Intranasal pediatric parainfluenza virus-vectored SARS-CoV-2 vaccine is protective in monkeys.

Pediatric SARS-CoV-2 vaccines are needed that elicit immunity directly in the airways as well as systemically. Building on pediatric parainfluenza virus vaccines in clinical development, we generated a live-attenuated parainfluenza-virus-vectored vaccine candidate expressing SARS-CoV-2 prefusion-stabilized spike (S) protein (B/HPIV3/S-6P) and evaluated its immunogenicity and protective efficacy in rhesus macaques. A single intranasal/intratracheal dose of B/HPIV3/S-6P induced strong S-specific airway mucosal immunoglobulin A (IgA) and IgG responses. High levels of S-specific antibodies were also induced in serum, which efficiently neutralized SARS-CoV-2 variants of concern of alpha, beta, and delta lineages, while their ability to neutralize Omicron sub-lineages was lower. Furthermore, B/HPIV3/S-6P induced robust systemic and pulmonary S-specific CD4+ and CD8+ T cell responses, including tissue-resident memory cells in the lungs. Following challenge, SARS-CoV-2 replication was undetectable in airways and lung tissues of immunized macaques. B/HPIV3/S-6P will be evaluated clinically as pediatric intranasal SARS-CoV-2/parainfluenza virus type 3 vaccine.

Live-attenuated pediatric parainfluenza vaccine expressing 6P-stabilized SARS-CoV-2 spike protein is protective against SARS-CoV-2 variants in hamsters.

The pediatric live-attenuated bovine/human parainfluenza virus type 3 (B/HPIV3)-vectored vaccine expressing the prefusion-stabilized SARS-CoV-2 spike (S) protein (B/HPIV3/S-2P) was previously evaluated in vitro and in hamsters. To improve its immunogenicity, we generated B/HPIV3/S-6P, expressing S further stabilized with 6 proline mutations (S-6P). Intranasal immunization of hamsters with B/HPIV3/S-6P reproducibly elicited significantly higher serum anti-S IgA/IgG titers than B/HPIV3/S-2P; hamster sera efficiently neutralized variants of concern (VoCs), including Omicron variants. B/HPIV3/S-2P and B/HPIV3/S-6P immunization protected hamsters against weight loss and lung inflammation following SARS-CoV-2 challenge with the vaccine-matched strain WA1/2020 or VoCs B.1.1.7/Alpha or B.1.351/Beta and induced near-sterilizing immunity. Three weeks post-challenge, B/HPIV3/S-2P- and B/HPIV3/S-6P-immunized hamsters exhibited a robust anamnestic serum antibody response with increased neutralizing potency to VoCs, including Omicron sublineages. B/HPIV3/S-6P primed for stronger anamnestic antibody responses after challenge with WA1/2020 than B/HPIV3/S-2P. B/HPIV3/S-6P will be evaluated as an intranasal vaccine to protect infants against both HPIV3 and SARS-CoV-2.

Evaluation of the Live-Attenuated Intranasal Respiratory Syncytial Virus (RSV) Vaccine RSV/6120/ΔNS2/1030s in RSV-Seronegative Young Children.

BACKGROUND: Respiratory syncytial virus (RSV) is the leading cause of pediatric lower respiratory illness (LRI) and a vaccine for immunization of children is needed. RSV/6120/ΔNS2/1030s is a cDNA-derived live-vaccine candidate attenuated by deletion of the interferon antagonist NS2 gene and the genetically stabilized 1030s missense polymerase mutation in the polymerase, conferring temperature sensitivity. METHODS: A single intranasal dose of RSV/6120/ΔNS2/1030s was evaluated in a double-blind, placebo-controlled trial (vaccine to placebo ratio, 2:1) at 105.7 plaque-forming units (PFU) in 15 RSV-seropositive 12- to 59-month-old children, and at 105 PFU in 30 RSV-seronegative 6- to 24-month-old children. RESULTS: RSV/6120/ΔNS2/1030s infected 100% of RSV-seronegative vaccinees and was immunogenic (geometric mean RSV plaque-reduction neutralizing antibody titer [RSV-PRNT], 1:91) and genetically stable. Mild rhinorrhea was detected more frequently in vaccinees (18/20 vaccinees vs 4/10 placebo recipients, P = .007), and LRI occurred in 1 vaccinee during a period when only vaccine virus was detected. Following the RSV season, 5 of 16 vaccinees had ≥4-fold rises in RSV-PRNT with significantly higher titers than 4 of 10 placebo recipients with rises (1:1992 vs 1:274, P = .02). Thus, RSV/6120/ΔNS2/1030s primed for substantial anamnestic neutralizing antibody responses following naturally acquired RSV infection. CONCLUSIONS: RSV/6120/ΔNS2/1030s is immunogenic and genetically stable in RSV-seronegative children, but the frequency of rhinorrhea in vaccinees exceeded that in placebo recipients. CLINICAL TRIALS REGISTRATION: NCT03387137.

