booster immunization, there was substantial vector replication and robust increases in serum RSV-PRNT, resulting in very high titers. In contrast, when RSV D46 was used as a boost, its replication was highly restricted and the increase in RSV-PRNT was significantly lower. Furthermore, the fold-increase in serum RSV-PRNT induced by the rB/HPIV3 vectors was greater for the high-quality RSV neutralizing serum antibodies measured without complement.

Comparison of booster immunizations in AGMs. Booster immunizations also were evaluated in AGMs that had previously been primed by a single RSV infection by the combined IN/IT routes (10⁶PFU per site). The viruses used in these primary immunizations were various attenuated derivatives of RSV strain A2, or the wt strain rRSV A/Maryland/001/11 (which, like strain A2, is from subgroup A). The priming RSVs are listed in Tables 8-10 and described below. Note that we treated all of these viruses as being equivalent with respect to priming, although we did distribute the animals that received the various viruses evenly between boosting groups. In three separate experiments (AGM experiments #1-#3), the previously-primed AGMs were given a single booster infection by the combined IN/IT routes with an attenuated RSV (10⁶PFU per site) or DS-Cav1/B3TMCT vector (10⁶TCID₅₀per site). In experiment #2, additional RSV-primed AGMs received DS-Cav1 vector (10⁶TCID₅₀per site). In all cases, the booster RSV was a live-attenuated vaccine candidate called RSV 276, which is a version of RSV strain A2 attenuated by deletion of most of the M2-2 ORF.

For 12 animals (AGM experiment #1, FIG. 15 and Table 8), the time interval between prime and boost was ˜2 months (specifically 51 days, which equal two months minus nine days). For 20 other animals (AGM experiment #2, FIG. 16 and Table 9), the time interval between prime and boost was ˜6 months (specifically 189 days, which equal six months plus nine days). For the remaining four animals (AGM experiment #3, FIG. 17 and Table 10), the time interval was ˜15 months (specifically 443 days, which equal 15 months minus seven days).

To monitor virus shedding following the boost, NP and TL were collected daily and every other day, respectively, for ten consecutive days, and virus titers were determined by immunoplaque assay (RSV) or limiting dilution (B/HPIV3 vectors). Sera were collected on one or more days prior to the day of the boost, on the day of the boost, and every seven days thereafter for four consecutive weeks, and PRNTs were determined.

AGM experiment #1: Booster immunization of AGMs ˜2 months (two months minus nine days) following priming with RSV. Twelve AGMs were available that had previously received a single priming immunization with one of three different RSVs (Table 8): (i) RSV LID/ΔNS1, an NS1 gene-deletion mutant; (ii) RSV LID/F1G2/ΔNS1, a derivative of the preceding virus in which the RSV F and G genes were moved to the 1^stand 2^ndgenome position in order to increase their expression; and (iii) the subgroup A wt strain rRSV A/Maryland/001/11. On Day 37 post-priming (two weeks before boosting) sera were collected and analyzed to determine RSV-PRNT in the presence of complement, and to confirm HPIV3-seronegativity. The 12 AGMs were re-distributed into two groups of six animals each organized so that the two groups had similar numbers of animals with high, medium, or low Day 37 serum RSV-PRNTs, had essentially identical group mean RSV-PRNTs, and were balanced with regard to the identities of the priming viruses and sex ratio (Table 8).

TABLE 8

Group assignments of AGMs that were boosted ~2 months following priming (experiment #1).

Mean pre-

Pre-boosting
boosting
Post-boosting

serum RSV-
RSV serum-
RSV serum RSV-

neutralizing
neutralizing
neutralizing

Animal

Boosting
titer
titer
titer

ID
Sex
Priming RSV
virus
(Log₂PRNT) ^a
(Log₂PRNT)
(Log₂PRNT) ^b

9131
M
LID/F1G2/ΔNS1
RSV 276
4
8.1
12.8

9128
F
LID/F1G2/ΔNS1
(n = 6)
8.1

13.6

9147
M
LID/ΔNS1

6.5

11.2

9136
F
LID/ΔNS1

8.8

13

9143
F
A/Maryland/001/11

10.3

12.4

9141
M
A/Maryland/001/11

10.8

11.9

9084
M
LID/F1G2/ΔNS1
DS-Cav1/
6.9
8.1
15.5

9135
F
LID/F1G2/ΔNS1
B3TMCT
5.5

15.3

9140
M
LID/ΔNS1
(n = 6)
7.8

13.1

9116
F
LID/ΔNS1

7.4

13.8

9161
M
A/Maryland/001/11

10.2

14.6

9173
F
A/Maryland/001/11

11

14.6

^aSera were collected two weeks before boosting and were analyzed with RSV PRN assay with added complement. Values are for individual animals.

^bSera were collected two weeks after boosting and were analyzed with RSV PRN assay with added complement. Values are for individual animals.

On Day 51, one group was boosted with RSV 276 and the other with DS-Cav1/B3TMCT vector. NP, TL, and serum specimens were collected to evaluate virus shedding and immunogenicity (FIGS. 15B-15E). Shedding of RSV 276 was detected only in trace amounts on a single day in the NP and was not detected in the TL. It was evident that the very low shedding of RSV 276 was due to restriction by RSV-specific immunity in the RSV-primed animals because, in a previous experiment, infection of four RSV-seronegative AGMs with RSV 276 resulted in substantial viral shedding over a period of 10 days, with the highest peak mean titers approaching 3.0 log₁₀PFU/ml in NP and TL (FIG. 19). In contrast, shedding of DS-Cav1/B3TMCT vector was substantial over 10 days in both the NP and TL, with peak mean titers of approximately 5.0 log₁₀TCID₅₀/ml in the NP and 3.4 log₁₀TCID₅₀/ml in the TL (FIGS. 15B and 15C).

