ABSTRACTRespiratory syncytial virus (RSV) infection may cause serious illness and lead to death in older adults (≥ 65 years), causing over 60,000 hospitalizations and 6,000 deaths each year in the United States. Seroprevalence rates vary by age and geographic location but are typically high due to the repeated nature of RSV infections. This study investigated the seroprevalence of antibodies against human RSV in a convenience sample of adults from Olmsted County, Minnesota. Of the 480 subjects studied, 83.8% tested RSV IgG positive, 8.1% tested RSV IgG negative, and 8.1% had equivocal results. Additional cohorts from recently vaccinated individuals or those with a recent diagnosis of RSV infection were tested for comparison, yielding antibody positivity rates of 95.8% and 97.5% respectively.

Rubella is typically a mild viral illness, but it can lead to severe complications when contracted during pregnancy, such as pregnancy loss or developmental defects in the fetus (congenital rubella syndrome). Therefore, it is crucial to develop and maintain protective immunity in women of childbearing age. In this study, we assessed the transcriptional factors associated with rubella-specific immune outcomes (IgG binding antibody and avidity, neutralizing antibody, and memory B cell ELISpot response) following a third MMR vaccine dose in women of reproductive age to identify key factors/signatures impacting the immune response. We identified baseline (Day 0) and differentially expressed (Day 28–Day 0) genes associated with several RV-specific immune outcomes, including the transferrin receptor 2 (TFR2), which is an important factor regulating iron homeostasis and macrophage functional activity, and a close functional homolog of TFR1, the cellular receptor of the New World hemorrhagic fever arenaviruses. We also identified enriched KEGG pathways, “cell adhesion molecules”, “antigen processing and presentation”, “natural killer cell-mediated cytotoxicity”, and “immune network for IgA production”, relevant to immune response priming and immune activation to be associated with RV-specific immune outcomes. This study provides novel insights into potential biomarkers of rubella-specific immunity in women of childbearing age.

The study examined the relationship between rubella virus (RV)-induced gene expression and proinflammatory cytokine/chemokine secretion in females previously vaccinated with MMR. Gene clusters enriched for functional pathways of innate immune system activation and antiviral responses were found to predict RV-specific MCP-1, MCP-4, IP-10, and IL-2 secretion.

OBJECTIVES:Respiratory syncytial virus (RSV) infection may cause serious illness and mortality in older adults (≥65 years). This study aimed to assess the seroprevalence of anti-RSV IgG antibodies in adults from a United States cohort and compare antibody levels among individuals with a history of recent RSV vaccination or infection. METHODS:A total of 475 subjects (ages 27-99) were randomly selected from the Mayo Clinic Biobank's residual sera repository (2022-2023). Samples were tested for anti-RSV IgG using an enzyme-linked immunosorbent assay (ELISA). Additional cohorts included individuals with documented RSV infection (n = 40) or recent RSV vaccination (n = 71). RESULTS:Among the seroprevalence cohort, 83.8% tested RSV IgG positive against RSV-A whole virus antigen. Males had significantly higher antibody titers than females (P < 0.001), and antibody titers increased post-RSV season (p = 0.02). Compared to the general cohort, RSV-seropositivity rates were higher in recently diagnosed (97.5%) and vaccinated (95.8%) individuals. CONCLUSIONS:This study demonstrates a high seroprevalence of RSV-A IgG in adults, with variations across sex and seasonality, and corroborates waning immunity following RSV infection. Recent infection or vaccination significantly boosts antibody levels, reinforcing the importance of continued surveillance of RSV immunity.