A single intranasal dose of a live-attenuated parainfluenza virus-vectored SARS-CoV-2 vaccine is protective in hamsters.

Single-dose vaccines with the ability to restrict SARS-CoV-2 replication in the respiratory tract are needed for all age groups, aiding efforts toward control of COVID-19. We developed a live intranasal vector vaccine for infants and children against COVID-19 based on replication-competent chimeric bovine/human parainfluenza virus type 3 (B/HPIV3) that express the native (S) or prefusion-stabilized (S-2P) SARS-CoV-2 S spike protein, the major protective and neutralization antigen of SARS-CoV-2. B/HPIV3/S and B/HPIV3/S-2P replicated as efficiently as B/HPIV3 in vitro and stably expressed SARS-CoV-2 S. Prefusion stabilization increased S expression by B/HPIV3 in vitro. In hamsters, a single intranasal dose of B/HPIV3/S-2P induced significantly higher titers compared to B/HPIV3/S of serum SARS-CoV-2-neutralizing antibodies (12-fold higher), serum IgA and IgG to SARS-CoV-2 S protein (5-fold and 13-fold), and IgG to the receptor binding domain (10-fold). Antibodies exhibited broad neutralizing activity against SARS-CoV-2 of lineages A, B.1.1.7, and B.1.351. Four weeks after immunization, hamsters were challenged intranasally with 104.5 50% tissue-culture infectious-dose (TCID50) of SARS-CoV-2. In B/HPIV3 empty vector-immunized hamsters, SARS-CoV-2 replicated to mean titers of 106.6 TCID50/g in lungs and 107 TCID50/g in nasal tissues and induced moderate weight loss. In B/HPIV3/S-immunized hamsters, SARS-CoV-2 challenge virus was reduced 20-fold in nasal tissues and undetectable in lungs. In B/HPIV3/S-2P-immunized hamsters, infectious challenge virus was undetectable in nasal tissues and lungs; B/HPIV3/S and B/HPIV3/S-2P completely protected against weight loss after SARS-CoV-2 challenge. B/HPIV3/S-2P is a promising vaccine candidate to protect infants and young children against HPIV3 and SARS-CoV-2.

Evaluation of Recombinant Live-Attenuated Respiratory Syncytial Virus (RSV) Vaccines RSV/ΔNS2/Δ1313/I1314L and RSV/276 in RSV-Seronegative Children.

BACKGROUND: This United States-based study compared 2 candidate vaccines: RSV/ΔNS2/Δ1313/I1314L, attenuated by NS2 gene-deletion and temperature-sensitivity mutation in the polymerase gene; and RSV/276, attenuated by M2-2 deletion. METHODS: RSV-seronegative children aged 6-24 months received RSV/ΔNS2/Δ1313/I1314L (106 plaque-forming units [PFU]), RSV/276 (105 PFU), or placebo intranasally. Participants were monitored for vaccine shedding, reactogenicity, and RSV serum antibodies, and followed over the subsequent RSV season. RESULTS: Enrollment occurred September 2017 to October 2019. During 28 days postinoculation, upper respiratory illness and/or fever occurred in 64% of RSV/ΔNS2/Δ1313/I1314L, 84% of RSV/276, and 58% of placebo recipients. Symptoms were generally mild. Cough was more common in RSV/276 recipients than RSV/ΔNS2/Δ1313/I1314L (48% vs 12%; P = .012) or placebo recipients (17%; P = .084). There were no lower respiratory illness or serious adverse events. Eighty-eight and 96% of RSV/ΔNS2/Δ1313/I1314L and RSV/276 recipients were infected with vaccine (shed vaccine and/or had ≥4-fold rises in RSV antibodies). Serum RSV-neutralizing titers and anti-RSV F IgG titers increased ≥4-fold in 60% and 92% of RSV/ΔNS2/Δ1313/I1314L and RSV/276 vaccinees, respectively. Exposure to community RSV during the subsequent winter was associated with strong anamnestic RSV-antibody responses. CONCLUSIONS: Both vaccines had excellent infectivity and were well tolerated. RSV/276 induced an excess of mild cough. Both vaccines were immunogenic and primed for strong anamnestic responses. CLINICAL TRIALS REGISTRATION: NCT03227029 and NCT03422237.