The mean pre-boost serum RSV-PRNT for all 12 animals measured with complement was 1:256 (FIG. 15D). Boosting with RSV 276 and the DS-Cav1/B3TMCT vector increased the serum RSV-PRNTs measured with complement by 22-fold and 91-fold over the pre-boost titer to peak mean titers of 1:5,793 and 1:23,170, respectively (occurring on day 14 post-boost), which were significantly different (FIG. 15D). The peak mean post-boost serum RSV-PRNT for the vector was 4-fold greater than for RSV 276.

Measured in the absence of complement, the mean pre-boost serum RSV-PRNT for all 12 animals was 1:12 (FIG. 15E). Boosting with RSV 276 and the DS-Cav1/B3TMCT vector increased the titer by 25-fold and 366-fold, respectively, to peak mean titers of 1:294 and 1:4,390, respectively (occurring on day 14 post-boost; FIG. 15E), which were significantly different. The peak mean post-boost serum RSV-PRNT for the vector was 15-fold greater than for RSV 276. Thus, the vector had a stronger boosting effect than RSV 276, particularly for “high-quality” neutralizing antibodies detected without complement.

AGM experiment #2: Booster immunization of AGMs ˜ 6 months (6 months plus nine days) following priming with RSV. Twenty other AGMs were available that had previously received a single priming immunization with one of five different RSVs (Table 9): (i) RSV D46/NS2/N/ΔM2-2-HindIII, a M2-2 deletion mutant; (ii) RSV LID/ΔNS2/1030s, a NS2 deletion mutant with a genetically stabilized temperature sensitivity (ts) mutation in the L polymerase; (iii) RSV LID/ΔNS1, an NS1 deletion mutant that also was one of the priming viruses in experiment #1, but in different AGMs; (iv) RSV LID/F1BBG2/ΔNS1/, a version of the preceding LID/ΔNS1 mutant in which the F gene was codon-optimized for increased translation and the F and G genes were moved to the 1^stand 2^ndgenome positions, respectively, for increased expression; and (v) RSV 276 (described above), which also is the attenuated ΔM2-2 RSV used in the boosts, as already noted.

TABLE 9

Group assignments of AGMs that were boosted ~6 months following priming (experiment #2).

Mean pre-

Pre-boosting
boosting
Post-boosting

serum RSV-
RSV serum-
RSV serum RSV-

neutralizing
neutralizing
neutralizing

Animal

Boosting
titer
titer
titer

ID
Sex
Priming RSV
virus
(Log₂PRNT)^a
(Log₂PRNT)
(Log₂PRNT) ^b

8926
M
D46/NS2/N/ΔM2-2-HindIII
RSV 276
7.1
6.0
11.4

8983
M
LID/ΔNS2/1030s
(n = 6)
8

11.4

8974
F
LID/F1BBG2/ΔNS1

6.9

13.3

8951
M
LID/ΔNS1

3.3

12

8960
F
LID/ΔNS1

5.2

13.3

8918
F
RSV 276

5.7

12.3

8938
F
D46/NS2/N/ΔM2-2-HindIII
DS-Cav1
6.7
6.0
14.8

8911
M
D46/NS2/N/ΔM2-2-HindIII
(n = 7)
4.3

16.2

8928
F
LID/ΔNS2/1030s

7.5

13.4

8994
M
LID/F1BBG2/ΔNS1

4.5

14.3

8966
M
LID/F1BBG2/ΔNS1

5.1

16.1

8992
F
LID/ΔNS1

6.6

14.5

8913
M
RSV 276

7.3

13.6

9041
F
D46/NS2/N/ΔM2-2-HindIII
DS-Cav1/
7.1
6.1
14.9

9045
M
LID/ΔNS2/1030s
B3TMCT
8.5

14.1

8940
F
LID/ΔNS2/1030s
(n = 7)
6.7

12.9

8922
F
LID/F1BBG2/ΔNS1

5.1

22.3

8904
M
LID/ΔNS1

7.1

15.2

8902
M
RSV 276

3.7

14.4

8952
M
RSV 276

4.7

15.5

^aSera collected 35 days before boosting were analyzed with RSV PRN assay with added complement. Values are for individual animals.

^bSera collected two weeks after boosting were analyzed with RSV PRN assay with added complement. Values are for individual animals.

Following the primary immunization, sera were collected on Days 28 and 154 and analyzed to measure RSV-PRNTs in the presence of complement as well as to confirm HPIV3-seronegativity (one animal, #AG 8960 [Table 9], was found to be HPIV3-seropositive and therefore was assigned to be boosted by RSV rather than a rB/HPIV3 vector). The 20 AGMs were re-distributed into three groups (n=6, 7, and 7) organized to balance the Day-154 serum RSV-PRNTs, the identities of the priming viruses, and sex ratio (Table 9). On Day 189 (6 months plus nine days) post-priming, the three groups were boosted with RSV 276, DS-Cav1 vector, or DS-Cav1/B3TMCT vector. NP, TL, and serum specimens were collected to evaluate virus shedding and immunogenicity (FIG. 16).

Shedding of RSV 276 was undetectable in the NP by immunoplaque assay (FIG. 161B), whereas there was low, sporadic detection of RSV 276 in the TL (FIG. 16C). Evaluation of TL specimens for a number of animals by RT-qPCR (that detected both positive- and negative-sense RSV M RNA) was more sensitive, yielding a greater number of samples positive for RSV RNA, with titers up to 450-5,500 RNA molecules/ml (FIG. 20). In contrast, shedding of the DS-Cav1 and DS-Cav1/B3TMCT vectors was detected abundantly by limiting dilution assay in the NP and TL over a period of 8 to 10 days, with similar peak mean titers of approximately 5.5 log₁₀TCID₅₀/ml in the NP and 5.0 log₁₀TCID₅₀/ml in the TL. The DS-Cav1/B3TMCT vector appeared to replicate slightly slower than the DS-Cav1 vector, but this difference was significant only on Days 2 and 4 in the TL (FIG. 16C). The two vectors eventually reached similar peak NP and TL titers (FIGS. 16B and 16C).

