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.

Rescue of codon-pair deoptimized respiratory syncytial virus by the emergence of genomes with very large internal deletions that complemented replication.

Recoding viral genomes by introducing numerous synonymous but suboptimal codon pairs-called codon-pair deoptimization (CPD)-provides new types of live-attenuated vaccine candidates. The large number of nucleotide changes resulting from CPD should provide genetic stability to the attenuating phenotype, but this has not been rigorously tested. Human respiratory syncytial virus in which the G and F surface glycoprotein ORFs were CPD (called Min B) was temperature-sensitive and highly restricted in vitro. When subjected to selective pressure by serial passage at increasing temperatures, Min B substantially regained expression of F and replication fitness. Whole-genome deep sequencing showed many point mutations scattered across the genome, including one combination of six linked point mutations. However, their reintroduction into Min B provided minimal rescue. Further analysis revealed viral genomes bearing very large internal deletions (LD genomes) that accumulated after only a few passages. The deletions relocated the CPD F gene to the first or second promoter-proximal gene position. LD genomes amplified de novo in Min B-infected cells were encapsidated, expressed high levels of F, and complemented Min B replication in trans This study provides insight on a variation of the adaptability of a debilitated negative-strand RNA virus, namely the generation of defective minihelper viruses to overcome its restriction. This is in contrast to the common "defective interfering particles" that interfere with the replication of the virus from which they originated. To our knowledge, defective genomes that promote rather than inhibit replication have not been reported before in RNA viruses.

Interprotomer disulfide-stabilized variants of the human metapneumovirus fusion glycoprotein induce high titer-neutralizing responses.

Human metapneumovirus (HMPV) is a major cause of respiratory disease worldwide, particularly among children and the elderly. Although there is no licensed HMPV vaccine, promising candidates have been identified for related pneumoviruses based on the structure-based stabilization of the fusion (F) glycoprotein trimer, with prefusion-stabilized F glycoprotein trimers eliciting significantly higher neutralizing responses than their postfusion F counterparts. However, immunization with HMPV F trimers in either prefusion or postfusion conformations has been reported to elicit equivalent neutralization responses. Here we investigate the impact of stabilizing disulfides, especially interprotomer disulfides (IP-DSs) linking protomers of the F trimer, on the elicitation of HMPV-neutralizing responses. We designed F trimer disulfides, screened for their expression, and used electron microscopy (EM) to confirm their formation, including that of an unexpected postfusion variant. In mice, IP-DS-stabilized prefusion and postfusion HMPV F elicited significantly higher neutralizing responses than non-IP-DS-stabilized HMPV Fs. In macaques, the impact of IP-DS stabilization was more measured, although IP-DS-stabilized variants of either prefusion or postfusion HMPV F induced neutralizing responses many times the average titers observed in a healthy human cohort. Serological and absorption-based analyses of macaque responses revealed elicited HMPV-neutralizing responses to be absorbed differently by IP-DS-containing and by non-IP-DS-containing postfusion Fs, suggesting IP-DS stabilization to alter not only the immunogenicity of select epitopes but their antigenicity as well. We speculate the observed increase in immunogenicity by IP-DS trimers to be related to reduced interprotomer flexibility within the HMPV F trimer.

Reversion mutations in phosphoprotein P of a codon-pair-deoptimized human respiratory syncytial virus confer increased transcription, immunogenicity, and genetic stability without loss of attenuation.