Optimization of the Codon Pair Usage of Human Respiratory Syncytial Virus Paradoxically Resulted in Reduced Viral Replication In Vivo and Reduced Immunogenicity.

We subjected various open reading frames (ORFs) in the genome of respiratory syncytial virus (RSV) to codon pair optimization (CPO) by increasing the content of codon pairs that are overrepresented in the human genome without changing overall codon usage and amino acid sequences. CPO has the potential to increase the expression of the encoded protein(s). Four viruses were made: Max A (with CPO of NS1, NS2, N, P, M, and SH ORFs), Max B (with CPO of G and F), Max L (with CPO of L), and Max FLC (with CPO of all ORFs except M2-1 and M2-2). Because of the possibility of increased viral replication, each CPO virus was attenuated by the inclusion of a codon deletion mutation (Δ1313) and a missense mutation (I1314L) in the L polymerase. CPO had no effect on multicycle virus replication in vitro, temperature sensitivity, or specific infectivity. Max A and L, which in common had CPO of one or more ORFs of proteins of the polymerase complex, exhibited global increases in viral protein synthesis. Max B alone exhibited decreased protein synthesis, and it alone had reduced single-cycle virus replication in vitro All CPO RSVs exhibited marginal reductions in replication in mice and hamsters. Surprisingly, the CPO RSVs induced lower levels of serum RSV-neutralizing antibodies in hamsters. This reduced immunogenicity might reflect reduced viral replication and possibly also the decrease in CpG and UpA dinucleotides as immune stimulators. Overall, our study describes paradoxical effects of CPO of an RNA virus on viral replication and the adaptive humoral immune response.IMPORTANCE Using computer algorithms and large-scale DNA synthesis, one or more ORFs of a microbial pathogen can be recoded by different strategies that involve the introduction of up to thousands of nucleotide changes without affecting amino acid coding. This approach has been used mostly to generate deoptimized viruses used as vaccine candidates. However, the effects of the converse approach of generating optimized viruses are still largely unknown. Here, various ORFs in the genome of respiratory syncytial virus (RSV) were codon pair optimized (CPO) by increasing the content of codon pairs that are overrepresented in the human genome. CPO did not affect RSV replication in multicycle replication experiments in vitro. However, replication was marginally reduced in two rodents models. In hamsters, CPO RSVs induced lower levels of serum RSV-neutralizing antibodies. Thus, CPO of an RNA virus for a mammalian host has paradoxical effects on virus replication and the adaptive humoral immune response.

Safety and Immunogenicity of the Respiratory Syncytial Virus Vaccine RSV/ΔNS2/Δ1313/I1314L in RSV-Seronegative Children.

BACKGROUND: Respiratory syncytial virus (RSV) is the leading global cause of severe pediatric acute respiratory tract illness, and a vaccine is needed. RSV/ΔNS2/Δ1313/I1314L contains 2 attenuating elements: (1) deletion of the interferon antagonist NS2 gene and (2) deletion of codon 1313 of the RSV polymerase gene and the stabilizing missense mutation I1314L. This live vaccine candidate was temperature-sensitive, genetically stable, replication restricted, and immunogenic in nonhuman primates. METHODS: A single intranasal dose of RSV/ΔNS2/Δ1313/I1314L was evaluated in a double-blind, placebo-controlled trial (vaccine-placebo ratio, 2:1) at 106 plaque-forming units (PFU) in 15 RSV-seropositive children and at 105 and 106 PFU in 21 and 30 RSV-seronegative children, respectively. RESULTS: In RSV-seronegative children, the 105 PFU dose was overattenuated, but the 106 PFU dose was well tolerated, infectious (RSV/ΔNS2/Δ1313/I1314L replication detected in 90% of vaccinees), and immunogenic (geometric mean serum RSV plaque-reduction neutralizing antibody titer, 1:64). After the RSV season, 9 of 20 vaccinees had increases in the RSV titer that were significantly greater than those in 8 of 10 placebo recipients (1:955 vs 1:69, respectively), indicating that the vaccine primed for anamnestic responses after natural RSV exposure. CONCLUSION: Rational design yielded a genetically stable candidate RSV vaccine that is attenuated yet immunogenic in RSV-seronegative children, warranting further evaluation. CLINICAL TRIALS REGISTRATION: NCT01893554.