When measured by neutralization assays with complement (FIG. 16D), the mean serum RSV-PRNT in the 20 AGMs induced by the primary immunization with the attenuated RSVs approached 10 Log₂(1:1,024) at Day 28 (FIG. 16D), and then decreased to 1:64 on Day 189 when the boost was given. Boosting with RSV 276, DS-Cav1 vector, and DS-Cav1/B3TMCT vector increased the peak mean serum RSV-PRNTs measured with complement by 78-fold, 260-fold, and 776-fold, respectively, to peak mean titers of 1:4,973, 1:16,615, and 1:49,667 (occurring 14 days post-boost; FIG. 16D), which were significantly different for each vector versus RSV 276. The peak mean post-boost serum RSV-PRNTs for the DS-Cav1 and DS-Cav1/B3TMCT vectors were 3-fold and 10-fold higher, respectively, than for RSV 276. The DS-Cav1/B3TMCT vector appeared to be slightly more immunogenic than the DS-Cav1 despite its slightly slower replication noted above, which might be due to the B3TMCT modification, but the difference was not statistically significant (FIG. 16D).

When measured by PRN assay without complement (FIG. 16E), the mean pre-boost serum RSV-PRNT in the 20 AGMs was 1:8. Boosting with RSV 276, DS-Cav1 vector, and DS-Cav1/B3TMCT vector increased the peak mean serum RSV-PRNT by 37-fold, 315-fold, and 446-fold, respectively, to titers of 1:294, 1:2,521, and 1:3,565 (occurring 14 days post-boost), which were significantly different for each vector versus RSV 276. The peak mean post-boost serum RSV-PRNTs for the DS-Cav1 and DS-Cav1/B3TMCT vectors were 9-fold and 12-fold higher than for RSV 276.

The ability of boosts with the DS-Cav1 and DS-Cav1/B3TMCT vectors to induce serum HPIV3-neutralizing antibodies was compared using HPIV3 neutralization assays without added complement (FIG. 21). Note that the animals were seronegative for HPIV3 before the boosts, and thus these inoculations were primary immunizations with respect to HPIV3. The two vectors induced very similar peak mean serum HPIV3-PRNTs (1:724 and 1:776, respectively, occurring on day 14 post-innoculation), indicating they were essentially equally immunogenic for HPIV3. This is in agreement with the observation that the two vectors replicated to similar peak titers in AGMs (FIGS. 16B and 16C).

The ability of the boosts with the DS-Cav1 and DS-Cav1/B3TMCT vectors versus RSV 276 to induce serum and mucosal IgA antibodies that bind RSV DS-Cav1 F protein was also assessed (FIGS. 16F and 16G). A highly sensitive DELFIA TRF immunoassay was used to detect monkey IgA binding to purified recombinant RSV DS-Cav1 F protein as antigen. The IgA titers are expressed as the log₂dilution yielding 400 fluorescence units in the DELFIA TRF assay. Boosting with the two vectors induced strong serum IgA responses: peak mean titers following boosts with the two vectors were identical (19.5 log₂) and were about 16-fold higher than those induced by boosting with RSV 276 (15.6 log₂). The peak serum IgA response was detected at 14 days post-boost, coinciding with the peak serum RSV-neutralizing antibody response. Since the respiratory mucosal antibody response is considered to be particularly effective in limiting RSV infection, we also evaluated the nasal mucosal IgA response to the boosts. Nasal mucosal lining fluid was collected using absorptive membranes (SAM strips). This method provides relatively concentrated mucosal samples, suitable to detect IgA (FIG. 16G). In all groups, the peaks of the mucosal IgA responses were at day 14 following the boosts (FIG. 16G), coinciding with the peaks of the serum IgA (FIG. 16F) and the serum RSV-neutralizing antibodies (FIGS. 16D and 16E). Again, the DS-Cav1 and DS-Cav1/B3TMCT vectors induced similarly strong responses (peak mean titers of 12.1 and 12.8 log₂, respectively), whereas the response to RSV 276 was about 8-fold lower (peak mean titer of 9.2 log₂). Thus, the DS-Cav1 and DS-Cav1/B3TMCT vectors significantly exceeded RSV 276 in their ability to boost the serum and mucosal IgA antibody responses to the RSV pre-F protein.

AGM experiment #3: Booster immunization of AGMs ˜ 15 months (15 months minus seven days) following priming with RSV. Four other AGMs were available that had previously received a single primary immunization with RSV 276 (Table 10). On Day 429 following the primary infection (and two weeks before boosting), sera were collected and analyzed to determine RSV-PRNT in the presence of complement, and to confirm HPIV3-seronegativity. The AGMs were distributed into two groups (n=2 each) that were balanced with regard to serum RSV-PRNTs and sex ratios (Table 10). On Day 443 (15 months minus seven days) following the primary infection, the two groups were boosted with RSV 276 or DS-Cav1/B3TMCT vector and viral replication and serological responses were monitored as described above.

TABLE 10

Group assignments of AGMs that were boosted ~15 months following priming (experiment #3).