Recoding viral genomes by introducing numerous synonymous nucleotide substitutions that create suboptimal codon pairs provides new live-attenuated vaccine candidates. Because recoding typically involves a large number of nucleotide substitutions, the risk of de-attenuation is presumed to be low. However, this has not been thoroughly studied. We previously generated human respiratory syncytial virus (RSV) in which the NS1, NS2, N, P, M and SH ORFs were codon-pair deoptimized (CPD) by 695 synonymous nucleotide changes (Min A virus). Min A exhibited a global reduction in transcription and protein synthesis, was restricted for replication in vitro and in vivo, and exhibited moderate temperature sensitivity. Here, we show that under selective pressure by serial passage at progressively increasing temperatures, Min A regained replication fitness and lost its temperature sensitivity. Whole-genome deep sequencing identified numerous missense mutations in several genes, in particular ones accumulating between codons 25 and 34 of the phosphoprotein (P), a polymerase cofactor and chaperone. When re-introduced into Min A, these P mutations restored viral transcription to wt level, resulting in increased protein expression and RNA replication. Molecular dynamic simulations suggested that these P mutations increased the flexibility of the N-terminal domain of P, which might facilitate its interaction with the nucleoprotein N, and increase the functional efficiency of the RSV transcription/replication complex. Finally, we evaluated the effect of the P mutations on Min A replication and immunogenicity in hamsters. Mutation P[F28V] paradoxically reduced Min A replication but not its immunogenicity. The further addition of one missense mutation each in M and L generated a version of Min A with increased genetic stability. Thus, this study provides further insight into the adaptability of large-scale recoded RNA viruses under selective pressure and identified an improved CPD RSV vaccine candidate.

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.

Live-attenuated Vaccines Prevent Respiratory Syncytial Virus-associated Illness in Young Children.

Rationale: Active immunization is needed to protect infants and young children against respiratory syncytial virus (RSV). Rationally designed live-attenuated RSV vaccines are in clinical development.Objectives: Develop preliminary estimates of vaccine efficacy, assess durability of antibody responses to vaccination and "booster" responses after natural RSV infection, and determine sample sizes needed for more precise estimates of vaccine efficacy.Methods: We analyzed data from seven phase 1 trials of live-attenuated RSV vaccines in 6- to 24-month-old children (n = 239).Measurements and Main Results: The five vaccine regimens that induced neutralizing antibody responses in ≥80% of vaccinees (defined post hoc as "more promising") protected against RSV-associated medically attended acute respiratory illness (RSV-MAARI) and medically attended acute lower respiratory illness (RSV-MAALRI) and primed for potent anamnestic responses upon natural exposure to wild-type RSV. Among recipients of "more promising" RSV vaccines, efficacy against RSV-MAARI was 67% (95% confidence interval [CI], 24 to 85; P = 0.008) and against RSV-MAALRI was 88% (95% CI, -9 to 99; P = 0.04). A greater than or equal to fourfold increase in RSV serum neutralizing antibody following vaccination was strongly associated with protection against RSV-MAARI (odds ratio, 0.26; 95% CI, 0.09 to 0.75; P = 0.014) and RSV-MAALRI; no child with a greater than or equal to fourfold increase developed RSV-MAALRI. Rates of RSV-MAARI and RSV-MAALRI in placebo recipients were 21% and 7%, respectively. Given these rates, a study of 540 RSV-naive children would have 90% power to demonstrate ≥55% efficacy against RSV-MAARI and ≥80% efficacy against RSV-MAALRI; if rates were 10% and 3%, a study of 1,300 RSV-naive children would be needed.Conclusions: Rapid development of a live-attenuated RSV vaccine could contribute substantially to reducing the global burden of RSV disease.

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.

A Parainfluenza Virus Vector Expressing the Respiratory Syncytial Virus (RSV) Prefusion F Protein Is More Effective than RSV for Boosting a Primary Immunization with RSV.

