Multivalent antigen display on nanoparticle immunogens increases B cell clonotype diversity and neutralization breadth to pneumoviruses.

Nanoparticles for multivalent display and delivery of vaccine antigens have emerged as a promising avenue for enhancing B cell responses to protein subunit vaccines. Here, we evaluated B cell responses in rhesus macaques immunized with prefusion-stabilized respiratory syncytial virus (RSV) F glycoprotein trimer compared with nanoparticles displaying 10 or 20 copies of the same antigen. We show that multivalent display skews antibody specificities and drives epitope-focusing of responding B cells. Antibody cloning and repertoire sequencing revealed that focusing was driven by the expansion of clonally distinct B cells through recruitment of diverse precursors. We identified two antibody lineages that developed either ultrapotent neutralization or pneumovirus cross-neutralization from precursor B cells with low initial affinity for the RSV-F immunogen. This suggests that increased avidity by multivalent display facilitates the activation and recruitment of these cells. Diversification of the B cell response by multivalent nanoparticle immunogens has broad implications for vaccine design.

COVID-19 vaccine mRNA-1273 elicits a protective immune profile in mice that is not associated with vaccine-enhanced disease upon SARS-CoV-2 challenge.

Vaccine-associated enhanced respiratory disease (VAERD) was previously observed in some preclinical models of severe acute respiratory syndrome (SARS) and MERS coronavirus vaccines. We used the SARS coronavirus 2 (SARS-CoV-2) mouse-adapted, passage 10, lethal challenge virus (MA10) mouse model of acute lung injury to evaluate the immune response and potential for immunopathology in animals vaccinated with research-grade mRNA-1273. Whole-inactivated virus or heat-denatured spike protein subunit vaccines with alum designed to elicit low-potency antibodies and Th2-skewed CD4+ T cells resulted in reduced viral titers and weight loss post challenge but more severe pathological changes in the lung compared to saline-immunized animals. In contrast, a protective dose of mRNA-1273 induced favorable humoral and cellular immune responses that protected from viral replication in the upper and lower respiratory tract upon challenge. A subprotective dose of mRNA-1273 reduced viral replication and limited histopathological manifestations compared to animals given saline. Overall, our findings demonstrate an immunological signature associated with antiviral protection without disease enhancement following vaccination with mRNA-1273.

Vaccination with prefusion-stabilized respiratory syncytial virus fusion protein induces genetically and antigenically diverse antibody responses.

An effective vaccine for respiratory syncytial virus (RSV) is an unrealized public health goal. A single dose of the prefusion-stabilized fusion (F) glycoprotein subunit vaccine (DS-Cav1) substantially increases serum-neutralizing activity in healthy adults. We sought to determine whether DS-Cav1 vaccination induces a repertoire mirroring the pre-existing diversity from natural infection or whether antibody lineages targeting specific epitopes predominate. We evaluated RSV F-specific B cell responses before and after vaccination in six participants using complementary B cell sequencing methodologies and identified 555 clonal lineages. DS-Cav1-induced lineages recognized the prefusion conformation of F (pre-F) and were genetically diverse. Expressed antibodies recognized all six antigenic sites on the pre-F trimer. We identified 34 public clonotypes, and structural analysis of two antibodies from a predominant clonotype revealed a common mode of recognition. Thus, vaccination with DS-Cav1 generates a diverse polyclonal response targeting the antigenic sites on pre-F, supporting the development and advanced testing of pre-F-based vaccines against RSV.

Immunological Lessons from Respiratory Syncytial Virus Vaccine Development.

Respiratory syncytial virus (RSV) has eluded active vaccination efforts for more than five decades and continues to cause substantial morbidity and mortality in infants, the immunocompromised, and older adults. Although newer approaches of passive antibody-mediated protection show promise, vaccines aimed at eliciting fusion protein (F)-targeting antibodies have repeatedly failed to meet pre-established, modest-efficacy goals. Newer candidates, including protein-based vaccines, live-attenuated viruses, and gene-based delivery platforms, incorporate structurally defined and stabilized versions of the prefusion form of the F glycoprotein and are advancing rapidly into critical efficacy studies in susceptible target populations. This review discusses the storied history of RSV vaccine development, immunological lessons learned along the way, and critical findings about protein structure that remodeled our understanding of protective immunity to this important pathogen.

Enterovirus virus-like-particle and inactivated poliovirus vaccines do not elicit substantive cross-reactive antibody responses.