Mean pre-

Pre-boosting
boosting
Post-boosting

serum RSV-
RSV serum-
RSV serum RSV-

neutralizing
neutralizing
neutralizing

Animal

Boosting
titer
titer
titer

ID
Sex
Priming RSV
virus
(Log₂PRNT) ^a
(Log₂PRNT)
(Log₂PRNT) ^b

8903
M
RSV 276
RSV 276
6.2
5.4
14

8986
F
RSV 276
(n = 6)
4.6

11.6

8900
M
RSV 276
DS-Cav1/B3TMCT
5.2
4.7
15.9

9025
F
RSV 276
(n = 6)
4.2

13.9

^aSera were collected two weeks before boosting and were analyzed with RSV PRN assay with added complement. Values are for individual animals.

^bSera were collected two weeks after boosting and were analyzed with RSV PRN assay with added complement. Values are for individual animals.

Replication of RSV 276 following the ˜15-month interval was highly restricted, with no virus detected by immunoplaque assay of the NP and TL, whereas replication of the DS-Cav1/B3TMCT vector was robust (FIGS. 17B and 17C).

The mean pre-boost serum RSV-PRNT for the four animals measured with complement was 1:33 (FIG. 17D). Boosting with RSV 276 and DS-Cav1/B3TMCT vector increased the peak mean serum RSV-PRNT measured in the presence of complement by 216-fold and 926-fold, respectively, to titers of 1:7,132 and 1:30,574 (occurring on day 14, FIG. 17D). The peak mean post-boost serum RSV-PRNT for the vector was 4-fold higher than for RSV 276.

The mean pre-boost serum RSV-PRNT for the four animals measured without complement was 1:6 (FIG. 17D). Boosting with RSV 276 and the DS-Cav1/B3TMCT vector increased the peak mean serum RSV-PRNTs by 171-fold and 1,274-fold, respectively, to titers of 1:1,024 and 1:7,643 (occurring on day 14, FIG. 17E). The peak mean post-boost serum RSV-PRNT for the vector was 7-fold higher than for RSV 276.

RSV serum antibodies suppressed DS-Cav1/B3TMCT vector replication in vitro. Because the replication of the DS-Cav1/B3TMCT vector in hamsters was significantly reduced in the NT by RSV-specific immunity from the primary infection (FIGS. 14B-14D), whether replication of this vector in vitro could be inhibited by RSV-neutralizing antibodies was investigated. LLC-MK2 cells were infected with the empty rB/HPIV3 vector or with the DS-Cav1 or DS-Cav1/B3TMCT vector at an MOI of 0.01, and then incubated with medium containing 10% heat-inactivated (56° C. for 30 min) serum from uninfected hamsters or from hamsters infected with RSV D46 or rB/HPIV3 (from the experiment in FIG. 14), in the absence of added complement (FIG. 18). Replication of the vectors in the presence of added hamster sera was monitored by taking a small aliquot of culture medium supernatant daily for three consecutive days for virus titration.

As expected, rB/HPIV3-immune serum completely inhibited the replication of all three vector constructs (FIGS. 18A-18C, black curve) while the pre-immune serum, collected prior to the primary immunization, did not affect the replication of any construct (FIGS. 18A-18C). RSV-immune serum had no effect on the replication of empty rB/HPIV3 vector (FIG. 18A) or DS-Cav1 vector (FIG. 18B), but it significantly reduced the replication of the DS-Cav1/B3TMCT vector by Days 2 and 3, the latter by 100-fold (FIG. 18C). These results indicated that RSV-specific serum antibodies inhibited replication of the DS-Cav1/B3TMCT vector, but not the DS-Cav1 vector.

DISCUSSION

Non-limiting approaches to developing a pediatric RSV vaccine include (i) attenuated derivatives of RSV, and (ii) rB/HPIV3 expressing the RSV F protein from an added gene. Both approaches have provided promising candidates presently under evaluation for primary immunization, but their usefulness for booster immunization was less clear. In previous pediatric clinical studies, live-attenuated RSVs as vaccine candidates were inefficient (at least with regard to inducing increases in RSV-specific serum antibodies) in booster immunizations administered in successive doses over several months. For example, when the live-attenuated RSV called MEDI-559 was given to infants and young children in three successive doses at 2-month intervals, the second and third doses generally were poorly infectious and inefficient in boosting serum antibody responses in those subjects who had a “take” with a previous dose (See Malkin et al. 2013. Safety and immunogenicity of a live attenuated RSV vaccine in healthy RSV-seronegative children 5 to 24 months of age. PLoS One 8:e77104). Similar findings were made when the live-attenuated RSV cpts248/404 was given to infants and young children in two successive doses at an interval of 4-6 weeks (See Wright et al. 2000. Evaluation of a live, cold-passaged, temperature-sensitive, respiratory syncytial virus vaccine candidate in infancy. J Infect Dis 182:1331-42). Similar findings also have been made with attenuated versions of PIV3 given in successive doses at intervals of up to 6 months. Thus, in a “homologous” prime-boost (i.e., successive doses of the same vaccine virus), an attenuated RSV (or PIV3) usually is infectious and substantially immunogenic for serum virus-neutralizing antibodies only for the first “take”, at least within a time interval of 6 months or less. Incidentally, the situation is different when the secondary infection is with RSV D46 rather than an attenuated RSV. Specifically, in vaccine trials in which subjects received an attenuated RSV during the summer and had natural exposure to community RSV D46 during the following winter, there were 20- to 40-fold boosts in serum RSV-PRNT (See Karron et al. 2015. A gene deletion that up-regulates viral gene expression yields an attenuated RSV vaccine with improved antibody responses in children. Sci Transl Med 7:312ra175). Thus, the poor immunogenicity of repeat doses of attenuated RSVs appears largely due to their attenuation.

