Administration sequence- and formation-dependent vaccination using acid-degradable polymeric nanoparticles with high antigen encapsulation capability.

Vaccines aim to efficiently and specifically activate the immune system via a cascade of antigen uptake, processing, and presentation by antigen-presenting cells (APCs) to CD4 and CD8 T cells, which in turn drive humoral and cellular immune responses. The specific formulation of vaccine carriers can not only shield the antigens from premature sequestering before reaching APCs but also favorably promote intracellular antigen presentation and processing. This study compares two different acid-degradable polymeric nanoparticles that are capable of encapsulating a moderately immunogenic antigen, GFP, at nearly full efficacy via electrostatic interactions or molecular affinity between His tag and Ni-NTA-conjugated monomners. This resulted in GFP-encapsulating NPs composed of ketal monomers and crosslinkers (KMX/GFP NPs) and NTA-conjugated ketal monomers and crosslinkers (NKMX/GFP NPs), respectively. Encapsulated GFP was found to be released more rapidly from NKMX/GFP NPs (electrostatic encapsulation) than from KMX/GFP NPs (affinity-driven encapsulation). In vivo vaccination studies demonstrated that while repeated injections of either NP formulation resulted in poorer generation of anti-GFP antibodies than injections of the GFP antigen itself, sequential injections of NPs and GFP as prime and booster vaccines, respectively, restored the humoral response. We proposed that NPs primarily assist APCs in antigen presentation by T cells, and B cells need to be further stimulated by free protein antigens to produce antibodies. The findings of this study suggest that the immune response can be modulated by varying the chemistry of vaccine carriers and the sequences of vaccination with free antigens and antigen-encapsulating NPs.

Tissue-specific sex differences in pediatric and adult immune cell composition and function.

Sex-based differences in immune cell composition and function can contribute to distinct adaptive immune responses. Prior work has quantified these differences in peripheral blood, but little is known about sex differences within human lymphoid tissues. Here, we characterized the composition and phenotypes of adaptive immune cells from male and female ex vivo tonsils and evaluated their responses to influenza antigens using an immune organoid approach. In a pediatric cohort, female tonsils had more memory B cells compared to male tonsils direct ex vivo and after stimulation with live-attenuated but not inactivated vaccine, produced higher influenza-specific antibody responses. Sex biases were also observed in adult tonsils but were different from those measured in children. Analysis of peripheral blood immune cells from in vivo vaccinated adults also showed higher frequencies of tissue homing CD4 T cells in female participants. Together, our data demonstrate that distinct memory B and T cell profiles are present in male vs. female lymphoid tissues and peripheral blood respectively and suggest that these differences may in part explain sex biases in response to vaccines and viruses.

Broad antibody and T cell responses to Ebola, Sudan, and Bundibugyo ebolaviruses using mono- and multi-valent adjuvanted glycoprotein vaccines.

Currently, there are two approved vaccine regimens designed to prevent Ebola virus (EBOV) disease (EVD). Both are virus-vectored, and concerns about cold-chain storage and pre-existing immunity to the vectors warrant investigating additional vaccine strategies. Here, we have explored the utility of adjuvanted recombinant glycoproteins (GPs) from ebolaviruses Zaire (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) for inducing antibody (Ab) and T cell cross-reactivity. Glycoproteins expressed in insect cells were administered to C57BL/6 mice as free protein or bound to the surface of liposomes, and formulated with toll-like receptor agonists CpG and MPLA (agonists for TLR 9 and 4, respectively), with or without the emulsions AddaVax or TiterMax. The magnitude of Ab cross-reactivity in binding and neutralization assays, and T cell cross-reactivity in antigen recall assays, correlated with phylogenetic relatedness. While most adjuvants screened induced IgG responses, a combination of CpG, MPLA and AddaVax emulsion ("IVAX-1") was the most potent and polarized in an IgG2c (Th1) direction. Breadth was also achieved by combining GPs into a trivalent (Tri-GP) cocktail with IVAX-1, which did not compromise antibody responses to individual components in binding and neutralizing assays. Th1 signature cytokines in T cell recall assays were undetectable after Tri-GP/IVAX-1 administration, despite a robust IgG2c response, although administration of Tri-GP on lipid nanoparticles in IVAX-1 elevated Th1 cytokines to detectable levels. Overall, the data indicate an adjuvanted trivalent recombinant GP approach may represent a path toward a broadly reactive, deployable vaccine against EVD.

