Equivalent immunogenicity across three RSVpreF vaccine lots in healthy adults 18-49 years of age: Results of a randomized phase 3 study.

BACKGROUND: Bivalent RSV prefusion F subunit vaccine (RSVpreF), comprised of equal quantities of stabilized prefusion F antigens from the major circulating subgroups (RSV A, RSV B), is licensed for prevention of RSV-associated lower respiratory tract illness (LRTI) in older adults and for maternal vaccination for prevention of RSV-associated LRTI in infants. To support licensure and large-scale manufacturing, this lot consistency study was conducted to demonstrate equivalence in immunogenicity across 3 RSVpreF lots. METHODS: This phase 3, multicenter, parallel-group, placebo-controlled, randomized (1:1:1:1), double-blind study evaluated immunogenicity, safety, and tolerability of RSVpreF in healthy 18-49-year-old adults. Participants received a single 120-µg injection of 1 of 3RSVpreF lots or placebo. Geometric mean ratio (GMR) of RSV serum 50 % neutralizing geometric mean titers obtained 1 month after vaccination were compared between each vaccine lot for RSV A and RSV B, separately. Equivalence between lots was defined using a 1.5-fold criterion (GMR 95 % CIs for every lot pair within the 0.667-1.5 interval). Safety and tolerability were assessed. RESULTS: Of 992participants vaccinated, 948 were included in the evaluable immunogenicity population. All 3 RSVpreF lots elicited strong immune responses, meeting the 1.5-fold equivalence criterion for all between-lot comparisons for both RSV A and RSV B. Across the 3 lots, RSV A and RSV B 50 % neutralizing geometric mean titers substantially increased from baseline (RSV A, 1671-1795; RSV B 1358-1429) to 1 month after RSVpreF vaccination (RSV A, 24,131-25,238; RSV B, 19,238-21,702), corresponding to ≥14-fold increases in 50 % neutralizing titers for both RSV A and RSV B from before to 1 month after vaccination. Single doses of RSVpreF were safe and well tolerated, with similar safety profiles across the 3 RSVpreF lots. CONCLUSIONS: These findings support the reproducibility of RSVpreF vaccine manufacturing with similar safety and reactogenicity profiles (NCT05096208).

Rational design of a highly immunogenic prefusion-stabilized F glycoprotein antigen for a respiratory syncytial virus vaccine.

Respiratory syncytial virus (RSV) is the leading, global cause of serious respiratory disease in infants and is an important cause of respiratory illness in older adults. No RSV vaccine is currently available. The RSV fusion (F) glycoprotein is a key antigen for vaccine development, and its prefusion conformation is the target of the most potent neutralizing antibodies. Here, we describe a computational and experimental strategy for designing immunogens that enhance the conformational stability and immunogenicity of RSV prefusion F. We obtained an optimized vaccine antigen after screening nearly 400 engineered F constructs. Through in vitro and in vivo characterization studies, we identified F constructs that are more stable in the prefusion conformation and elicit ~10-fold higher serum-neutralizing titers in cotton rats than DS-Cav1. The stabilizing mutations of the lead construct (847) were introduced onto F glycoprotein backbones of strains representing the dominant circulating genotypes of the two major RSV subgroups, A and B. Immunization of cotton rats with a bivalent vaccine formulation of these antigens conferred complete protection against RSV challenge, with no evidence of disease enhancement. The resulting bivalent RSV prefusion F investigational vaccine has recently been shown to be efficacious against RSV disease in two pivotal phase 3 efficacy trials, one for passive protection of infants by immunization of pregnant women and the second for active protection of older adults by direct immunization.

Neutralization of Omicron sublineages and Deltacron SARS-CoV-2 by three doses of BNT162b2 vaccine or BA.1 infection.