Live-Attenuated Respiratory Syncytial Virus Vaccine With M2-2 Deletion and With Small Hydrophobic Noncoding Region Is Highly Immunogenic in Children.

BACKGROUND: Respiratory syncytial virus (RSV) is the leading viral cause of severe pediatric respiratory illness, and vaccines are needed. Live RSV vaccine D46/NS2/N/ΔM2-2-HindIII, attenuated by deletion of the RSV RNA regulatory protein M2-2, is based on previous candidate LID/ΔM2-2 but incorporates prominent differences from MEDI/ΔM2-2, which was more restricted in replication in phase 1. METHODS: RSV-seronegative children aged 6-24 months received 1 intranasal dose (105 plaque-forming units [PFUs] of D46/NS2/N/ΔM2-2-HindIII [n = 21] or placebo [n = 11]) and were monitored for vaccine shedding, reactogenicity, RSV-antibody responses and RSV-associated medically attended acute respiratory illness (RSV-MAARI) and antibody responses during the following RSV season. RESULTS: All 21 vaccinees were infected with vaccine; 20 (95%) shed vaccine (median peak titer, 3.5 log10 PFUs/mL with immunoplaque assay and 6.1 log10 copies/mL with polymerase chain reaction). Serum RSV-neutralizing antibodies and anti-RSV fusion immunoglobulin G increased ≥4-fold in 95% and 100% of vaccines, respectively. Mild upper respiratory tract symptoms and/or fever occurred in vaccinees (76%) and placebo recipients (18%). Over the RSV season, RSV-MAARI occurred in 2 vaccinees and 4 placebo recipients. Three vaccinees had ≥4-fold increases in serum RSV-neutralizing antibody titers after the RSV season without RSV-MAARI. CONCLUSIONS: D46/NS2/N/ΔM2-2-HindIII had excellent infectivity and immunogenicity and primed vaccine recipients for anamnestic responses, encouraging further evaluation of this attenuation strategy. CLINICAL TRIALS REGISTRATION: NCT03102034 and NCT03099291.

Live Respiratory Syncytial Virus Attenuated by M2-2 Deletion and Stabilized Temperature Sensitivity Mutation 1030s Is a Promising Vaccine Candidate in Children.

BACKGROUND: The safety and immunogenicity of live respiratory syncytial virus (RSV) candidate vaccine, LID/ΔM2-2/1030s, with deletion of RSV ribonucleic acid synthesis regulatory protein M2-2 and genetically stabilized temperature-sensitivity mutation 1030s in the RSV polymerase protein was evaluated in RSV-seronegative children. METHODS: Respiratory syncytial virus-seronegative children ages 6-24 months received 1 intranasal dose of 105 plaque-forming units (PFU) of LID/ΔM2-2/1030s (n = 21) or placebo (n = 11). The RSV serum antibodies, vaccine shedding, and reactogenicity were assessed. During the following RSV season, medically attended acute respiratory illness (MAARI) and pre- and postsurveillance serum antibody titers were monitored. RESULTS: Eighty-five percent of vaccinees shed LID/ΔM2-2/1030s vaccine (median peak nasal wash titers: 3.1 log10 PFU/mL by immunoplaque assay; 5.1 log10 copies/mL by reverse-transcription quantitative polymerase chain reaction) and had ≥4-fold rise in serum-neutralizing antibodies. Respiratory symptoms and fever were common (60% vaccinees and 27% placebo recipients). One vaccinee had grade 2 wheezing with rhinovirus but without concurrent LID/ΔM2-2/1030s shedding. Five of 19 vaccinees had ≥4-fold increases in antibody titers postsurveillance without RSV-MAARI, indicating anamnestic responses without significant illness after infection with community-acquired RSV. CONCLUSIONS: LID/ΔM2-2/1030s had excellent infectivity without evidence of genetic instability, induced durable immunity, and primed for anamnestic antibody responses, making it an attractive candidate for further evaluation.

Genetic stability of genome-scale deoptimized RNA virus vaccine candidates under selective pressure.