Human enteroviruses are the most common human pathogen with over 300 distinct genotypes. Previous work with poliovirus has suggested that it is possible to generate antibody responses in humans and animals that can recognize members of multiple enterovirus species. However, cross protective immunity across multiple enteroviruses is not observed epidemiologically in humans. Here we investigated whether immunization of mice or baboons with inactivated poliovirus or enterovirus virus-like-particles (VLPs) vaccines generates antibody responses that can recognize enterovirus D68 or A71. We found that mice only generated antibodies specific for the antigen they were immunized with, and repeated immunization failed to generate cross-reactive antibody responses as measured by both ELISA and neutralization assay. Immunization of baboons with IPV failed to generate neutralizing antibody responses against enterovirus D68 or A71. These results suggest that a multivalent approach to enterovirus vaccination is necessary to protect against enterovirus disease in vulnerable populations.

Vaccination with prefusion-stabilized respiratory syncytial virus fusion protein elicits antibodies targeting a membrane-proximal epitope.

IMPORTANCE: Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants, infecting all children by age 5. RSV also causes substantial morbidity and mortality in older adults, and a vaccine for older adults based on a prefusion-stabilized form of the viral F glycoprotein was recently approved by the FDA. Here, we investigate a set of antibodies that belong to the same public clonotype and were isolated from individuals vaccinated with a prefusion-stabilized RSV F protein. Our results reveal that these antibodies are highly potent and recognize a previously uncharacterized antigenic site on the prefusion F protein. Vaccination with prefusion RSV F proteins appears to boost the elicitation of these neutralizing antibodies, which are not commonly elicited by natural infection.

RSV neutralizing antibodies in dried blood.

BACKGROUND: The key correlate of protection of respiratory syncytial virus (RSV) vaccines and monoclonal antibodies (mAb) is virus neutralization, measured using sera obtained through venipuncture. Dried blood obtained with a finger prick can simplify acquisition, processing, storage, and transport in trials, and thereby reduce costs. In this study we validate an assay to measure RSV neutralization in dried capillary blood. METHODS: Functional antibodies were compared between matched serum and dried blood samples from a phase I trial with RSM01, an investigational anti-RSV Prefusion F mAb. Hep-2 cells were infected with a serial dilution of sample-virus mixture using RSV-A2-mKate to determine half-maximal inhibitory concentration. Stability of dried blood was evaluated over time and during temperature stress. RESULTS: Functional antibodies in dried blood were highly correlated with serum (R2 = 0.98, p < 0.0001). The precision of the assay for dried blood was similar to serum. The function of mAb remained stable for 9 months at room temperature and frozen dried blood samples. INTERPRETATION: We demonstrated the feasibility of measuring RSV neutralization using dried blood as a patient-centered solution that may replace serology testing in trials against RSV or other viruses, such as influenza and SARS-CoV-2.

Recurrent Respiratory Syncytial Virus Infection in a CD14-Deficient Patient.

BACKGROUND: Recurrent respiratory syncytial virus (RSV) infection requiring hospitalization is rare and the underlying mechanism is unknown. We aimed to determine the role of CD14-mediated immunity in the pathogenesis of recurrent RSV infection. METHODS: We performed genotyping and longitudinal immunophenotyping of the first patient with a genetic CD14 deficiency who developed recurrent RSV infection. We analyzed gene expression profiles and interleukin (IL)-6 production by patient peripheral blood mononuclear cells in response to RSV pre- and post-fusion (F) protein. We generated CD14-deficient human nasal epithelial cells cultured at air-liquid interface (HNEC-ALI) of patient-derived cells and after CRISPR-based gene editing of control cells. We analyzed viral replication upon RSV infection. RESULTS: Sanger sequencing revealed a homozygous single-nucleotide deletion in CD14, resulting in absence of the CD14 protein in the index patient. In vitro, viral replication was similar in wild-type and CD14-/- HNEC-ALI. Loss of immune cell CD14 led to impaired cytokine and chemokine responses to RSV pre- and post-F protein, characterized by absence of IL-6 production. CONCLUSIONS: We report an association of recurrent RSV bronchiolitis with a loss of CD14 function in immune cells. Lack of CD14 function led to defective immune responses to RSV pre- and post-F protein without a change in viral replication.

T cell immunity to SARS-CoV-2 following natural infection and vaccination.