Grandparents' Experiences of Childhood Cancer: A Qualitative Study.

A child's cancer diagnosis has a significant impact on the lives of grandparents. Grandparents experience the stress of worrying about both their adult children and their grandchildren. Our study aimed to explore the lived experience of grandparents of children diagnosed with cancer. A qualitative design involving semi-structured interviews was used and data were analyzed using reflexive thematic analysis. Twenty grandparents aged 41 to 77 years were interviewed. Six themes were identified: (a) Diagnosis: changing everything; (b) Aspects of treatment: A different world; (c) Sandwich generation; (d) Family: Worrying about everyone; (e) Balancing work; and (f) It's like suddenly a door opens. Our study demonstrates the life-changing impact of having a grandchild diagnosed with cancer. It expands on existing knowledge and shows that, due to an aging population and demographic changes, some grandparents must juggle the demands of caring for aging family members and working while supporting adult children and grandchildren.

Multivalent vaccines demonstrate immunogenicity and protect against Coxiella burnetii aerosol challenge.

Vaccines are among the most cost-effective public health measures for controlling infectious diseases. Coxiella burnetii is the etiological agent of Q fever, a disease with a wide clinical spectrum that ranges from mild symptoms, such as fever and fatigue, to more severe disease, such as pneumonia and endocarditis. The formalin-inactivated whole-cell vaccine Q-VAX® contains hundreds of antigens and confers lifelong protection in humans, but prior sensitization from infection or vaccination can result in deleterious reactogenic responses to vaccination. Consequently, there is great interest in developing non-reactogenic alternatives based on adjuvanted recombinant proteins. In this study, we aimed to develop a multivalent vaccine that conferred protection with reduced reactogenicity. We hypothesized that a multivalent vaccine consisting of multiple antigens would be more immunogenic and protective than a monovalent vaccine owing to the large number of potential protective antigens in the C. burnetii proteome. To address this, we identified immunogenic T and B cell antigens, and selected proteins were purified to evaluate with a combination adjuvant (IVAX-1), with or without C. burnetii lipopolysaccharide (LPS) in immunogenicity studies in vivo in mice and in a Hartley guinea pig intratracheal aerosol challenge model using C. burnetii strain NMI RSA 493. The data showed that multivalent vaccines are more immunogenic than monovalent vaccines and more closely emulate the protection achieved by Q-VAX. Although six antigens were the most immunogenic, we also discovered that multiplexing beyond four antigens introduces detectable reactogenicity, indicating that there is an upper limit to the number of antigens that can be safely included in a multivalent Q-fever vaccine. C. burnetii LPS also demonstrates efficacy as a vaccine antigen in conferring protection in an otherwise monovalent vaccine formulation, suggesting that its addition in multivalent vaccines, as demonstrated by a quadrivalent formulation, would improve protective responses.

Influenza vaccine format mediates distinct cellular and antibody responses in human immune organoids.

Highly effective vaccines elicit specific, robust, and durable adaptive immune responses. To advance informed vaccine design, it is critical that we understand the cellular dynamics underlying responses to different antigen formats. Here, we sought to understand how antigen-specific B and T cells were activated and participated in adaptive immune responses within the mucosal site. Using a human tonsil organoid model, we tracked the differentiation and kinetics of the adaptive immune response to influenza vaccine and virus modalities. Each antigen format elicited distinct B and T cell responses, including differences in their magnitude, diversity, phenotype, function, and breadth. These differences culminated in substantial changes in the corresponding antibody response. A major source of antigen format-related variability was the ability to recruit naive vs. memory B and T cells to the response. These findings have important implications for vaccine design and the generation of protective immune responses in the upper respiratory tract.