Recoding viral genomes by numerous synonymous but suboptimal substitutions provides live attenuated vaccine candidates. These vaccine candidates should have a low risk of deattenuation because of the many changes involved. However, their genetic stability under selective pressure is largely unknown. We evaluated phenotypic reversion of deoptimized human respiratory syncytial virus (RSV) vaccine candidates in the context of strong selective pressure. Codon pair deoptimized (CPD) versions of RSV were attenuated and temperature-sensitive. During serial passage at progressively increasing temperature, a CPD RSV containing 2,692 synonymous mutations in 9 of 11 ORFs did not lose temperature sensitivity, remained genetically stable, and was restricted at temperatures of 34 °C/35 °C and above. However, a CPD RSV containing 1,378 synonymous mutations solely in the polymerase L ORF quickly lost substantial attenuation. Comprehensive sequence analysis of virus populations identified many different potentially deattenuating mutations in the L ORF as well as, surprisingly, many appearing in other ORFs. Phenotypic analysis revealed that either of two competing mutations in the virus transcription antitermination factor M2-1, outside of the CPD area, substantially reversed defective transcription of the CPD L gene and substantially restored virus fitness in vitro and in case of one of these two mutations, also in vivo. Paradoxically, the introduction into Min L of one mutation each in the M2-1, N, P, and L proteins resulted in a virus with increased attenuation in vivo but increased immunogenicity. Thus, in addition to providing insights on the adaptability of genome-scale deoptimized RNA viruses, stability studies can yield improved synthetic RNA virus vaccine candidates.

Differential Responses by Human Respiratory Epithelial Cell Lines to Respiratory Syncytial Virus Reflect Distinct Patterns of Infection Control.

Respiratory syncytial virus (RSV) infects small foci of respiratory epithelial cells via infected droplets. Infection induces expression of type I and III interferons (IFNs) and proinflammatory cytokines, the balance of which may restrict viral replication and affect disease severity. We explored this balance by infecting two respiratory epithelial cell lines with low doses of recombinant RSV expressing green fluorescent protein (rgRSV). A549 cells were highly permissive, whereas BEAS-2B cells restricted infection to individual cells or small foci. After infection, A549 cells expressed higher levels of IFN-ß-, IFN-λ-, and NF-κB-inducible proinflammatory cytokines. In contrast, BEAS-2B cells expressed higher levels of antiviral interferon-stimulated genes, pattern recognition receptors, and other signaling intermediaries constitutively and after infection. Transcriptome analysis revealed that constitutive expression of antiviral and proinflammatory genes predicted responses by each cell line. These two cell lines provide a model for elucidating critical mediators of local control of viral infection in respiratory epithelial cells.IMPORTANCE Airway epithelium is both the primary target of and the first defense against respiratory syncytial virus (RSV). Whether RSV replicates and spreads to adjacent epithelial cells depends on the quality of their innate immune responses. A549 and BEAS-2B are alveolar and bronchial epithelial cell lines, respectively, that are often used to study RSV infection. We show that A549 cells are permissive to RSV infection and express genes characteristic of a proinflammatory response. In contrast, BEAS-2B cells restrict infection and express genes characteristic of an antiviral response associated with expression of type I and III interferons. Transcriptome analysis of constitutive gene expression revealed patterns that may predict the response of each cell line to infection. This study suggests that restrictive and permissive cell lines may provide a model for identifying critical mediators of local control of infection and stresses the importance of the constitutive antiviral state for the response to viral challenge.

Live Respiratory Syncytial Virus (RSV) Vaccine Candidate Containing Stabilized Temperature-Sensitivity Mutations Is Highly Attenuated in RSV-Seronegative Infants and Children.

Background: Respiratory syncytial virus (RSV) is the most important viral cause of severe respiratory illness in young children and lacks a vaccine. RSV cold-passage/stabilized 2 (RSVcps2) is a modification of a previously evaluated vaccine candidate in which 2 major attenuating mutations have been stabilized against deattenuation. Methods: RSV-seronegative 6-24-month-old children received an intranasal dose of 105.3 plaque-forming units (PFU) of RSVcps2 (n = 34) or placebo (n = 16) (International Maternal Pediatric Adolescent AIDS Clinical Trials protocol P1114 and companion protocol CIR285). RSV serum neutralizing antibody titers before and 56 days after vaccination, vaccine virus infectivity (defined as vaccine virus shedding detectable in nasal wash and/or a ≥4-fold rise in serum antibodies), reactogenicity, and genetic stability were assessed. During the following RSV transmission season, participants were monitored for respiratory illness, with serum antibody titers measured before and after the season. Results: A total of 85% of vaccinees were infected with RSVcps2 (median peak titer, 0.5 log10 PFU/mL by culture and 2.9 log10 copies/mL by polymerase chain reaction analysis); 77% shed vaccine virus, and 59% developed a ≥4-fold rise in RSV-serum neutralizing antibody titers. Respiratory tract and/or febrile illness occurred at the same rate (50%) in the vaccine and placebo groups. Deattenuation was not detected at either of 2 stabilized mutation sites. Conclusions: RSVcps2 was well tolerated and moderately immunogenic and had increased genetic stability in 6-24-month-old RSV-seronegative children. Clinical Trials Registration: NCT01852266 and NCT01968083.