SARS-CoV-2 first emerged in the human population in late 2019 in Wuhan, China, and in a matter of months, spread across the globe resulting in the Coronavirus Disease 19 (COVID-19) pandemic and substantial economic fallout. SARS-CoV-2 is transmitted between humans via respiratory particles, with infection presenting a spectrum of clinical manifestations ranging from asymptomatic to respiratory failure with multiorgan dysfunction and death in severe cases. Prior experiences with human pathogenic coronaviruses and respiratory virus diseases in general have revealed an important role for cellular immunity in limiting disease severity. Here, we review some of the key mechanisms underlying cell-mediated immunity to respiratory viruses and summarize our current understanding of the functional capacity and role of SARS-CoV-2-specific T cells following natural infection and vaccination.

OMIP-061: 20-Color Flow Cytometry Panel for High-Dimensional Characterization of Murine Antigen-Presenting Cells.

This 20-color flow cytometry panel was designed to resolve the cellular heterogeneity of antigen-presenting cells and was optimized for lymph node tissue. Reagents were carefully selected and optimized for identification of B cells (B220), neutrophils (Ly6G), monocytes and macrophages (Ly6C, CD169, F4/80), and dendritic cells (XCR1, CD172a, CD11c, I-A/I-E, CD24, CD64, pDCA-1, CD103, CD11b). Inclusion of additional functional markers involved in cell migration (CCR7), co-stimulation (CD80), and adhesion (ICAM-1) enabled further phenotypic characterization. Finally, this panel has been tested and is compatible with fluorescently labeled antigens such as Alexa Fluor 488 (Ax488) for the study of antigen-bearing cells in vivo. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.

Pulmonary Dendritic Cell Subsets Shape the Respiratory Syncytial Virus-Specific CD8+ T Cell Immunodominance Hierarchy in Neonates.

Young infants are generally more susceptible to viral infections and experience more severe disease than do adults. CD8+ T cells are important for viral clearance, and although often ineffective in neonates they can be protective when adequately stimulated. Using a murine CB6F1/J hybrid model of respiratory syncytial virus (RSV) infection, we previously demonstrated that the CD8+ T cell immunodominance hierarchy to two RSV-derived epitopes, KdM282-90 and DbM187-195, was determined by the age at infection. To determine whether age-dependent RSV-specific CD8+ T cell responses could be modified through enhanced innate signaling, we used TLR4 or TLR9 agonist treatment at the time of infection, which remarkably changed the neonatal codominant response to an adult-like KdM282-90 CD8+ T cell immunodominant response. This shift was associated with an increase in the number of conventional dendritic cells, CD11b+ and CD103+ dendritic cells, in the lung-draining lymph node, as well as increased expression of the costimulatory molecule CD86. The magnitude of the KdM282-90 CD8+ T cell response in TLR agonist-treated neonates could be blocked with Abs against CD80 and CD86. These studies demonstrate the age-dependent function of conventional dendritic cells, their role in determining immunodominance hierarchy, and epitope-specific CD8+ T cell requirements for costimulation, all of which influence the immune response magnitude. The unique impact of TLR agonists on neonatal T cell responses is important to consider for RSV vaccines designed for young infants.

Quantitative and qualitative deficits in neonatal lung-migratory dendritic cells impact the generation of the CD8+ T cell response.

CD103+ and CD11b+ populations of CD11c+MHCIIhi murine dendritic cells (DCs) have been shown to carry antigens from the lung through the afferent lymphatics to mediastinal lymph nodes (MLN). We compared the responses of these two DC populations in neonatal and adult mice following intranasal infection with respiratory syncytial virus. The response in neonates was dominated by functionally-limited CD103+ DCs, while CD11b+ DCs were diminished in both number and function compared to adults. Infecting mice at intervals through the first three weeks of life revealed an evolution in DC phenotype and function during early life. Using TCR transgenic T cells with two different specificities to measure the ability of CD103+ DC to induce epitope-specific CD8+ T cell responses, we found that neonatal CD103+ DCs stimulate proliferation in a pattern distinct from adult CD103+ DCs. Blocking CD28-mediated costimulatory signals during adult infection demonstrated that signals from this costimulatory pathway influence the hierarchy of the CD8+ T cell response to RSV, suggesting that limited costimulation provided by neonatal CD103+ DCs is one mechanism whereby neonates generate a distinct CD8+ T cell response from that of adults.

Thermoresponsive Polymer Nanoparticles Co-deliver RSV F Trimers with a TLR-7/8 Adjuvant.