Distinct SARS-CoV-2 Omicron sublineages have evolved showing increased fitness and immune evasion than the original Omicron variant BA.1. Here, we report the neutralization activity of sera from BNT162b2 vaccinated individuals or unimmunized Omicron BA.1-infected individuals against Omicron sublineages and "Deltacron" variant (XD). BNT162b2 post-dose 3 immune sera neutralized USA-WA1/2020, Omicron BA.1-, BA.2-, BA.2.12.1-, BA.3-, BA.4/5-, and XD-spike SARS-CoV-2s with geometric mean titres (GMTs) of 1335, 393, 298, 315, 216, 103, and 301, respectively; thus, BA.4/5 SARS-CoV-2 spike variant showed the highest propensity to evade vaccine neutralization compared to the original Omicron variants BA.1. BA.1-convalescent sera neutralized USA-WA1/2020, BA.1-, BA.2-, BA.2.12.1-, BA.3-, BA.4/5-, and Deltacron-spike SARS-CoV-2s with GMTs of 15, 430, 110, 109, 102, 25, and 284, respectively. The unique mutation F486V in the BA.4/5 spike contributes to the increased evasion of antibody neutralization by sublineage BA.4/5. The low neutralization titres of vaccinated sera or convalescent sera from BA.1 infected individuals against the emerging and rapidly spreading Omicron BA.4/5 variants provide important results for consideration in the selection of an updated vaccine in the current Omicron wave.Trial registration: ClinicalTrials.gov; identifier: NCT04368728.

A Phase 1/2 Study of a Respiratory Syncytial Virus Prefusion F Vaccine With and Without Adjuvant in Healthy Older Adults.

BACKGROUND: Respiratory syncytial virus (RSV) is an important cause of disease in older adults. We evaluated the safety and immunogenicity of a stabilized RSV prefusion F subunit (RSVpreF) vaccine candidate with/without adjuvant in adults aged 65-85 years. METHODS: Primary cohort participants were equally randomized to 1 of 7 RSVpreF formulations: 60 µg with either Al(OH)3 or CpG/Al(OH)3, 120 µg with either Al(OH)3 or CpG/Al(OH)3, 240 µg with either Al(OH)3 or CpG/Al(OH)3, 240 µg unadjuvanted, or placebo, administered concomitantly with high-dose seasonal inactivated influenza vaccine (SIIV). Participants in the month 0,2 cohort were randomized to RSVpreF 240 µg with CpG/Al(OH)3 or placebo, administered at months 0 and 2. RESULTS: All RSVpreF vaccine candidates elicited robust and persistent serum neutralizing responses when administered alone or with SIIV. There was no notable difference in neutralizing response between the formulations, including those containing CpG. In the month 0,2 cohort, there was no booster effect of dose 2. SIIV responses were similar or slightly lower with concomitant administration of RSVpreF. Most systemic and local reactions were mild and more frequent after RSVpreF than placebo. CONCLUSIONS: RSVpreF formulations were well tolerated and elicited robust neutralizing responses in older adults; however, CpG/Al(OH)3 did not further enhance responses. Clinical Trials Registration. NCT03572062.

Prefusion F Protein-Based Respiratory Syncytial Virus Immunization in Pregnancy.