Live-Attenuated Respiratory Syncytial Virus Vaccine Candidate With Deletion of RNA Synthesis Regulatory Protein M2-2 is Highly Immunogenic in Children.

Background: Live respiratory syncytial virus (RSV) candidate vaccine LIDΔM2-2 is attenuated by deletion of the RSV RNA regulatory protein M2-2, resulting in upregulated viral gene transcription and antigen expression but reduced RNA replication. Methods: RSV-seronegative children ages 6-24 months received a single intranasal dose of 105 plaque forming units (PFU) of LIDΔM2-2 (n = 20) or placebo (n = 9) (NCT02237209, NCT02040831). RSV serum antibodies, vaccine infectivity, and reactogenicity were assessed. During the following RSV season, participants were monitored for respiratory illness and pre- and post-RSV season serum antibodies. Results: Vaccine virus was shed by 95% of vaccinees (median peak titers of 3.8 log10 PFU/mL by quantitative culture and 6.3 log10 copies/mL by PCR); 90% had ≥4-fold rise in serum neutralizing antibodies. Respiratory symptoms and fever were common in vaccine (95%) and placebo (78%). One vaccinee had grade 2 rhonchi concurrent with vaccine shedding, rhinovirus, and enterovirus. Eight of 19 vaccinees versus 2 of 9 placebo recipients had substantially increased RSV antibody titers after the RSV season without medically attended RSV disease, indicating anamnestic vaccine responses to wild-type RSV without significant illness. Conclusion: LIDΔM2-2 had excellent infectivity and immunogenicity, encouraging further study of vaccine candidates attenuated by M2-2 deletion. Clinical Trials Registration: NCT02237209, NCT02040831.

Attenuation of human respiratory syncytial virus by genome-scale codon-pair deoptimization.

Human respiratory syncytial virus (RSV) is the most important viral agent of serious pediatric respiratory-tract disease worldwide. A vaccine or generally effective antiviral drug is not yet available. We designed new live attenuated RSV vaccine candidates by codon-pair deoptimization (CPD). Specifically, viral ORFs were recoded by rearranging existing synonymous codons to increase the content of underrepresented codon pairs. Amino acid coding was completely unchanged. Four CPD RSV genomes were designed in which the indicated ORFs were recoded: Min A (NS1, NS2, N, P, M, and SH), Min B (G and F), Min L (L), and Min FLC (all ORFs except M2-1 and M2-2). Surprisingly, the recombinant CPD viruses were temperature-sensitive for replication in vitro (level of sensitivity: Min FLC > Min L > Min B > Min A). All of the CPD mutants grew less efficiently in vitro than recombinant wild-type (WT) RSV, even at the typically permissive temperature of 32 °C (growth efficiency: WT > Min L > Min A > Min FLC > Min B). CPD of the ORFs for the G and F surface glycoproteins provided the greatest restrictive effect. The CPD viruses exhibited a range of restriction in mice and African green monkeys comparable with that of two attenuated RSV strains presently in clinical trials. This study provided a new type of attenuated RSV and showed that CPD can rapidly generate vaccine candidates against nonsegmented negative-strand RNA viruses, a large and expanding group that includes numerous pathogens of humans and animals.

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.