The present study investigated a “heterologous” prime-boost strategy. In this strategy, a primary immunization with RSV was boosted by a secondary immunization with rB/HPIV3 expressing RSV F protein from an added gene. The RSV F protein was substantially stabilized in the pre-F conformation by the DS-Cav1 mutations and, in the case of the DS-Cav1/B3TMCT vector used in each experiment, contained the B3TMCT modification that mediates efficient packaging of RSV F into the vector virion. While the rB/HPIV3 vector itself is antigenically distinct from RSV, it does express an RSV antigen, in this case the F protein. This might be targeted by RSV-specific immunity including RSV-specific serum antibodies, as measured in the present study, as well as other effectors that historically have been less well characterized, including RSV-specific mucosal antibodies and cellular immunity. With regard to RSV-specific antibodies, binding to RSV F expressed by the vector probably would not directly block vector infection and spread because the RSV F in this study was largely non-functional for fusion due to the DS-Cav1 mutations and therefore presumably would have minimal or no contribution to vector replication and spread. However, the expression of RSV F in vector-infected cells might target those cells for destruction by cytotoxic T cells or antibody-dependent cell-mediated cytotoxicity. In addition, as noted, the DS-Cav1/B3TMCT vector used in all of experiments in the present study expressed the B3TMCT version of RSV F protein that was efficiently packaged in the vector virion, and this might target the vector virion for destruction such as by opsonization. Binding of antibodies to packaged RSV F might also create steric hindrance that interferes with the vector HN and F proteins during attachment and entry. It also was possible that antibody responses to RSV F might be suppressed by pre-existing F-specific antibodies.

Homologous and heterologous boosting was assessed in hamsters. When hamsters were given a primary infection with RSV D46 and a homologous booster infection with RSV D46 ˜6 weeks later, there was no detectable infectious RSV in harvested NT and lung tissue, consistent with the expectation that RSV-specific immunity would severely restrict replication of an RSV boost. However, there was a significant, ˜3-fold increase in serum RSV-PRNT measured with or without added complement, suggesting that some infection and antigen expression had occurred. In hamsters boosted with the empty rB/HPIV3 vector, there was no difference in virus titers in the NT and lungs between RSV-primed versus unprimed animals, showing that RSV-specific immunity indeed did not restrict the rB/HPIV3 backbone. In contrast, the replication of the DS-Cav1 and DS-Cav1/B3TMCT vectors exhibited a modest amount of restriction in RSV-primed versus unprimed animals, although the difference was significant only in the case of the DS-Cav1/B3TMCT vector in the NT. This suggests that expression of the unpackaged version of the RSV F protein resulted in a marginal restriction of the vectors by RSV-specific immunity, and expression of the packaged version of RSV F protein resulted in a somewhat greater restriction. This was associated with a significant decrease in the induction of serum HPIV3-neutralizing antibodies by the DS-Cav1/B3TMCT vector (but not the DS-Cav1 vector) in RSV-primed versus unprimed hamsters, providing another indication of reduced DS-Cav1/B3TMCT vector replication. In the RSV-primed hamsters, the DS-Cav1 and DS-Cav1/B3TMCT vectors induced increases in serum RSV-PRNT assayed with complement of 8- and 6-fold versus 3-fold for RSV D46; when assayed without complement, the increases were 9-and 18-fold versus 3-fold, respectively. The resulting peak mean post-boost serum RSV-PRNTs assayed with and without complement were significantly greater for the vectors than for RSV D46. Thus, these results in hamsters showed that (i) homologous boosts with RSV D46 were highly restricted and modestly immunogenic; (ii) the empty rB/HPIV3 vector was unrestricted by RSV-specific immunity; (iii) expression of RSV F by rB/HPIV3 conferred a low level of sensitivity to restriction by RSV-specific immunity that became significant when RSV F was packaged into the vector virion, and (iv) boosts with the vectors were significantly more immunogenic than with RSV D46.

Boosting also was evaluated in AGMs that previously had a primary RSV infection. Three different time intervals between the primary RSV infection and the boost were evaluated: ˜2, ˜6, and ˜15 months. It was anticipated that pre-boost host immunity might diminish with increasing interval of time, which might affect the efficiency of the boost. Indeed, the mean pre-boost serum RSV-PRNTs diminished progressively in the ˜2-, ˜6-, and ˜15-month groups, with respective titers of 1:256, 1:64, and 1:33 measured with complement, and 1:12, 1:8, and 1:6 measured without complement. It's reasonable to suppose that other immune effectors induced by the primary RSV infection, such as mucosal antibodies and cellular immunity, also diminished with time.

The replication of the RSV 276 boost was highly and nearly-equally restricted following each time interval, with only sporadic traces of shed infectious virus. Thus, there was no difference between the time intervals in the ability to strongly restrict this attenuated RSV. Although shedding of infectious RSV was highly restricted, there presumably was some viral infection and antigen expression leading to the observed secondary immune response. This is supported by the RT-qPCR performed in experiment #2 that detected shedding of viral nucleic acid, which may have included progeny virus that was neutralized by secretory antibodies and not detected by an infectivity assay.