Alterations of gene expression by miRNAs contribute substantially to genetic regulation and cellular functions. We conducted a comprehensive study in 53 individuals before and after seasonal inactivated influenza vaccine to characterize lymphocyte-specific miRNA expression (in purified B cells, CD4+ T cells, CD8+ T cells, and NK cells) and its effect on influenza vaccine-induced immune outcomes (hemagglutination inhibition antibody titers/HAI, viral neutralizing antibody titers /VNA, and memory B cell ELISPOT). Overall, we observed relatively stable miRNA expression before and after influenza vaccination. Statistical analysis uncovered three baseline miRNAs (miR-150-3p, miR-629-5p, and miR-4443) that were significantly correlated with influenza vaccine-induced immune outcomes in different cell types. Predictive modeling of influenza vaccine-induced HAI/VNA titers identified a set of specific baseline miRNAs in CD4+ T cells as factors predictive of antibody responses. A pathway enrichment analysis on the putative target genes revealed several regulated signaling pathways and functions: TGF-β signaling, PI3K-Akt signaling, p53 signaling, MAPK signaling, TNF signaling, and C-type lectin receptor signaling, as well as cell adhesion and adherens junctions, and antiviral host response. In conclusion, our study offers evidence for the role of epigenetic modification (miRNAs) on influenza vaccine-induced immunity. After validation, identified miRNAs may serve as potential biomarkers of immune response after influenza vaccination.

BACKGROUND:Antibody responses to MF59-adjuvanted (MF59Flu) and high-dose (HDFlu) influenza vaccines have been well-characterized in older adults, yet corresponding cellular immune response data remain limited. METHODS:Blood samples were collected from 106 MF59Flu recipients and 112 HDFlu recipients at 4 time points: before vaccination (day 0) and on days 1, 8, and 28 postvaccination. Antibody responses were assessed in serum samples using a hemagglutination inhibition assay. Eight proinflammatory cytokines and chemokines, including IFN-α2a, IFN-γ, IP-10, MCP-1, MIP-1α, IL-1β, IL-6, and IL-8, were quantified from peripheral blood mononuclear cells using a multiplex assay. Associations between cytokine/chemokine secretion levels and antibody responses were examined, along with the effect of sex, age, body mass index (BMI), and cytomegalovirus infection status. RESULTS:Age was positively correlated with IL-8 levels on day 1 (r = 0.24, P < .001) and the change in IL-8 levels from day 0 to day 1 (r = 0.16, P = .021). Notably, the change in IL-8 levels was negatively associated with day 28 hemagglutination inhibition titers (r = -0.15, P = .026). Causal mediation analysis demonstrated that IL-8 exerted significant causal mediation effects on antibody responses at both day 8 (P = .046) and day 28 (P = .0045). No significant effects of other cytokines and chemokines, vaccine type, sex, or BMI on antibody responses were observed. CONCLUSIONS:Our findings underscore IL-8 as a potential mediator of antibody responses to influenza vaccination in older adults, suggesting that IL-8 inhibition could serve as a molecular intervention to improve antibody responses in this high-risk population.

Importance:India's goal of measles elimination remains unmet, as evidenced by significant recent outbreaks. Objective:To identify seroprevalence rates among vaccinated children and to examine demographic factors that influence antibody responses. Design, Setting, and Participants:This cross-sectional study included a highly vaccinated cohort of Indian children from the states of Kerala and Tamil Nadu between 2018 and 2023. Eligible children had received at least 2 doses of measles-containing vaccines; their mothers were recruited from various community settings, including local nursery and secondary schools, residential associations, and hospitals. Exposure:Measles-specific antibodies were quantified in serum samples. Main Outcomes and Measures:Measles-specific immunoglobulin G (IgG) and neutralizing antibodies were quantified in both children and mothers. Measles-specific IgM was also measured in children. Results:The total cohort comprised 684 children (median age, 9 years [range, 4-18 years]; 348 male [50.1%]) and 544 mothers. All children received at least 2 doses of measles-containing vaccines; 435 children (63.6%) received a third dose, and 7 (1.0%) received a fourth dose. Among the children, 621 (90.8%) had positive measles-specific IgG, and 623 (91.5%) had protective neutralizing antibodies titers, with a strong correlation between measles-specific IgG and neutralizing antibodies (r = 0.73; P < .001). Female children exhibited significantly higher titers of both measles-specific IgG and neutralizing antibodies compared with male children. While IgG and neutralizing antibody titers remained stable over time and were not associated with the number of vaccine doses in children, neutralizing antibody titers increased with age in mothers, likely due to repeated viral exposure. Notably, in 20 families with at least 2 children, differential measles-specific IgM profiles were observed between siblings despite high IgG and neutralizing antibody titers, suggesting ongoing breakthrough infections. Conclusions and Relevance:In this cross-sectional study, substantial measles immunity gaps were found despite high vaccine coverage with evidence of breakthrough infections, posing significant challenges to India's measles elimination efforts. These results underscore the urgent need to strengthen India's immunization program and investigate the mechanisms driving suboptimal responses to measles vaccination; without addressing these immune response deficiencies, achieving measles elimination through increased vaccine coverage alone may remain unattainable in India.