Human respiratory syncytial virus (RSV) is the leading viral cause of lower respiratory tract disease in infants and children worldwide. In previous work to develop point mutations in RSV with improved genetic stability, we observed that an attenuating mutation at amino acid position 1321 in the L polymerase protein was subject to deattenuation by a spontaneous second-site compensatory mutation at position 1313 (C. Luongo, C. C. Winter, P. L. Collins, and U. J. Buchholz, J. Virol. 86:10792-10804, 2012). In the present study, we found that deletion of position 1313 (Δ1313), irrespective of the presence of an attenuating mutation at position 1321, provided a new attenuating mutation. RSV bearing Δ1313 replicated in cell culture as efficiently as wild-type virus at 32°C, was restricted for replication at 37°C, and was restricted 50-fold and 150-fold in the upper and lower respiratory tracts, respectively, of mice. We combined the Δ1313 deletion with the previously described, attenuating NS2 gene deletion (ΔNS2) to produce the recombinant live-attenuated RSV vaccine candidate ΔNS2/Δ1313. During in vitro stress tests involving serial passage at incrementally increasing temperatures, a second-site compensatory mutation was detected in close proximity of Δ1313, namely, I1314T. This site was genetically and phenotypically stabilized by an I1314L substitution. Combination of I1314L with ΔNS2/Δ1313 yielded a virus, ΔNS2/Δ1313/1314L, with genetic stability at physiological temperature. This stabilized vaccine candidate was moderately temperature sensitive and had a level of restriction in chimpanzees comparable to that of MEDI-559, a promising RSV vaccine candidate that presently is in clinical trials but lacks stabilized attenuating mutations. The level of attenuation and genetic stability identify ΔNS2/Δ1313/1314L as a promising candidate for evaluation in pediatric phase I studies.

Mutations in the F protein of the live-attenuated respiratory syncytial virus vaccine candidate ΔNS2/Δ1313/I1314L increase the stability of infectivity and content of prefusion F protein.

Respiratory syncytial virus (RSV) is the leading viral cause of bronchiolitis and pneumonia in infants and toddlers, but there currently is no licensed pediatric vaccine. A leading vaccine candidate that has been evaluated for intranasal immunization in a recently completed phase 1/2 clinical trial is an attenuated version of RSV strain A2 called RSV/ΔNS2/Δ1313/I1314L (hereafter called ΔNS2). ΔNS2 is attenuated by deletion of the interferon antagonist NS2 gene and introduction into the L polymerase protein gene of a codon deletion (Δ1313) that confers temperature-sensitivity and is stabilized by a missense mutation (I1314L). Previously, introduction of four amino acid changes derived from a second RSV strain "line 19" (I79M, K191R, T357K, N371Y) into the F protein of strain A2 increased the stability of infectivity and the proportion of F protein in the highly immunogenic pre-fusion (pre-F) conformation. In the present study, these four "line 19" assignments were introduced into the ΔNS2 candidate, creating ΔNS2-L19F-4M. During in vitro growth in Vero cells, ΔNS2-L19F-4M had growth kinetics and peak titer similar to the ΔNS2 parent. ΔNS2-L19F-4M exhibited an enhanced proportion of pre-F protein, with a ratio of pre-F/total F that was 4.5- to 5.0-fold higher than that of the ΔNS2 parent. The stability of infectivity during incubation at 4°C, 25°C, 32°C and 37°C was greater for ΔNS2-L19F-4M; for example, after 28 days at 32°C, its titer was 100-fold greater than ΔNS2. ΔNS2-L19F-4M exhibited similar levels of replication in human airway epithelial (HAE) cells as ΔNS2. The four "line 19" F mutations were genetically stable during 10 rounds of serial passage in Vero cells. In African green monkeys, ΔNS2-L19F-4M and ΔNS2 had similar growth kinetics, peak titer, and immunogenicity. These results suggest that ΔNS2-L19F-4M is an improved live attenuated vaccine candidate whose enhanced stability may simplify its manufacture, storage and distribution, which merits further evaluation in a clinical trial in humans.

Mucosal prime-boost immunization with live murine pneumonia virus-vectored SARS-CoV-2 vaccine is protective in macaques.

Immunization via the respiratory route is predicted to increase the effectiveness of a SARS-CoV-2 vaccine. Here, we evaluate the immunogenicity and protective efficacy of one or two doses of a live-attenuated murine pneumonia virus vector expressing SARS-CoV-2 prefusion-stabilized spike protein (MPV/S-2P), delivered intranasally/intratracheally to male rhesus macaques. A single dose of MPV/S-2P is highly immunogenic, and a second dose increases the magnitude and breadth of the mucosal and systemic anti-S antibody responses and increases levels of dimeric anti-S IgA in the airways. MPV/S-2P also induces S-specific CD4+ and CD8+ T-cells in the airways that differentiate into large populations of tissue-resident memory cells within a month after the boost. One dose induces substantial protection against SARS-CoV-2 challenge, and two doses of MPV/S-2P are fully protective against SARS-CoV-2 challenge virus replication in the airways. A prime/boost immunization with a mucosally-administered live-attenuated MPV vector could thus be highly effective in preventing SARS-CoV-2 infection and replication.