In contrast to RSV, the DS-Cav1/B3TMCT vector replicated robustly. The peak mean titers of vector in the NP and TLs were very similar for experiments #2 and #3, ranging from 4.8 to 5.4 log₁₀TCID₅₀/ml. This level of shedding of DS-Cav1/B3TMCT vector was similar to that observed previously in RSV- and HPIV3-seronegative rhesus macaques (See Liang et al. 2017. Improved Prefusion Stability, Optimized Codon Usage, and Augmented Virion Packaging Enhance the Immunogenicity of Respiratory Syncytial Virus Fusion Protein in a Vectored-Vaccine Candidate. J Virol 91), suggesting that there was little or no restriction of the vector by pre-existing immunity to RSV following the ˜6-month and ˜15-month time intervals. In contrast, for experiment #1 (˜2-month time interval), the titers of shed DS-Cav1/B3TMCT vector were lower by approximately 0.5 log₁₀TCID₅₀/ml in the NP and by 1.4 to 1.8 log₁₀TCID₅₀/ml in the TL (Table 11). This might indicate that expression of RSV F by this vector, and the packaging of RSV F into the vector virion, conferred a moderate amount of sensitivity to restriction by RSV-specific immunity following the shortest interval, similar to the restriction of the DS-Cav1/B3TMCT vector in RSV-primed versus unprimed hamsters. Since cellular immunity induced by a primary infection may persist only for several months, as noted above for the hamster model, this may have contributed to the restriction of the DS-Cav1/B3TMCT vector observed at ˜2 months but not at ˜6 and ˜15 months. In any event, however, the restriction following the ˜2-month interval was small and not statistically significant.

TABLE 11

Pre-boost serum RSV-neutralizing titers in AGMs boosted with DS-Cav1/B3TMCT

and peak mean titers of post-boosting replication and RSV-neutralizing

antibodies in 2-month, 6-month, and 15-month study cohorts

Pre-boosting

Post-boosting
Post-boosting

serum RSV-

serum RSV-
serkum RSV-

neutralizing titer
Peak mean
Peak mean
neutralizing titer
neutralizing titer

Study cohort
(with complement)^a
titer in NP^b
titer in TL^b
(with complement)^c
(without complement)^c

~2-month
8.1 ± 2.1
4.9 ± 1.0
3.4 ± 1.2
14.5
12.9

~6-month
6.1 ± 1.7
5.4 ± 0.5
5.2 ± 0.9
15.6
11.8

~15-month
4.7 ± 0.7
5.4 ± 2.3
4.8 ± 1.1
14.9
12.1

^aSera were collected two weeks (~2-month and ~15-month studies) or 35 days (~6-month study) before boosting and were analyzed by RSV PRN assay with added complement and expressed as mean log₂, PRNT.

^bNP and TL were collected daily and every second day, respectively, for 10 days post-boost and titrated by limiting dilution. Titers are in TCID₅₀per ml. Means were calculated for each day, and the peak mean titer for each virus is shown.

^cSera were collected two weeks following boosting and analyzed by RSV PRN assay with or without complement, as weeks following boosting and analyzed by RSV PRN assay with or without complement, as indicated, with titers expressed as mean log₂PRNT.

To investigate possible restriction in AGMs associated with packaging of RSV F protein into the vector virion, the post-boost replication of the DS-Cav1/B3TMCT vector (which efficiently packages RSV F) was compared to that of the DS-Cav1 vector (which is very inefficient for packaging) in AGM experiment #2 (˜6-month interval following priming). The replication of the DS-Cav1/B3TMCT vector hypothetically might have been reduced compared to the DS-Cav1 vector by (i) interference in vector replication due to packaging the RSV F protein into the vector virion, and/or (ii) restriction due to RSV-specific immunity targeted to the packaged RSV F protein, as already noted. The kinetics of replication of the DS-Cav1/B3TMCT vector were slightly slower than for the DS-Cav1 vector, but the difference was modest and was significant only for Days 2 and 4 in the TL, and the peak titers for the two vectors were similar. We did find that RSV-specific hamster antiserum inhibited the replication of the DS-Cav1/B3TMCT vector, and not the DS-Cav1 or empty vector, in cell culture (in the absence of complement). In this in vitro setting, the inhibition presumably was due to the binding of antibodies to RSV F packaged in the vector virions, and may not fully recapitulate the mechanism(s) that might operate in vivo.

Boosting with RSV 276 following intervals of ˜2, ˜6, and ˜15 months resulted in increases in peak mean serum RSV-PRNT measured with complement of, respectively, 22-fold, 78-fold, and 216-fold, resulting in peak mean titers of 1:5,793, 1:4,973, and 1:7,132. When measured without complement, the respective increases in peak mean serum RSV-PRNT were 25-fold, 37-fold, and 171-fold, and resulted in peak mean titers of 1:294, 1:294, and 1:1,024. Thus, the post-boost serum RSV-PRNTs were generally similar among the different time intervals except that the titers following the ˜15-month interval were slightly higher (1-fold with complement and 3.5-fold without complement) compared to the other intervals.

For the DS-Cav1/B3TMCT vector, boosting following intervals of ˜2, ˜6, and ˜15 months resulted in increases in peak mean serum RSV-PRNT measured with complement of, respectively, 91-fold, 776-fold, and 926-fold, resulting in peak mean post-boost serum RSV-PRNTs of 1:23,170, 1:49,667, and 1:30,574. When measured without complement, the respective increases were 366-fold, 446-fold, and 1,274-fold, resulting in peak mean post-boost serum RSV-PRNTs of 1:4,390, 1:3,565, and 1:7,643. Thus, the post-boost titers were generally similar among the different time intervals, with differences that were no greater than 2-fold and were not consistent for any interval.

When compared in AGM experiment #2, boosting with the DS-Cav1/B3TMCT vector versus the DS-Cav1 vector induced a 3-fold greater serum RSV-PRNT measured with complement, and a 1.4-fold greater titer measured without complement. In the hamster study, boosting with the DS-Cav1/B3TMCT vector versus the DS-Cav1 vector was greater only for serum RSV-PRNT measured without complement, and only 2-fold.