Structure-based vaccine design has been used to develop immunogens that display conserved neutralization sites on pathogens such as HIV-1, respiratory syncytial virus (RSV), and influenza. Improving the immunogenicity of these designed immunogens with adjuvants will require formulations that do not alter protein antigenicity. Here, we show that nanoparticle-forming thermoresponsive polymers (TRP) allow for co-delivery of RSV fusion (F) protein trimers with Toll-like receptor 7 and 8 agonists (TLR-7/8a) to enhance protective immunity. Although primary amine conjugation of TLR-7/8a to F trimers severely disrupted the recognition of critical neutralizing epitopes, F trimers site-selectively coupled to TRP nanoparticles retained appropriate antigenicity and elicited high titers of prefusion-specific, TH1 isotype anti-RSV F antibodies following vaccination. Moreover, coupling F trimers to TRP delivering TLR-7/8a resulted in ∼3-fold higher binding and neutralizing antibody titers than soluble F trimers admixed with TLR-7/8a and conferred protection from intranasal RSV challenge. Overall, these data show that TRP nanoparticles may provide a broadly applicable platform for eliciting neutralizing antibodies to structure-dependent epitopes on RSV, influenza, HIV-1, or other pathogens.

Bioorthogonal deprotection on the dendritic cell surface for chemical control of antigen cross-presentation.

The activation of CD8(+) T-cells requires the uptake of exogenous polypeptide antigens and proteolytic processing of these antigens to octamer or nonamer peptides, which are loaded on MHC-I complexes and presented to the T-cell. By using an azide as a bioorthogonal protecting group rather than as a ligation handle, masked antigens were generated-antigens that are not recognized by their cognate T-cell unless they are deprotected on the cell using a Staudinger reduction.

Consequences of immature and senescent immune responses for infection with respiratory syncytial virus.

Infants in the first 6 months of life and older adults (>65 years of age) are disproportionately burdened with respiratory syncytial virus (RSV)-associated morbidity and mortality. While other factors play a role in the risk these groups assume, shortcomings of the immune response make a substantial contribution to the predisposition to severe disease. Ineffectual antibody production with misdirected cytokine responses and excess inflammation in the airways are common to both groups. However, the mechanisms underlying these immune responses differ between infants and older adults and need to be better understood. Preventative approaches to decreasing the burden of disease are preferable to therapeutic intervention and effective vaccination strategies will need to target the strengths of the immune responses in these populations.

Antibody responses to respiratory syncytial virus: a population-based cross-sectional serological study in Southern China, 2021.

OBJECTIVES: With remarkable progress in the field of respiratory syncytial virus (RSV) prophylaxis, it is critical to understand population immunity against RSV. We aim to describe the RSV pre-F IgG antibodies across all age groups in Southern China and to evaluate the risk factors associated with lower antibody levels. METHODS: We performed a community-based cross-sectional sero-epidemiological study in Anhua County, Hunan Province, Southern China, from July 15, 2021, to November 5, 2021. Serum samples were tested for IgG antibodies against the RSV prefusion F (pre-F) protein using an enzyme-linked immunosorbent assay. We estimated the geometric mean titres (GMTs) and seropositivity rates across all age groups. The generalized linear models were built to identify factors associated with antibody levels. RESULTS: A total of 890 participants aged 4 months to older than 89 years were enrolled. The lowest RSV pre-F IgG GMTs were observed in infants and toddlers aged 4 months to younger than 2 years (3.0; 95% CI, 2.6-3.5). With increasing age, the RSV pre-F IgG GMT increased to 4.3 (95% CI, 4.1-4.4) between the ages of 2 and younger than 5 years and then stabilized at high levels throughout life. All the children had serological evidence of RSV infection by the age of 5 years. Age was associated with RSV pre-F antibody levels in children, with an estimated 1.8-fold (95% CI, 1.1-2.9) increase in titre per year before 5 years of age, although it was not significantly associated with antibody levels in adults aged older than 60 years. DISCUSSION: Our findings could provide a comprehensive understanding of the gaps in RSV immunity at the population level and inform the prioritization of immunization platforms.

Antibody levels against respiratory syncytial virus fusion protein conformations and lack of association with life-threatening infection in previously healthy infants.