Sural nerve biopsy utility by masked assessment of individual histologic preparations.

BACKGROUND AND AIMS: Comprehensive study of sural nerve biopsy utility based on individual histopathologic preparations is lacking. We aimed to quantify the value of different histologic preparations in diagnosis. METHODS: One hundred consecutive sural nerves were studied by standard histological preparations plus graded teased nerve fibers (GTNF), immunohistochemistry, and epoxy-semithin morphometry. Three examiners scored the individual preparations separately by a questionnaire of neuropathic and interstitial abnormalities, masked to the biopsy number, versus a gold-standard of all preparations. Multivariate modeling was utilized to determine best approach versus the gold-standard. RESULTS: Highest confidence (range 8-9 of 10) and inter-rater reliability (99%) for fiber abnormalities came from GTNF, and interstitial abnormalities from paraffin stains (range 7-8, 99%). Vasculitic neuropathy associated with GTNF axonal degeneration (moderate to severe 79%) with OR 3.8, 95% CI (1.001-14.7), p = .04, but not significantly with the other preparations. Clinicopathologic diagnoses associated with teased fiber abnormalities in chronic inflammatory demyelinating polyradiculoneuropathy, 80% (8/10); amyloidosis, 50% (1/2); adult-onset polyglucosan disease 100% (1/1). GTNF and paraffin stains significantly correlated with fiber density determined by morphometric analysis (GTNF: OR 9.9, p < .0001, paraffin: OR 3.8, p = .03). GTNF combined with paraffin sections had highest accuracy for clinicopathologic diagnoses and fiber density with 0.86 C-stat prediction versus morphometric analysis. Pathological results lead to initiation or changes of immunotherapy in 70% (35/50; initiation n = 22, reduction n = 9, escalation n = 4) with the remaining having alternative intervention or no change. INTERPRETATION: Nerve biopsy paraffin stains combined with GTNF have highest diagnostic utility, confidence, inter-rater reliability, improving accuracy for a pathologic diagnosis aiding treatment recommendations. Immunostains and epoxy preparations are also demonstrated useful supporting consensus guidelines. This study provides class II evidence for individual nerve preparation utility.

Composite nerve conduction scores and signs for diagnosis and somatic staging of diabetic polyneuropathy: Mid North American ethnic cohort survey.

INTRODUCTION/AIMS: In the Diabetes Control and Complications Trial (DCCT), the minimal nerve conduction (NC) criterion for diabetic sensorimotor polyneuropathy (DSPN) was abnormality of NC in more than one peripheral nerve without specifying the attributes of NCs to be evaluated. In the present study, we assess individual and composite scores of NCs meeting the DCCT criterion and signs for improved diagnosis and assessment of DSPN severity. METHODS: Evaluated were 13 attributes and 6 composite NC scores and signs and symptoms in 395 healthy subjects (HS) and 388 persons with diabetes (DM). RESULTS: Percent abnormality between subjects with DM and HS was remarkably different among individual attributes and the six composite NC scores. For diagnosis of DSPN using the DCCT criterion, assessment of conduction velocities (CVs) and distal latencies (DLs) provided sensitive diagnoses of DSPN. NC amplitudes provided stronger measures of severity. In studied cohorts, DSPN was staged: N0, no NC abnormality using NC score 2 (CVs and DLs), 60.0%; N1, NC abnormality only, 18.4%; N2, NC abnormality and signs of feet or legs, 16.3%; and N3, NC abnormality and signs of thighs, 5.3%. DISCUSSION: For sensitive and standard diagnosis of DSPN using the DCCT NC criterion, specifically defined composite scores of CVs and DLs, e.g., score 2, is recommended. A composite score of amplitudes, e.g., score 4, provides a stronger measure of neuropathy severity. Also, provided are HS reference values of evaluated attributes of NCs and estimates of staged severity of DSPN of mid North American DM cohorts.