In the AGM experiments, the peak mean post-boost serum RSV-PRNTs for the vectors were exceptionally high: up to 1:49,667 and 1:7,643 assayed with and without complement, respectively. This raises the possibility that some of the greater instances of boosting with the vectors might have been blunted due to limitations on the magnitude of the immune response. The titers for the vectors were higher than for RSV 276 when assayed with complement (3- to 10-fold), and the difference was even greater when assayed without complement (7-to 15-fold). Both vectors also induced significantly greater responses of serum and mucosal IgA compared to RSV 276, with little difference between the two vectors. The greater immunogenicity of the boosts by the vectors compared to RSV likely reflects both their higher level of replication (and resulting greater antigen expression) and the DS-Cav1 and B3TMCT modifications to the RSV F protein.

Because immune responses to RSV are reduced during infancy, it could be important to boost RSV-specific immunity following primary immunization with a pediatric RSV vaccine. In this study, boosting with an attenuated PIV vector expressing RSV F with DS-Cav1 and B3TMCT modifications was substantially more immunogenic than boosting with an attenuated RSV, particularly for “high quality” RSV-neutralizing antibodies that neutralize in vitro without added complement. The results were similar whether the interval between priming and boosting was ˜2, ˜6, or ˜15 months. This suggests that a PIV vector could be effective in boosting during the same vaccination season or, alternatively, during the following year. In addition, a PIV-vectored RSV vaccine may be well-suited for primary immunization of young infants who have passive serum RSV-neutralizing antibodies from maternal transfer, including following maternal immunization, or from passive antibody immunoprophylaxis, including the use of antibody engineered for increased half-life. These passive antibodies might restrict an attenuated RSV, but not a PIV-vectored RSV vaccine. The use of an attenuated PIV3 vector also provides immunization against HPIV3, which is second only to RSV as an agent of severe acute pediatric respiratory disease.

Materials and Methods

Viruses. The RSVs in this example were recombinant (r) wild type (wt) or attenuated versions of the subgroup A strain A2 (Genbank KT992094) prepared using reverse genetics (Collins et al. 1995. Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5′ proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development. Proc Natl Acad Sci USA 92:11563-7). The attenuated RSVs are described in the Results. One additional RSV was rRSV A/Maryland/001/11, which is a recombinant version of the wt subgroup A strain RSV A/Maryland/001/11 that was isolated from a nasal wash collected in 2011 from an adult with acute respiratory illness. The F and G proteins of the Maryland/001/11 strain have 97% and 86% amino acid sequence identity with strain A2. The rB/HPIV3 constructs were previously described (Liang et al. 2017. Improved Prefusion Stability, Optimized Codon Usage, and Augmented Virion Packaging Enhance the Immunogenicity of Respiratory Syncytial Virus Fusion Protein in a Vectored-Vaccine Candidate. J Virol 91) and express modified versions of the F protein of RSV strain A2 (Genbank KT992094) from an added gene in the second gene position. The vector constructs were: (i) rB/HPIV3, which is the empty vector; (ii) rB/HPIV3/DS-Cav1 (abbreviated as DS-Cav1 vector), which expresses RSV F protein with increased stability in the pre-F conformation due to the DS and Cav1 mutations (McLellan et al. 2013. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 342:592-8); and (iii) rB/HPIV3/DS-Cav1/B3TMCT (abbreviated as DS-Cav1/B3TMCT vector), which expresses RSV F with the DS-Cav1 mutations and with its TM and CT domains replaced by those of BPIV3 F. In addition, as previously described, the RSV F ORF used in the vector constructs had been modified by codon optimization (GenScript, Piscataway, N.J.) and had been further modified by two amino acid substitutions called HEK (K66E and Q101P) that make F identical at the amino acid level to an early passage of RSV strain A2.

RSVs and rB/HPIV3 vectors were grown at 32° C. in Vero and LLC-MK2 cells, respectively. The complete genome sequences of all viruses were confirmed by automated Sanger sequencing analysis of un-cloned RT-PCR products to be free of adventitious mutations detectable above background (except for ˜30 and ˜120 nucleotides at the 3′ and 5′ ends, respectively, which include the primer binding sites and were not sequenced).

Titration of infectious virus and serum antibodies. Titers of RSV preparations were determined by plaque assay in Vero cells with immunostaining using a mixture of three F-specific monoclonal antibodies, as described previously (Luongo et al. 2013. Respiratory syncytial virus modified by deletions of the NS2 gene and amino acid S1313 of the L polymerase protein is a temperature-sensitive, live-attenuated vaccine candidate that is phenotypically stable at physiological temperature. J Virol 87:1985-96); titers are reported as log₁₀plaque forming units (PFU) per ml or g. Titers of rB/HPIV3 vectors were determined by limiting dilution in LLC-MK2 cells, with virus-positive wells detected by hemadsorption with guinea pig erythrocytes, as described previously (Durbin et al. 1999. Mutations in the C, D, and V open reading frames of human parainfluenza virus type 3 attenuate replication in rodents and primates. Virology 261:319-30); titers are reported as log₁₀50% tissue culture infectious doses (TCID₅₀) per ml or g.

Serum RSV- or HPIV3-neutralizing antibody titers were determined by 60% plaque reduction neutralization (PRN) assays on Vero cells using RSV or HPIV3 expressing green fluorescent protein (GFP) (Liang et al. 2014. Chimeric bovine/human parainfluenza virus type 3 expressing respiratory syncytial virus (RSV) F glycoprotein: effect of insert position on expression, replication, immunogenicity, stability, and protection against RSV infection. J Virol 88:4237-50). The assays were performed in the presence (RSV) or absence (RSV, HPIV3) of 5% added guinea pig complement (Cedarlane, Burlington, N.C.) as noted. The 60% plaque reduction neutralization titers (PRNTs) are reported in log₂and/or arithmetic values. Prior to primary infection, animals were confirmed to be RSV- or HPIV3-seronegative by, respectively, a PRN assay with complement or a hemagglutination-inhibition assay using guinea pig erythrocytes (Coates et al. 1966. An antigenic analysis of respiratory syncytial virus isolates by a plaque reduction neutralization test. Am J Epidemiol 83:299-313; van Wyke Coelingh et al. 1988. Attenuation of bovine parainfluenza virus type 3 in nonhuman primates and its ability to confer immunity to human parainfluenza virus type 3 challenge. J Infect Dis 157:655-62).