Background Multiple sclerosis (MS) is a clinically and biologically heterogenous disease with currently unpredictable progression and relapse. After the development and success of neurofilament as a cerebrospinal fluid (CSF) biomarker, there is reinvigorated interest in identifying other markers of or contributors to disease. The objective of this study is to probe the predictive potential of a panel of brain-enriched proteins on MS disease progression and subtype. Methods This study includes 40 individuals with MS and 14 headache controls. The MS cohort consists of 20 relapsing remitting (RR) and 20 primary progressive (PP) patients. The CSF of all individuals was analyzed for 63 brain enriched proteins using a method of liquid-chromatography tandem mass spectrometry. Wilcoxon rank sum test, Kruskal-Wallis one-way ANOVA, logistic regression, and Pearson correlation were used to refine the list of candidates by comparing relative protein concentrations as well as relation to known imaging and molecular biomarkers. Results We report 30 proteins with some relevance to disease, clinical subtype, or severity. Strikingly, we observed widespread protein depletion in the disease CSF as compared to control. We identified numerous markers of relapsing disease, including KLK6 (kallikrein 6, OR = 0.367, p < 0.05), which may be driven by active disease as defined by MRI enhancing lesions. Other oligodendrocyte-enriched proteins also appeared at reduced levels in relapsing disease, namely CNDP1 (carnosine dipeptidase 1), LINGO1 (leucine rich repeat and Immunoglobin-like domain-containing protein 1), MAG (myelin associated glycoprotein), and MOG (myelin oligodendrocyte glycoprotein). Finally, we identified three proteins-CNDP1, APLP1 (amyloid beta precursor like protein 1), and OLFM1 (olfactomedin 1)-that were statistically different in relapsing vs. progressive disease raising the potential for use as an early biomarker to discriminate clinical subtype. Conclusions We illustrate the utility of targeted mass spectrometry in generating potential targets for future biomarker studies and highlight reductions in brain-enriched proteins as markers of the relapsing remitting disease stage.

The responsiveness/cross-binding of vaccine-induced memory B cells/MBCs to previous and emerging divergent SARS-CoV-2 variants (e.g., Omicron) is understudied. In this longitudinal study subjects receiving two or three doses of monovalent ancestral strain-containing COVID-19 mRNA vaccine were evaluated. In contrast to others, we observed significantly lower frequencies of MBCs reactive to the receptor-binding domain/RBD, the N-terminal domain/NTD, and the S1 of Omicron/BA.1, compared to Wuhan and Delta, even after a 3rd vaccine dose/booster. Our study is a proof of concept that MBC cross-reactivity to variants with greater sequence divergence from the vaccine strain may be overestimated and suggests that these variants may exhibit immune escape with reduced recognition by circulating pre-existing MBCs upon infection.