Live-attenuated pediatric vaccines for intranasal administration are being developed for human respiratory syncytial virus (RSV), an important worldwide pediatric respiratory pathogen that lacks a licensed vaccine or suitable antiviral drug. We evaluated a prime-boost strategy in which primary immunization with RSV was boosted by secondary immunization with RSV or with a chimeric recombinant bovine/human parainfluenza virus type 3 (rB/HPIV3) vector expressing the RSV fusion F protein. The vector-expressed F protein had been engineered (DS-Cav1 mutations) for increased stability in the highly immunogenic prefusion (pre-F) conformation, with or without replacement of its transmembrane and cytoplasmic tail domains with their counterparts from bovine parainfluenza virus type 3 (BPIV3) F protein to direct incorporation into the vector virion for increased immunogenicity. In hamsters that received a primary infection with RSV, a booster infection with RSV ∼6 weeks later was completely restricted for producing infectious virus but induced a significant increase in the serum RSV-plaque-reduction neutralizing antibody titer (RSV-PRNT). Boosting instead with the rB/HPIV3-RSV-pre-F vectors resulted in efficient replication and induced significantly higher RSV-PRNTs than RSV. In African green monkeys that received a primary infection with RSV, a booster infection with RSV ∼2, ∼6, or ∼15 months later was highly restricted, whereas booster infections with the vectors had robust replication. Compared with RSV, boosts with the vectors induced 7- to 15-fold higher titers of RSV-specific serum antibodies with high neutralizing activity, as well as significantly higher titers of RSV-specific mucosal IgA antibodies. These findings support further development of this heterologous prime-boost strategy.IMPORTANCE Immune responses to RSV in infants can be reduced due to immunological immaturity and immunosuppression by RSV-specific maternal antibodies. In infants and young children, two infections with wild-type RSV typically are needed to achieve the titers of RSV-specific serum antibodies and protection against illness that are observed in adults. Therefore, a boost might substantially improve the performance of live pediatric RSV vaccines presently being developed. Hamsters and African green monkeys received a primary intranasal infection with RSV and were given a boost with RSV or a parainfluenza virus (PIV) vector expressing RSV fusion protein engineered for enhanced immunogenicity. The RSV boost was highly restricted but induced a significant increase in serum RSV-neutralizing antibodies. The PIV vectors replicated efficiently and induced significantly higher antibody responses. The use of an attenuated PIV vector expressing RSV antigen to boost a primary immunization with an attenuated RSV warrants further evaluation.

The alpha-1 subunit of the Na+,K+-ATPase (ATP1A1) is required for macropinocytic entry of respiratory syncytial virus (RSV) in human respiratory epithelial cells.

Human respiratory syncytial virus (RSV) is the leading viral cause of acute pediatric lower respiratory tract infections worldwide, with no available vaccine or effective antiviral drug. To gain insight into virus-host interactions, we performed a genome-wide siRNA screen. The expression of over 20,000 cellular genes was individually knocked down in human airway epithelial A549 cells, followed by infection with RSV expressing green fluorescent protein (GFP). Knockdown of expression of the cellular ATP1A1 protein, which is the major subunit of the Na+,K+-ATPase of the plasma membrane, had one of the strongest inhibitory effects on GFP expression and viral titer. Inhibition was not observed for vesicular stomatitis virus, indicating that it was RSV-specific rather than a general effect. ATP1A1 formed clusters in the plasma membrane very early following RSV infection, which was independent of replication but dependent on the attachment glycoprotein G. RSV also triggered activation of ATP1A1, resulting in signaling by c-Src-kinase activity that transactivated epidermal growth factor receptor (EGFR) by Tyr845 phosphorylation. ATP1A1 signaling and activation of both c-Src and EGFR were found to be required for efficient RSV uptake. Signaling events downstream of EGFR culminated in the formation of macropinosomes. There was extensive uptake of RSV virions into macropinosomes at the beginning of infection, suggesting that this is a major route of RSV uptake, with fusion presumably occurring in the macropinosomes rather than at the plasma membrane. Important findings were validated in primary human small airway epithelial cells (HSAEC). In A549 cells and HSAEC, RSV uptake could be inhibited by the cardiotonic steroid ouabain and the digitoxigenin derivative PST2238 (rostafuroxin) that bind specifically to the ATP1A1 extracellular domain and block RSV-triggered EGFR Tyr845 phosphorylation. In conclusion, we identified ATP1A1 as a host protein essential for macropinocytic entry of RSV into respiratory epithelial cells, and identified PST2238 as a potential anti-RSV drug.