Increased genetic and phenotypic stability of a promising live-attenuated respiratory syncytial virus vaccine candidate by reverse genetics.

Human respiratory syncytial virus (RSV) is the most important viral cause of serious pediatric respiratory illness worldwide. Currently, the most promising live-attenuated vaccine candidate is a temperature-sensitive (ts) cDNA-derived virus named rA2cp248/404/1030ΔSH, in reference to its set of attenuating mutations. In a previous clinical study, more than one-third of postvaccination nasal wash isolates exhibited partial loss of the ts phenotype. Most of this instability appeared to be due to reversion at a missense point mutation called 1030. This 1030 mutation is a single-nucleotide tyrosine-to-asparagine substitution at position 1321 (Y1321N) of the polymerase L protein that contributes to the ts and attenuation phenotypes of the vaccine candidate. The goals of the present study were to identify a reversion-resistant codon at position 1321 conferring a comparable level of attenuation and to use this to develop a genetically stable version of the vaccine virus. We modified wild-type (wt) RSV to insert each of the 20 possible amino acids at position 1321; 19 viruses were recoverable. We also investigated small deletions at or near this position, but these viruses were not recoverable. Phenotypic analysis identified alternative attenuating amino acids for position 1321. Several of these amino acids were predicted, based on the genetic code, to be refractory to deattenuation. Classical genetics, using temperature stress tests in vitro combined with nucleotide sequencing, confirmed this stability but identified a second site with a compensatory mutation at position 1313. It was possible to stabilize the 1313 site as well, providing a stable 1030 mutation. Further stress tests identified additional incidental mutations, but these did not reverse the ts/attenuation phenotype. An improved version of the vaccine candidate virus was constructed and validated in vitro by temperature stress tests and in vivo by evaluation of attenuation in seronegative chimpanzees. In addition to developing an improved version of this promising live-attenuated RSV vaccine candidate, this study demonstrated the propensity of an RNA virus to escape from attenuation but also showed that, through systematic analysis, genetics can be used to cut off the routes of escape.

Intranasal murine pneumonia virus-vectored SARS-CoV-2 vaccine induces mucosal and serum antibodies in macaques.

Next-generation SARS-CoV-2 vaccines are needed that induce systemic and mucosal immunity. Murine pneumonia virus (MPV), a murine homolog of respiratory syncytial virus, is attenuated by host-range restriction in nonhuman primates and has a tropism for the respiratory tract. We generated MPV vectors expressing the wild-type SARS-CoV-2 spike protein (MPV/S) or its prefusion-stabilized form (MPV/S-2P). Both vectors replicated similarly in cell culture and stably expressed S. However, only S-2P was associated with MPV particles. After intranasal/intratracheal immunization of rhesus macaques, MPV/S and MPV/S-2P replicated to low levels in the airways. Despite its low-level replication, MPV/S-2P induced high levels of mucosal and serum IgG and IgA to SARS-CoV-2 S or its receptor-binding domain. Serum antibodies from MPV/S-2P-immunized animals efficiently inhibited ACE2 receptor binding to S proteins of variants of concern. Based on its attenuation and immunogenicity in macaques, MPV/S-2P will be further evaluated as a live-attenuated vaccine for intranasal immunization against SARS-CoV-2.

Mucosal prime-boost immunization with live murine pneumonia virus-vectored SARS-CoV-2 vaccine is protective in macaques.

Immunization via the respiratory route is predicted to increase the effectiveness of a SARS-CoV-2 vaccine. We evaluated the immunogenicity and protective efficacy of one or two doses of a live-attenuated murine pneumonia virus vector expressing SARS-CoV-2 prefusion-stabilized spike protein (MPV/S-2P), delivered intranasally/intratracheally to rhesus macaques. A single dose of MPV/S-2P was highly immunogenic, and a second dose increased the magnitude and breadth of the mucosal and systemic anti-S antibody responses and increased levels of dimeric anti-S IgA in the airways. MPV/S-2P also induced S-specific CD4+ and CD8+ T-cells in the airways that differentiated into large populations of tissue-resident memory cells within a month after the boost. One dose induced substantial protection against SARS-CoV-2 challenge, and two doses of MPV/S-2P were fully protective against SARS-CoV-2 challenge virus replication in the airways. A prime/boost immunization with a mucosally-administered live-attenuated MPV vector could thus be highly effective in preventing SARS-CoV-2 infection and replication.