Vaccination against SARS-CoV-2 using extracellular blebs derived from spike protein-expressing dendritic cells.

COVID-19 has caused significant morbidity and mortality worldwide but also accelerated the clinical use of emerging vaccine formulations. To address the current shortcomings in the prevention and treatment of SARS-CoV-2 infection, this study developed a novel vaccine platform that closely mimics dendritic cells (DCs) in antigen presentation and T-cell stimulation in a cell-free and tunable manner. Genetically engineered DCs that express the SARS-CoV-2 spike protein (S) were chemically converted into extracellular blebs (EBs). The resulting EBs elicited potentially protective humoral immunity in vivo, indicated by the production of antibodies that potently neutralized S-pseudotyped virus, presenting EBs as a promising and safe vaccine.

Fathers' Experiences of Childhood Cancer: A Phenomenological Qualitative Study.

Research has shown differences in how fathers and mothers respond to a child's cancer diagnosis. Previous studies have highlighted that sociocultural norm shape fathers' experiences of their child's cancer diagnosis. Our phenomenological qualitative study aimed to examine the lived experiences of fathers whose children have been diagnosed with cancer and explore the impact of sociocultural gender roles. Fathers whose children were currently receiving treatment or had completed treatment in the previous 15 months were recruited from across Australia. Twenty-one fathers were interviewed. Five themes were identified: (a) Your world falls apart: Diagnosis and treatment; (b) Care for the child: Just the way it is; (c) Keeping strong: Finding ways to cope; (d) Employment: Practical and emotional support at work; and (e) Guilt, relief, and grief: Facing death. This study demonstrates the profound impact of a child's diagnosis on fathers and demonstrates that societal-cultural norms influence fathers' experience of childhood cancer.

Protein Nanoparticle-Mediated Delivery of Recombinant Influenza Hemagglutinin Enhances Immunogenicity and Breadth of the Antibody Response.

The vast majority of seasonal influenza vaccines administered each year are derived from virus propagated in eggs using technology that has changed little since the 1930s. The immunogenicity, durability, and breadth of response would likely benefit from a recombinant nanoparticle-based approach. Although the E2 protein nanoparticle (NP) platform has been previously shown to promote effective cell-mediated responses to peptide epitopes, it has not yet been reported to deliver whole protein antigens. In this study, we synthesized a novel maleimido tris-nitrilotriacetic acid (NTA) linker to couple protein hemagglutinin (HA) from H1N1 influenza virus to the E2 NP, and we evaluated the HA-specific antibody responses using protein microarrays. We found that recombinant H1 protein alone is immunogenic in mice but requires two boosts for IgG to be detected and is strongly IgG1 (Th2) polarized. When conjugated to E2 NPs, IgG2c is produced leading to a more balanced Th1/Th2 response. Inclusion of the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) significantly enhances the immunogenicity of H1-E2 NPs while retaining the Th1/Th2 balance. Interestingly, broader homo- and heterosubtypic cross-reactivity is also observed for conjugated H1-E2 with MPLA, compared to unconjugated H1 with or without MPLA. These results highlight the potential of an NP-based delivery of HA for tuning the immunogenicity, breadth, and Th1/Th2 balance generated by recombinant HA-based vaccination. Furthermore, the modularity of this protein-protein conjugation strategy may have utility for future vaccine development against other human pathogens.

A Protein Microarray-Based Respiratory Viral Antigen Testing Platform for COVID-19 Surveillance.