Intermediate Monocytes in Acute Alcoholic Hepatitis Are Functionally Activated and Induce IL-17 Expression in CD4+ T Cells.

In humans, the three main circulating monocyte subsets are defined by their relative cell surface expression of CD14 and CD16. They are all challenging to study because their characteristics are strongly context specific, and this has led to a range of conflicting reports about their function, which is especially so for CD14++CD16+ (intermediate) monocytes. Ex vivo cultures are also often confounded by the concomitant use of immunosuppressive drugs. We therefore sought to characterize the phenotype and function of intermediate monocytes in the setting of acute inflammation prior to treatment in a cohort of 41 patients with acute alcoholic hepatitis (AH). Circulating intermediate monocytes were enriched in patients with AH and had an activated phenotype with enhanced expression of CCR2 and CD206 compared with healthy controls. Proinflammatory cytokine expression, including IL-1ß and IL-23, was also higher than in healthy controls, but both classical (CD14++CD16-) and intermediate monocytes in AH were refractory to TLR stimulation. Compared with healthy controls, both AH monocyte subsets had greater phagocytic capacity, enhanced ability to drive memory T cell proliferation in coculture, and skewed CD4+ T cells to express an increased ratio of IL-17/IFN-γ. Furthermore, liver tissue from AH patients demonstrated an enrichment of monocytes including the intermediate subset compared with controls. These data demonstrate that intermediate monocytes are expanded, functionally activated, induce CD4+ T cell IL-17 expression, and are enriched in the liver of patients with AH.

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.

Lack of Activation Marker Induction and Chemokine Receptor Switch in Human Neonatal Myeloid Dendritic Cells in Response to Human Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) infects and causes disease in infants and reinfects with reduced disease throughout life without significant antigenic change. In contrast, reinfection by influenza A virus (IAV) largely requires antigenic change. The adaptive immune response depends on antigen presentation by dendritic cells (DC), which may be too immature in young infants to induce a fully protective immune response against RSV reinfections. We therefore compared the ability of RSV and IAV to activate primary human cord blood (CB) and adult blood (AB) myeloid DC (mDC). While RSV and IAV infected with similar efficiencies, RSV poorly induced maturation and cytokine production in CB and AB mDC. This difference between RSV and IAV was more profound in CB mDC. While IAV activated CB mDC to some extent, RSV did not induce CB mDC to increase the maturation markers CD38 and CD86 or CCR7, which directs DC migration to lymphatic tissue. Low CCR7 surface expression was associated with high expression of CCR5, which keeps DC in inflamed peripheral tissues. To evaluate a possible inhibition by RSV, we subjected RSV-inoculated AB mDC to secondary IAV inoculation. While RSV-inoculated AB mDC responded to secondary IAV inoculation by efficiently upregulating activation markers and cytokine production, IAV-induced CCR5 downregulation was slightly inhibited in cells exhibiting robust RSV infection. Thus, suboptimal stimulation and weak and mostly reversible inhibition seem to be responsible for inefficient mDC activation by RSV. The inefficient mDC stimulation and immunological immaturity in young infants may contribute to reduced immune responses and incomplete protection against RSV reinfection.IMPORTANCE Respiratory syncytial virus (RSV) causes disease early in life and can reinfect symptomatically throughout life without undergoing significant antigenic change. In contrast, reinfection by influenza A virus (IAV) requires antigenic change. The adaptive immune response depends on antigen presentation by dendritic cells (DC). We used myeloid DC (mDC) from cord blood and adult blood donors to evaluate whether immunological immaturity contributes to the inability to mount a fully protective immune response to RSV. While IAV induced some activation and chemokine receptor switching in cord blood mDC, RSV did not. This appeared to be due to a lack of activation and a weak and mostly reversible inhibition of DC functions. Both viruses induced a stronger activation of mDC from adults than mDC from cord blood. Thus, inefficient stimulation of mDC by RSV and immunological immaturity may contribute to reduced immune responses and increased susceptibility to RSV disease and reinfection in young infants.