BACKGROUND: Respiratory syncytial virus (RSV), a major cause of illness and death in infants worldwide, could be prevented by vaccination during pregnancy. The efficacy, immunogenicity, and safety of a bivalent RSV prefusion F protein-based (RSVpreF) vaccine in pregnant women and their infants are uncertain. METHODS: In a phase 2b trial, we randomly assigned pregnant women, at 24 through 36 weeks' gestation, to receive either 120 or 240 µg of RSVpreF vaccine (with or without aluminum hydroxide) or placebo. The trial included safety end points and immunogenicity end points that, in this interim analysis, included 50% titers of RSV A, B, and combined A/B neutralizing antibodies in maternal serum at delivery and in umbilical-cord blood, as well as maternal-to-infant transplacental transfer ratios. RESULTS: This planned interim analysis included 406 women and 403 infants; 327 women (80.5%) received RSVpreF vaccine. Most postvaccination reactions were mild to moderate; the incidence of local reactions was higher among women who received RSVpreF vaccine containing aluminum hydroxide than among those who received RSVpreF vaccine without aluminum hydroxide. The incidences of adverse events in the women and infants were similar in the vaccine and placebo groups; the type and frequency of these events were consistent with the background incidences among pregnant women and infants. The geometric mean ratios of 50% neutralizing titers between the infants of vaccine recipients and those of placebo recipients ranged from 9.7 to 11.7 among those with RSV A neutralizing antibodies and from 13.6 to 16.8 among those with RSV B neutralizing antibodies. Transplacental neutralizing antibody transfer ratios ranged from 1.41 to 2.10 and were higher with nonaluminum formulations than with aluminum formulations. Across the range of assessed gestational ages, infants of women who were immunized had similar titers in umbilical-cord blood and similar transplacental transfer ratios. CONCLUSIONS: RSVpreF vaccine elicited neutralizing antibody responses with efficient transplacental transfer and without evident safety concerns. (Funded by Pfizer; ClinicalTrials.gov number, NCT04032093.).

A Randomized Phase 1/2 Study of a Respiratory Syncytial Virus Prefusion F Vaccine.

BACKGROUND: Protection against human respiratory syncytial virus (RSV) remains an unmet need potentially addressable by maternal immunization. This phase 1/2 study evaluated a bivalent prefusion F vaccine (RSVpreF) with antigens from RSV subgroups A and B. METHODS: Adults 18-49 years old (N = 618) were randomized to receive placebo or 60, 120, or 240 µg RSVpreF with or without Al(OH)3. Safety and immunogenicity were evaluated. RESULTS: RSVpreF recipients more frequently reported local reactions and systemic events than placebo recipients; these were mostly mild or moderate. No vaccine-related serious adverse events occurred through 12 months postvaccination. All RSVpreF formulations induced 1-month postvaccination virus-neutralizing titers higher than those associated with protection of high-risk infants by palivizumab, the only prophylactic currently available for RSV. Geometric mean fold rises (GMFRs) across RSVpreF doses/formulations were 10.6-16.9 for RSV A and 10.3-19.8 for RSV B at 1 month postvaccination, greater than those historically elicited by postfusion F vaccines. GMFRs were 3.9-5.2 and 3.7-5.1, respectively, at 12 months postvaccination. CONCLUSIONS: RSVpreF formulations were safe, well tolerated, and induced robust neutralizing responses in adults. These findings support development of RSVpreF, which is being evaluated in a pivotal phase 3 study for maternal immunization. CLINICAL TRIALS REGISTRATION: NCT03529773.

Chlamydia trachomatis polymorphic membrane protein D is an oligomeric autotransporter with a higher-order structure.

Chlamydia trachomatis is a globally important obligate intracellular bacterial pathogen that is a leading cause of sexually transmitted disease and blinding trachoma. Effective control of these diseases will likely require a preventative vaccine. C. trachomatis polymorphic membrane protein D (PmpD) is an attractive vaccine candidate as it is conserved among C. trachomatis strains and is a target of broadly cross-reactive neutralizing antibodies. We show here that immunoaffinity-purified native PmpD exists as an oligomer with a distinct 23-nm flower-like structure. Two-dimensional blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that the oligomers were composed of full-length PmpD (p155) and two proteolytically processed fragments, the p73 passenger domain (PD) and the p82 translocator domain. We also show that PmpD undergoes an infection-dependent proteolytic processing step late in the growth cycle that yields a soluble extended PD (p111) that was processed into a p73 PD and a novel p30 fragment. Interestingly, soluble PmpD peptides possess putative eukaryote-interacting functional motifs, implying potential secondary functions within or distal to infected cells. Collectively, our findings show that PmpD exists as two distinct forms, a surface-associated oligomer exhibiting a higher-order flower-like structure and a soluble form restricted to infected cells. We hypothesize that PmpD is a multifunctional virulence factor important in chlamydial pathogenesis and could represent novel vaccine or drug targets for the control of human chlamydial infections.