BACKGROUND:Blood volume (BV) profiles vary markedly in patients with heart failure (HF), but how HF phenotypes and patient sex impact volume profiles remain to be explored. The aim of the study was to differentiate BV, plasma volume, and red blood cell mass profiles by phenotypes of preserved and reduced left ventricular ejection fractions and assess the impact of patient sex on profile heterogeneity. METHODS:Retrospective analysis of clinical and BV data was undertaken in patients with chronic New York Heart Association II-III heart failure. BV was quantitated using the nuclear medicine indicator-dilution methodology. RESULTS:A total of 530 BV analyses (360 HF with reduced ejection fraction and 170 HF with preserved ejection fraction) were identified in 395 unique patients. Absolute BV was greater in HF with reduced ejection fraction (6.7±1.8 versus 5.9±1.6 liters: P<0.001); however, large variability in frequency distribution of volume profiles was observed in both phenotypes (-22% deficit to +109% excess relative to normal volumes). HF with reduced ejection fraction was characterized by a higher prevalence of BV expansion ≥+25% of normal (39% versus 26%; P=0.003), and HF with preserved ejection fraction was characterized a by more frequent normal BV (42% versus 24%; P<0.001). Male sex in both phenotypes was associated with a larger absolute BV (7.0±1.6 versus 5.1±1.3 liters; P<0.001) and higher frequency of large BV and plasma volume expansions above normal (both P<0.001), while females in both phenotypes demonstrated a higher prevalence of normal BV and plasma volume (both P<0.001). CONCLUSIONS:Findings support significant differences in BV, plasma volume, and red blood cell mass profile distributions between heart failure phenotypes, driven in large part by sex-specific factors. This underscores the importance of identifying and distinguishing individual patient volume profiles to help guide volume management strategies.

Older age (>= 65 years) is associated with impaired responses to influenza vaccination, leading to the preferential recommendation of MF59-adjuvanted (MF59Flu) or high-dose (HDFlu) influenza vaccines for this age group in the United States. Herein, we characterized transcriptomic profiles of CD4(+) T cells isolated from 234 recipients (>= 65 years) of either MF59Flu or HDFlu vaccine, prior to vaccination and 28 days thereafter. We identified 412 and 645 differentially expressed genes (DEGs) in CD4(+ )T cells of older adults after receiving MF59Flu and HDFlu, respectively. DEGs in CD4(+) T cells of MF59Flu recipients were enriched in 14 KEGG pathways, all of which were downregulated. DEGs in CD4(+) T cells of HDFlu recipients were enriched in 11 upregulated pathways and 20 downregulated pathways. CD4(+) T cells in both vaccine groups shared 50 upregulated genes and 75 downregulated genes, all of which were enriched in 7 KEGG pathways. The remaining 287 and 520 DEGs were specifically associated with MF59Flu and HDFlu, respectively. Unexpectedly, none of these DEGs was significantly correlated with influenza A/H3N2-specific HAI titers, suggesting these DEGs at the individual level may have a limited role in protection against influenza. Our findings emphasize the need for further investigation into other factors influencing immunity against influenza in older adults.

