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.

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.

Evaluation of a Live-Attenuated Human Parainfluenza Virus Type 2 Vaccine in Adults and Children.

We conducted a phase I clinical trial of the live-attenuated recombinant human parainfluenza virus type 2 (HPIV2) vaccine candidate rHPIV2-15C/948L/∆1724 sequentially in adults, HPIV2-seropositive children, and HPIV2-seronegative children, the target population for vaccination. rHPIV2-15C/948L/∆1724 was appropriately restricted in replication in adults and HPIV2-seropositive children but was overattenuated for HPIV2-seronegative children.

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.

Correction: Generation by Reverse Genetics of an Effective, Stable, Live-Attenuated Newcastle Disease Virus Vaccine Based on a Currently Circulating, Highly Virulent Indonesian Strain.

[This corrects the article DOI: 10.1371/journal.pone.0052751.].

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.

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.

Ebola vaccine-induced protection in nonhuman primates correlates with antibody specificity and Fc-mediated effects.

Although substantial progress has been made with Ebola virus (EBOV) vaccine measures, the immune correlates of vaccine-mediated protection remain uncertain. Here, five mucosal vaccine vectors based on human and avian paramyxoviruses provided nonhuman primates with varying degrees of protection, despite expressing the same EBOV glycoprotein (GP) immunogen. Each vaccine produced antibody responses that differed in Fc-mediated functions and isotype composition, as well as in magnitude and coverage toward GP and its conformational and linear epitopes. Differences in the degree of protection and comprehensive characterization of the response afforded the opportunity to identify which features and functions were elevated in survivors and could therefore serve as vaccine correlates of protection. Pairwise network correlation analysis of 139 immune- and vaccine-related parameters was performed to demonstrate relationships with survival. Total GP-specific antibodies, as measured by biolayer interferometry, but not neutralizing IgG or IgA titers, correlated with survival. Fc-mediated functions and the amount of receptor binding domain antibodies were associated with improved survival outcomes, alluding to the protective mechanisms of these vaccines. Therefore, functional qualities of the antibody response, particularly Fc-mediated effects and GP specificity, rather than simply magnitude of the response, appear central to vaccine-induced protection against EBOV. The heterogeneity of the response profile between the vaccines indicates that each vaccine likely exhibits its own protective signature and the requirements for an efficacious EBOV vaccine are complex.

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.

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.

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.

Human parainfluenza virus type 3 expressing the respiratory syncytial virus pre-fusion F protein modified for virion packaging yields protective intranasal vaccine candidates.

Human respiratory syncytial virus (RSV) and parainfluenza virus type 3 (HPIV3) are among the most common viral causes of childhood bronchiolitis and pneumonia worldwide, and lack effective antiviral drugs or vaccines. Recombinant (r) HPIV3 was modified to express the RSV fusion (F) glycoprotein, the major RSV neutralization and protective antigen, providing a live intranasal bivalent HPIV3/RSV vaccine candidate. This extends previous studies using a chimeric bovine-human PIV3 vector (rB/HPIV3). One advantage is that rHPIV3 expresses all of the HPIV3 antigens compared to only two for rB/HPIV3. In addition, the use of rHPIV3 as vector should avoid excessive attenuation following addition of the modified RSV F gene, which may occur with rB/HPIV3. To enhance its immunogenicity, RSV F was modified (i) to increase the stability of the prefusion (pre-F) conformation and (ii) by replacement of its transmembrane (TM) and cytoplasmic tail (CT) domains with those of HPIV3 F (H3TMCT) to increase incorporation in the vector virion. RSV F (+/- H3TMCT) was expressed from the first (F/preN) or the second (F/N-P) gene position of rHPIV3. The H3TMCT modification dramatically increased packaging of RSV F into the vector virion and, in hamsters, resulted in significant increases in the titer of high-quality serum RSV-neutralizing antibodies, in addition to the increase conferred by pre-F stabilization. Only F-H3TMCT/preN replication was significantly attenuated in the nasal turbinates by the RSV F insert. F-H3TMCT/preN, F/N-P, and F-H3TMCT/N-P provided complete protection against wt RSV challenge. F-H3TMCT/N-P exhibited the most stable and highest expression of RSV F, providing impetus for its further development.

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.

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.

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.

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.