High-throughput and rapid screening testing is highly desirable to effectively combat the rapidly evolving COVID-19 pandemic co-presents with influenza and seasonal common cold epidemics. Here, we present a general workflow for iterative development and validation of an antibody-based microarray assay for the detection of a respiratory viral panel: (a) antibody screening to quickly identify optimal reagents and assay conditions, (b) immunofluorescence assay design including signal amplification for low viral titers, (c) assay characterization with recombinant proteins, inactivated viral samples and clinical samples, and (d) multiplexing to detect a panel of common respiratory viruses. Using RT-PCR-confirmed SARS-CoV-2 positive and negative pharyngeal swab samples, we demonstrated that the antibody microarray assay exhibited a clinical sensitivity and specificity of 77.2% and 100%, respectively, which are comparable to existing FDA-authorized antigen tests. Moreover, the microarray assay is correlated with RT-PCR cycle threshold (Ct) values and is particularly effective in identifying high viral titers. The multiplexed assay can selectively detect SARS-CoV-2 and influenza virus, which can be used to discriminate these viral infections that share similar symptoms. Such protein microarray technology is amenable for scale-up and automation and can be broadly applied as a both diagnostic and research tool.

'Torn in two': Experiences of Mothers Who Are Pregnant when Their Child Is Diagnosed With Cancer.

Mothers of children diagnosed with cancer have been shown to experience high rates of psychological distress and poor physical health. Pregnancy further increases the healthcare needs of mothers due to the marked physiological changes and psychological adaptations. Our study aimed to explore the experiences of mothers who were pregnant and/or had a baby while their older child was receiving treatment for cancer. Our study employed a qualitative description methodology using semi-structured interviews. Participants were recruited from across Australia via notices on social media sites and the distribution of flyers. The sample comprised 13 mothers who were pregnant and/or had a baby and had a child diagnosed with cancer who was under 17 years old. Thematic analysis was used to analyse the data from which six themes were identified: (1) an impossible balancing act, (2) mother's health and well-being, (3) creating certainty: birthing plans, (4) a bit of sunshine and a time to rest, (5) challenges of caring for the baby and (6) an unenviable position: doing my best versus feeling guilty. Our study demonstrates the additional challenges faced by mothers who are pregnant while their child is receiving cancer treatment. There is a need for a comprehensive and coordinated program that provides pregnant mothers with practical and psychological support. The implementation of such a program has the potential to improve health outcomes for all family members, particularly the mother and their newborn.

Magnitude and breadth of antibody cross-reactivity induced by recombinant influenza hemagglutinin trimer vaccine is enhanced by combination adjuvants.

The effects of adjuvants for increasing the immunogenicity of influenza vaccines are well known. However, the effect of adjuvants on increasing the breadth of cross-reactivity is less well understood. In this study we have performed a systematic screen of different toll-like receptor (TLR) agonists, with and without a squalene-in-water emulsion on the immunogenicity of a recombinant trimerized hemagglutinin (HA) vaccine in mice after single-dose administration. Antibody (Ab) cross-reactivity for other variants within and outside the immunizing subtype (homosubtypic and heterosubtypic cross-reactivity, respectively) was assessed using a protein microarray approach. Most adjuvants induced broad IgG profiles, although the response to a combination of CpG, MPLA and AddaVax (termed 'IVAX-1') appeared more quickly and reached a greater magnitude than the other formulations tested. Antigen-specific plasma cell labeling experiments show the components of IVAX-1 are synergistic. This adjuvant preferentially stimulates CD4 T cells to produce Th1>Th2 type (IgG2c>IgG1) antibodies and cytokine responses. Moreover, IVAX-1 induces identical homo- and heterosubtypic IgG and IgA cross-reactivity profiles when administered intranasally. Consistent with these observations, a single-cell transcriptomics analysis demonstrated significant increases in expression of IgG1, IgG2b and IgG2c genes of B cells in H5/IVAX-1 immunized mice relative to naïve mice, as well as significant increases in expression of the IFNγ gene of both CD4 and CD8 T cells. These data support the use of adjuvants for enhancing the breath and durability of antibody responses of influenza virus vaccines.