Effects of Alterations to the CX3C Motif and Secreted Form of Human Respiratory Syncytial Virus (RSV) G Protein on Immune Responses to a Parainfluenza Virus Vector Expressing the RSV G Protein.

Human respiratory syncytial virus (RSV) is a major pediatric respiratory pathogen. The attachment (G) and fusion (F) glycoproteins are major neutralization and protective antigens. RSV G is expressed as membrane-anchored (mG) and -secreted (sG) forms, both containing a central fractalkine-like CX3C motif. The CX3C motif and sG are thought to interfere with host immune responses and have been suggested to be omitted from a vaccine. We used a chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) vector to express RSV wild-type (wt) G and modified forms, including sG alone, mG alone, mutants with ablated CX3C, and G with enhanced packaging into vector virions. In hamsters, these viruses replicated to similar titers. When assayed with a complement-enhanced neutralization assay in Vero cells, sG did not reduce the serum RSV- or PIV3-neutralizing antibody (NAb) responses, whereas ablating CX3C drastically reduced the RSV NAb response. Protective efficacy against RSV challenge was not reduced by sG but was strongly dependent on the CX3C motif. In ciliated human airway epithelial (HAE) cells, NAbs induced by wt G, but not by wt F, completely blocked RSV infection in the absence of added complement. This activity was dependent on the integrity of the CX3C motif. In hamsters, the rB/HPIV3 expressing wt G conferred better protection against RSV challenge than that expressing wt F. Codon optimization of the wt G further increased its immunogenicity and protective efficacy. This study showed that ablation of the CX3C motif or sG in an RSV vaccine, as has been suggested previously, would be ill advised.IMPORTANCE Human RSV is the leading viral cause of severe pediatric respiratory illness. An RSV vaccine is not yet available. The RSV attachment protein G is an important protective and neutralization antigen. G contains a conserved fractalkine-like CX3C motif and is expressed in mG and sG forms. sG and the CX3C motif are thought to interfere with host immune responses, but this remains poorly characterized. Here, we used an attenuated chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) vector to express various modified forms of RSV G. We demonstrated that strong antibody and protective responses could be induced by G alone, and that this was highly dependent on the integrity of the CX3C motif. There was no evidence that sG or the CX3C motif impaired immune responses against RSV G or the rB/HPIV3 vector. rB/HPIV3 expressing wt RSV G provides a bivalent vaccine against RSV and HPIV3.

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.

Murine Pneumonia Virus Expressing the Fusion Glycoprotein of Human Respiratory Syncytial Virus from an Added Gene Is Highly Attenuated and Immunogenic in Rhesus Macaques.

Human respiratory syncytial virus (RSV) continues to be the leading viral cause of severe acute lower respiratory tract disease in infants and children worldwide. A licensed vaccine or antiviral drug suitable for routine use remains unavailable. Like RSV, Murine pneumonia virus (MPV) is a member of the genus Orthopneumovirus, family Pneumoviridae Humans are not normally exposed to MPV, and MPV is not cross-protective with RSV. We evaluated MPV as an RSV vaccine vector expressing the RSV fusion (F) glycoprotein. The RSV F open reading frame (ORF) was codon optimized, and the encoded RSV F protein was made identical to an early passage of RSV strain A2. The RSV F ORF was placed under the control of MPV transcription signals and inserted at the first (rMPV-F1), third (rMPV-F3), or fourth (rMPV-F4) gene position of a version of the MPV genome that contained a codon-pair-optimized polymerase (L) gene. The recovered viruses replicated in vitro as efficiently as the empty vector, with stable expression of RSV F protein. Replication and immunogenicity of rMPV-F1 and rMPV-F3 were evaluated in rhesus macaques following intranasal and intratracheal administration. Both viruses replicated at low levels in the upper and lower respiratory tracts, maintained stable RSV F expression, and induced RSV-neutralizing serum antibodies at high levels similar to those induced by wild-type RSV replicating to a 5- to 25-fold-higher titer. In conclusion, this study demonstrated that rMPV provides a highly attenuated yet immunogenic vector for the expression of RSV F protein, with potential application in RSV-naive and RSV-experienced populations.IMPORTANCE Human respiratory syncytial virus (RSV) is an important human pathogen that lacks a licensed vaccine or antiviral drug suitable for routine use. We describe here the evaluation of recombinant murine pneumonia virus (rMPV) as a live-attenuated vector that expresses the RSV F protein, the major RSV neutralization antigen, as an experimental RSV vaccine. The rMPV-RSV-F vectors expressing RSV F from the first, third, or fourth gene position were genetically stable and were not restricted for replication in vitro In contrast, the vectors exhibited highly attenuated replication in the respiratory tract of rhesus macaques, maintained stable RSV F expression, and induced RSV-neutralizing serum antibodies at high titers similar to those conferred by wild-type RSV. Given the lack of preexisting immunity to MPV in humans and the lack of cross-neutralization and cross-protection between MPV and RSV, an rMPV-vectored RSV vaccine should be immunogenic in both RSV-naive children and RSV-experienced adults.