BACKGROUND: Humoral immune response against the pre-fusion (pre-F) conformation of respiratory syncytial virus (RSV) F protein has been proposed to play a protective role against infection. An RSV pre-F maternal vaccine has been recently approved in several countries to protect young infants against RSV. We aimed to assess serum IgG titers against the pre-F and post-F conformations of RSV F protein and their association with life-threatening RSV disease (LTD) in previously healthy infants. METHODS: A prospective cohort study including hospitalized infants <12 months with a first RSV infection was conducted during 2017-2019. Patients with LTD required intensive care and mechanical respiratory assistance. RSV pre-F exclusive and post-F antibody responses were determined by post-F competition and non-competition immunoassays, respectively, and neutralizing activity was measured by plaque reduction neutralization test. RESULTS: Fifty-eight patients were included; the median age was 3.5 months and 41 % were females. Fifteen patients developed LTD. RSV F-specific antibody titers positively correlated with neutralizing antibody titers in acute and convalescent phases but, importantly, they did not associate with LTD. Acute RSV pre-F exclusive and post-F IgG titers negatively correlated with patient age (P = 0.0007 and P < 0.0001), while a positive correlation was observed between the fold changes in RSV F-specific antibody titers between convalescent and acute phase and patient age (P = 0.0014 and P < 0.0001). Infants ≤2 months exhibited significantly lower fold-changes in RSV F-specific and neutralizing antibody titers between convalescence and acute phase than older infants. Additionally, acute RSV antibody titers showed no correlation with nasal RSV load and, furthermore, nasal viral load was not associated with the development of LTD. CONCLUSIONS: This study highlights that protection against life-threatening RSV disease is not necessarily antibody-dependent. Further characterization of the immune response against RSV and its role in protection against severe disease is important for the development of the safest possible preventive strategies.

Neonatal CD8 T-cell hierarchy is distinct from adults and is influenced by intrinsic T cell properties in respiratory syncytial virus infected mice.

Following respiratory syncytial virus infection of adult CB6F1 hybrid mice, a predictable CD8+ T cell epitope hierarchy is established with a strongly dominant response to a K(d)-restricted peptide (SYIGSINNI) from the M2 protein. The response to K(d)M2(82-90) is ∼5-fold higher than the response to a subdominant epitope from the M protein (NAITNAKII, D(b)M(187-195)). After infection of neonatal mice, a distinctly different epitope hierarchy emerges with codominant responses to K(d)M2(82-90) and D(b)M(187-195). Adoptive transfer of naïve CD8+ T cells from adults into congenic neonates prior to infection indicates that intrinsic CD8+ T cell factors contribute to age-related differences in hierarchy. Epitope-specific precursor frequency differs between adults and neonates and influences, but does not predict the hierarchy following infection. Additionally, dominance of K(d)M2(82-90)-specific cells does not correlate with TdT activity. Epitope-specific Vß repertoire usage is more restricted and functional avidity is lower in neonatal mice. The neonatal pattern of codominance changes after infection at 10 days of age, and rapidly shifts to the adult pattern of extreme K(d)M2(82-90)-dominance. Thus, the functional properties of T cells are selectively modified by developmental factors in an epitope-specific and age-dependent manner.

The road to approved vaccines for respiratory syncytial virus.

After decades of work, several interventions to prevent severe respiratory syncytial virus (RSV) disease in high-risk infant and older adult populations have finally been approved. There were many setbacks along the road to victory. In this review, I will discuss the impact of RSV on human health and how structure-based vaccine design set the stage for numerous RSV countermeasures to advance through late phase clinical evaluation. While there are still many RSV countermeasures in preclinical and early-stage clinical trials, this review will focus on products yielding long-awaited efficacy results. Finally, I will discuss some challenges and next steps needed to declare a global victory against RSV.

EV-D68 virus-like particle vaccines elicit cross-clade neutralizing antibodies that inhibit infection and block dissemination.

Enterovirus D68 (EV-D68) causes severe respiratory illness in children and can result in a debilitating paralytic disease known as acute flaccid myelitis. No treatment or vaccine for EV-D68 infection is available. Here, we demonstrate that virus-like particle (VLP) vaccines elicit a protective neutralizing antibody against homologous and heterologous EV-D68 subclades. VLP based on a B1 subclade 2014 outbreak strain elicited comparable B1 EV-D68 neutralizing activity as an inactivated viral particle vaccine in mice. Both immunogens elicited weaker cross-neutralization against heterologous viruses. A B3 VLP vaccine elicited more robust neutralization of B3 subclade viruses with improved cross-neutralization. A balanced CD4+ T helper response was achieved using a carbomer-based adjuvant, Adjuplex. Nonhuman primates immunized with this B3 VLP Adjuplex formulation generated robust neutralizing antibodies against homologous and heterologous subclade viruses. Our results suggest that both vaccine strain and adjuvant selection are critical elements for improving the breadth of protective immunity against EV-D68.