Multimericity Amplifies the Synergy of BCR and TLR4 for B Cell Activation and Antibody Class Switching.

Sustained signaling through the B cell antigen receptor (BCR) is thought to occur only when antigen(s) crosslink or disperse multiple BCR units, such as by multimeric antigens found on the surfaces of viruses or bacteria. B cell-intrinsic Toll-like receptor (TLR) signaling synergizes with the BCR to induce and shape antibody production, hallmarked by immunoglobulin (Ig) class switch recombination (CSR) of constant heavy chains from IgM/IgD to IgG, IgA or IgE isotypes, and somatic hypermutation (SHM) of variable heavy and light chains. Full B cell differentiation is essential for protective immunity, where class switched high affinity antibodies neutralize present pathogens, memory B cells are held in reserve for future encounters, and activated B cells also serve as semi-professional APCs for T cells. But the rules that fine-tune B cell differentiation remain partially understood, despite their being essential for naturally acquired immunity and for guiding vaccine development. To address this in part, we have developed a cell culture system using splenic B cells from naive mice stimulated with several biotinylated ligands and antibodies crosslinked by streptavidin reagents. In particular, biotinylated lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) agonist, and biotinylated anti-IgM were pre-assembled (multimerized) using streptavidin, or immobilized on nanoparticles coated with streptavidin, and used to active B cells in this precisely controlled, high throughput assay. Using B cell proliferation and Ig class switching as metrics for successful B cell activation, we show that the stimuli are both synergistic and dose-dependent. Crucially, the multimerized immunoconjugates are most active over a narrow concentration range. These data suggest that multimericity is an essential requirement for B cell BCR/TLRs ligands, and clarify basic rules for B cell activation. Such studies highlight the importance in determining the choice of single vs multimeric formats of antigen and PAMP agonists during vaccine design and development.

Parents' Experiences of Childhood Cancer During the COVID-19 Pandemic: An Australian Perspective.

INTRODUCTION: COVID-19 has had far-reaching impacts including changes in work, travel, social structures, education, and healthcare. OBJECTIVE: This study aimed to explore the experiences of parents of children receiving treatment for cancer during the COVID-19 pandemic. METHODS: Parents whose children were currently in treatment for childhood cancer or had completed treatment in the previous 12 months, participated in semi-structured interviews, face-to-face or via teleconferencing. Thematic analysis was used to analyze the data. RESULTS: The sample consisted of 34 participants (17 fathers and 17 mothers) from all states across Australia. Median age 37.5 years (range 29-51, years, SD = 6.3). Five main themes were identified: "Welcome to the Club"; "Remote Work and Study"; "Silver Linings"; "The Loneliest Experience" with three sub-themes "Immediate Family"; "Friends"; and "Overseas Family" and "Lack of Support" with two sub-themes: "Community Support" and "Organized Support." CONCLUSION: These findings revealed contrasting experiences of the impact of the COVID-19 pandemic. For parents whose children were neutropenic, the pandemic provided benefits in increased community understanding of infection control. Parents also reflected that the movement to remote work made it easier to earn an income. In contrast, some parents observed that restrictions on visitors and family intensified feelings of isolation. Parents also described how the COVID-19 reduced access to support services. These findings contribute to an understanding of the multifaceted impacts of the COVID-19 pandemic on families of children with cancer.

Variable differences of nerve conduction amplitudes versus velocities and distal latencies of healthy subjects assessed in ethnic cohorts.