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.

Attenuated Human Parainfluenza Virus Type 1 Expressing the Respiratory Syncytial Virus (RSV) Fusion (F) Glycoprotein from an Added Gene: Effects of Prefusion Stabilization and Packaging of RSV F.

Human respiratory syncytial virus (RSV) is the most prevalent worldwide cause of severe respiratory tract infection in infants and young children. Human parainfluenza virus type 1 (HPIV1) also causes severe pediatric respiratory illness, especially croup. Both viruses lack vaccines. Here, we describe the preclinical development of a bivalent RSV/HPIV1 vaccine based on a recombinant HPIV1 vector, attenuated by a stabilized mutation, that expresses RSV F protein modified for increased stability in the prefusion (pre-F) conformation by previously described disulfide bond (DS) and hydrophobic cavity-filling (Cav1) mutations. RSV F was expressed from the first or second gene position as the full-length protein or as a chimeric protein with its transmembrane and cytoplasmic tail (TMCT) domains substituted with those of HPIV1 F in an effort to direct packaging in the vector particles. All constructs were recovered by reverse genetics. The TMCT versions of RSV F were packaged in the rHPIV1 particles much more efficiently than their full-length counterparts. In hamsters, the presence of the RSV F gene, and in particular the TMCT versions, was attenuating and resulted in reduced immunogenicity. However, the vector expressing full-length RSV F from the pre-N position was immunogenic for RSV and HPIV1. It conferred complement-independent high-quality RSV-neutralizing antibodies at titers similar to those of wild-type RSV and provided protection against RSV challenge. The vectors exhibited stable RSV F expression in vitro and in vivo In conclusion, an attenuated rHPIV1 vector expressing a pre-F-stabilized form of RSV F demonstrated promising immunogenicity and should be further developed as an intranasal pediatric vaccine.IMPORTANCE RSV and HPIV1 are major viral causes of acute pediatric respiratory illness for which no vaccines or suitable antiviral drugs are available. The RSV F glycoprotein is the major RSV neutralization antigen. We used a rHPIV1 vector, bearing a stabilized attenuating mutation, to express the RSV F glycoprotein bearing amino acid substitutions that increase its stability in the pre-F form, the most immunogenic form that elicits highly functional virus-neutralizing antibodies. RSV F was expressed from the pre-N or N-P gene position of the rHPIV1 vector as a full-length protein or as a chimeric form with its TMCT domain derived from HPIV1 F. TMCT modification greatly increased packaging of RSV F into the vector particles but also increased vector attenuation in vivo, resulting in reduced immunogenicity. In contrast, full-length RSV F expressed from the pre-N position was immunogenic, eliciting complement-independent RSV-neutralizing antibodies and providing protection against RSV challenge.