Animal studies. Animal studies were carried out in accordance with the Guide for Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the NIAID Animal Care and Use Committee.

A single prime-boost experiment was performed in Syrian Golden hamsters as described in the legend to FIG. 14 and the accompanying Results section.

Three prime-boost experiments were performed in African green monkeys (AGM Experiments #1-#3, Tables 8-10, and FIGS. 15-17, respectively). The primary AGM infections with RSV were performed using a number of different RSVs that mostly were attenuated derivatives of RSV strain A2 and are described in the Results and Tables 8-10. In those studies, RSV was administered by the combined IN/intratracheal (IT) routes at a dose per site of 10⁶PFU in 1 ml of L-15 medium. Booster infections with RSV or the DS-Cav/B3TMCT or DS-Cav1 vectors in AGMs in the present study were administered in the same way, by the combined IN/IT routes at a dose per site of 10⁶PFU (RSV) or 10⁶TCID₅₀(vectors) in 1 ml of L-15 medium. Following inoculation, nasopharyngeal swabs (NP) and tracheal lavages (TL) were collected daily and every other day, respectively, for 10 days, and were flash-frozen on dry ice and stored at −80° C. until titration. Sera were collected prior to inoculation on the day of the boost and every seven days thereafter for four consecutive weeks.

In AGM Experiment #2, nasal mucosal lining fluid was sampled on the day of boosting and on days 14, 21, and 28 post-boosting using synthetic adsorptive matrix (SAM) strips (Nasosorption FXi, Hunt Developments, UK). SAM strips were gently placed on the nasal mucosa of the lower turbinate of one nostril, held in place for 30 seconds, and placed into a microtube pre-filled with 300 μl of phosphate buffered saline buffer (PBS) containing 3% bovine serum albumin (BSA) and 0.1% Tween 20. Samples were flash frozen on dry ice and kept at −80° C. until use. Mucosal and serum IgA titers were determined by dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) as described below.

RSV RNA copy numbers in AGM TL samples were quantified by reverse transcription-quantitative PCR assay (RT-qPCR) that was specific for both positive- and negative-sense RSV M gene sequence. Frozen AGM TL samples were briefly thawed in a 37° C. water bath and immediately kept on ice. After clarification by centrifugation at 800 g for 5 min at 4° C., the total RNA in TL samples was extracted using QIAamp Viral RNA Mini Kit (Qiagen). cDNA was generated by reverse transcription using Applied Biosystem RT kit (ThermoFisher Scientific) with random hexamer primers, and analyzed by the M-specific qPCR. For comparison, a standard curve was generated using a DNA plasmid encoding the full-length genome of wt RSV A2 strain (LID) analyzed by the same qPCR assay. The copy number of reverse-transcribed RSV RNA in TL samples was interpolated from the standard curve. Statistical analyses were performed using GraphPad Prism version 8, GraphPad Software, San Diego, Calif. USA.

Detection of IgA antibodies using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA). Serum and mucosal IgA were detected by DELFIA immunoassay. Briefly, recombinant RSV DS-Cav1 F protein was expressed in HEK293 cells and purified as described previously (McLellan et al. 2013. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 342:592-8). Ninety-six-well DELFIA yellow plates (Perkin Elmer, Waltham Mass.) were coated with 100ng/well of purified RSV DS-Cav1 F protein diluted in 100 μl of carbonate-bicarbonate buffer (Millipore Sigma, St. Louis Mo.) and incubated at 4° C. overnight. All washes in the assay were performed once with 200 μl/well of wash buffer (1×DELFIA wash concentrate, Perkin Elmer, Waltham Mass.). After overnight antigen coating, plates were washed and incubated with 100 μl/well of blocking solution (PBS containing 0.1% Tween-20 and 3% BSA) at 37° C. for 1 h. Plates were washed and 100 μl of serum samples in 4-fold dilution series in blocking solution were added and incubated at 4° C. overnight. Following a wash, 100 μl/well of anti-monkey IgA biotin conjugate (Alpha Diagnostic, San Antonio, Tex.) diluted 1:5000 in blocking solution was added and incubated at 37° C. for 1 h. Plates were washed and 100 μl/well of Europium (Eu)-conjugated streptavidin diluted 1:2000 in DELFIA assay buffer (Perkin Elmer, Waltham, Mass.), was added. After incubation at 37° C. for 1 h, plates were washed and incubated with 100 μl/well of DELFIA enhancement solution (Perkin Elmer) at room temperature with gentle shaking for 20 minutes. Fluorescence was measured using a Eu-specific time-resolved fluorescence (TRF) program (340 nm excitation, 615 nm emission) in a Synergy Neo 2 plate reader (BioTek, Winooski, Vt.). The blocking solution was used to generate blank values, and an average of 24 blank values plus 3 standard deviations was used as a cut-off (corresponding to 400 fluorescence units). Test sample dilutions corresponding to 400 fluorescence units were interpolated from sigmoidal standard curves using GraphPad Prism version 8 (GraphPad Software, San Diego, Calif. USA) and were expressed as log₂values.

It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described embodiments. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

LIVE ATTENUATED RESPIRATORY SYNCYTIAL VIRUS

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