Variable differences of nerve conduction amplitudes vs velocities and distal latencies (DLs) of healthy subjects assessed in ethnic cohorts. INTRODUCTION/AIMS: The variables affecting reference compound muscle (CMAP) and sensory nerve action potential (SNAP) amplitudes as compared to ones affecting conduction velocities and DLs have not been adequately evaluated in previous studies. In this report, this subject is studied in healthy subject cohorts mainly of Northern European extraction, Northern Plains Indians, and Latinos. METHODS: Nineteen variables and 18 attributes of nerve conductions (NCs) were assessed using highly standard testing conditions and techniques. Classification and Regression Tree analyses were used to assess variable differences among amplitudes, conduction velocities, and DLs. RESULTS: The most important variable affecting CMAP and SNAP amplitudes was age. For conduction velocities (CVs) and DLs, the variables were height, ethnic cohort, and age. DISCUSSION: The variables affecting attributes of NCs were similar for the three ethnic cohorts evaluated. The differences of variables affecting amplitudes compared to CVs and DLs need to be taken into account in interpretation of NC results and in setting reference limits for use in medical practice, epidemiology surveys, and therapeutic trials. Scores of CMAP and SNAP amplitudes are suitable measures of sensorimotor polyneuropathy severity, whereas conduction velocities and DLs reflect physiologic/pathologic abnormality of nerve fibers.

Administration of Multivalent Influenza Virus Recombinant Hemagglutinin Vaccine in Combination-Adjuvant Elicits Broad Reactivity Beyond the Vaccine Components.

Combining variant antigens into a multivalent vaccine is a traditional approach used to provide broad coverage against antigenically variable pathogens, such as polio, human papilloma and influenza viruses. However, strategies for increasing the breadth of antibody coverage beyond the vaccine are not well understood, but may provide more anticipatory protection. Influenza virus hemagglutinin (HA) is a prototypic variant antigen. Vaccines that induce HA-specific neutralizing antibodies lose efficacy as amino acid substitutions accumulate in neutralizing epitopes during influenza virus evolution. Here we studied the effect of a potent combination adjuvant (CpG/MPLA/squalene-in-water emulsion) on the breadth and maturation of the antibody response to a representative variant of HA subtypes H1, H5 and H7. Using HA protein microarrays and antigen-specific B cell labelling, we show when administered individually, each HA elicits a cross-reactive antibody profile for multiple variants within the same subtype and other closely-related subtypes (homosubtypic and heterosubtypic cross-reactivity, respectively). Despite a capacity for each subtype to induce heterosubtypic cross-reactivity, broader coverage was elicited by simply combining the subtypes into a multivalent vaccine. Importantly, multiplexing did not compromise antibody avidity or affinity maturation to the individual HA constituents. The use of adjuvants to increase the breadth of antibody coverage beyond the vaccine antigens may help future-proof vaccines against newly-emerging variants.

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes.

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens. This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera. Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

Plantar Ulcers and Neuropathic Arthropathies: Associated Diseases, Polyneuropathy Correlates, and Risk Covariates.

OBJECTIVE: To evaluate the associated diseases, polyneuropathy correlates, and risk covariates of neuropathic plantar ulcers (PUs) and neuropathic arthropathies (NAs). DESIGN: The authors conducted a retrospective, observational study over 3.5 years of 69 patients with neuropathy, NA, or PU seen in a wound clinic who also had a comprehensive neurologic evaluation and neurophysiologic testing. Comparisons were made to a population representative cohort of patients with diabetes mellitus (DM; n = 259). RESULTS: Of the 69 wound clinic patients, 32 had PUs, 14 had NAs, and 23 had both. Of the 61 adequately assessed patients, 37 (61%) had DM, 22 (36%) had no known associated disease, and 2 (3%) had hereditary sensory and autonomic neuropathy. Of the 37 patients with DM, 35 had distal polyneuropathy, and 2 did not. In 22 patients with chronic idiopathic axonal polyneuropathy, 20 had distal polyneuropathy. CONCLUSIONS: Although DM was the disease most commonly associated with PUs and NAs, chronic hyperglycemia may not have been the major underlying risk factor. The major risk covariates are sensation loss from polyneuropathy, old age, obesity, repetitive foot injury, and inadequate foot care or treatment. Physicians and other healthcare providers can help by identifying patients at risk and instituting measures such as adequate foot care to decrease these risks.