Chlamydia trachomatis species-specific induction of ezrin tyrosine phosphorylation functions in pathogen entry.

Chlamydia trachomatis is an obligate intracellular pathogen of humans that exhibits species-specific biological characteristics in its early interactions with host cells that are likely important to pathogenesis. One such characteristic is the tyrosine phosphorylation (Tyr-P) of an approximately 70-kDa polypeptide that occurs only after infection of mammalian cells by human strains. We sought to identify this protein because of its potential significance to the pathogenesis of human chlamydial infections. Using an immunoproteomic approach we identified the host protein ezrin, a member of the ezrin-radixin-moesin (ERM) protein family that serves as a physical link between host cell receptors and the actin cytoskeleton. Confocal microscopy studies showed colocalization of ezrin and actin at the tips and crypts of microvilli, the site of chlamydial attachment and entry, respectively. To demonstrate a functional role for ezrin we infected cells with a dominant-negative (DN) ezrin phenotype or treated cells with ezrin-specific small interfering RNA (siRNA). We found that both DN and siRNA-treated cells were significantly less susceptible to infection by human chlamydial strains. Moreover, we demonstrated that inhibition of infection in ezrin DN cells occurred at the stage of chlamydial entry. We hypothesize that the C. trachomatis-specific Tyr-P of ezrin might relate to an undefined species-specific mechanism of pathogen entry that involves chlamydial specific ligand(s) and host cell coreceptor usage.

The phenotype and function of lung dendritic cells.

Dendritic cells (DCs) are central to the integration of innate and adaptive immunity. In contrast to B and T lymphocytes, DCs have retained many of the pattern recognition receptors and are thus uniquely able to sense stimuli such as tissue damage, necrosis, and bacterial and viral infection. Also, immature DCs respond to danger signals in the environment, which leads to their maturation, upon which DCs differentiate and acquire the ability to direct the development of the primary immune response. The ability of lung DCs to elicit specific CD4 and CD8 T lymphocyte responses have made them attractive targets for vaccine development strategies in the treatment and prevention of diseases such as allograft rejection responses, allergy, and asthma, as well as autoimmune disease and cancer.

Flt3-ligand, IL-4, GM-CSF, and adherence-mediated isolation of murine lung dendritic cells: assessment of isolation technique on phenotype and function.

Lung dendritic cells (DCs) are difficult to study due to their limited quantities and the complexities required for isolation. Although many procedures have been used to overcome this challenge, the effects of isolation techniques on lung DCs have not been reported. The current study shows that freshly isolated DCs (CD11c+) have limited ability to induce proliferation in allogeneic T cells, and are immature as indicated by low cell surface expression of costimulatory molecules compared with liver or splenic DCs. DCs isolated after overnight culture or from mice treated with Flt3L are phenotypically mature and potent stimulators of allogeneic T cells. DCs could not be propagated from lung mononuclear cells in response to IL-4 and GM-CSF. Contrary to data reported for nonpulmonary DCs, expression of CCR6 was decreased on mature lung DCs, and only a subset of mature DCs expressed higher levels of CCR7. Absence of CD8alpha expression indicates that freshly isolated DCs are myeloid-type, whereas mature DCs induced by overnight culture are both "lymphoid" (CD8alpha+) and "myeloid" (CD8alpha-). DCs from mice genetically deficient in CD8alpha expression were strong simulators of allogeneic T cells which was consistent with data showing that CD8alpha- DCs from CD8alpha-sufficient mice are better APCs compared with CD8alpha+ DCs from the same mice. These data show that freshly isolated lung DCs are phenotypically and functionally distinct, and that the isolation technique alters the biology of these cells. Therefore, lung DC phenotype and function must be interpreted relative to the technique used for isolation.