Objective This study assessed the seroprevalence of SARS-CoV-2 antibodies at two timepoints to evaluate the effectiveness of COVID-19 precautions in two colleges.Participants The study enrolled students, faculty, and staff from Covenant and Geneva Colleges.Methods During the fall of 2020 the Mayo Clinic Vaccine Research Group partnered with Vibrant America to hand out self-administered finger stick antibody tests at the start of the fall semester and again two months later.Results The study enrolled 305 participants from Covenant and 671 from Geneva College at timepoint 1 and 198 participants from Covenant and 554 from Geneva College at timepoint 2. Seroprevalence rates were 2.3% and 1.6% at timepoint 1, and 0.5% and 3.97% at timepoint 2 at Covenant and Geneva Colleges, respectively.Conclusion The results of this study suggest that implementation of strict preventive measures may have kept the spread of COVID-19 low at these colleges during the study period.### Competing Interest StatementDr. Poland offers consultative advice to Johnson & Johnson/Janssen Global Services LLC, and is the chair of a Safety Evaluation Committee for novel investigational vaccine trials being conducted by Merck Research Laboratories. Dr. Poland also offers consultative advice on vaccine development to Merck & Co., Medicago, GlaxoSmithKline, Sanofi Pasteur, Emergent Biosolutions, Dynavax, Genentech, Eli Lilly and Company, Kentucky Bioprocessing Inc, Bavarian Nordic, AstraZeneca, Exelixis, Regeneron, Janssen, Vyriad, Moderna, and Genevant Sciences, Inc. Drs. Poland and Ovsyannikova hold patents related to vaccinia and measles peptide vaccines. Drs. Kennedy, Poland, and Ovsyannikova hold a patent related to vaccinia peptide vaccines. Drs. Poland, Kennedy and Ovsyannikova hold a patent related to the impact of single nucleotide polymorphisms on measles vaccine immunity. Drs. Poland, Kennedy, and Ovsyannikova have received grant funding from ICW Ventures for preclinical studies on a peptide-based COVID-19 vaccine. Dr. Kennedy has received funding from Merck Research Laboratories to study waning immunity to mumps vaccine. Dr. Kennedy also offers consultative advice on vaccine development to Merck & Co. and Sanofi Pasteur. These activities have been reviewed by the Mayo Clinic Conflict of Interest Review Board and are conducted in compliance with Mayo Clinic Conflict of Interest policies. This research has been reviewed by the Mayo Clinic Conflict of Interest Review Board and was conducted in compliance with Mayo Clinic Conflict of Interest policies.### Funding StatementThis study did not receive any external funding### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Ethics committee/IRB of Mayo Clinic Rochester, MN gave ethical approval for this work Ethics committee/IRB of Geneva College gave ethical approval for this work Ethics committee/IRB of Covenant College gave ethical approval for this workI confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

Background:In the vaccine era, individuals receive multiple vaccines in their lifetime. Host gene expression in response to antigenic stimulation is usually virus-specific; however, identifying shared pathways of host response across a wide spectrum of vaccine pathogens can shed light on the molecular mechanisms/components which can be targeted for the development of broad/universal therapeutics and vaccines. Method:We isolated PBMCs, monocytes, B cells, and CD8+ T cells from the peripheral blood of healthy donors, who received both seasonal influenza vaccine (within <1 year) and smallpox vaccine (within 1 - 4 years). Each of the purified cell populations was stimulated with either influenza virus or vaccinia virus. Differentially expressed genes (DEGs) relative to unstimulated controls were identified for each in vitro viral infection, as well as for both viral infections (shared DEGs). Pathway enrichment analysis was performed to associate identified DEGs with KEGG/biological pathways. Results:We identified 2,906, 3,888, 681, and 446 DEGs in PBMCs, monocytes, B cells, and CD8+ T cells, respectively, in response to influenza stimulation. Meanwhile, 97, 120, 20, and 10 DEGs were identified as gene signatures in PBMCs, monocytes, B cells, and CD8+ T cells, respectively, upon vaccinia stimulation. The majority of DEGs identified in PBMCs were also found in monocytes after either viral stimulation. Of the virus-specific DEGs, 55, 63, and 9 DEGs occurred in common in PBMCs, monocytes, and B cells, respectively, while no DEGs were shared in infected CD8+ T cells after influenza and vaccinia. Gene set enrichment analysis demonstrated that these shared DEGs were over-represented in innate signaling pathways, including cytokine-cytokine receptor interaction, viral protein interaction with cytokine and cytokine receptor, Toll-like receptor signaling, RIG-I-like receptor signaling pathways, cytosolic DNA-sensing pathways, and natural killer cell mediated cytotoxicity. Conclusion:Our results provide insights into virus-host interactions in different immune cells, as well as host defense mechanisms against viral stimulation. Our data also highlights the role of monocytes as a major cell population driving gene expression in ex vivo PBMCs in response to viral stimulation. The immune response signaling pathways identified in this study may provide specific targets for the development of novel virus-specific therapeutics and improved vaccines for vaccinia and influenza. Although influenza and vaccinia viruses have been selected in this study as pathogen models, this approach could be applicable to other pathogens.

The measles-mumps-rubella (MMR) vaccine has been widely used in the US, but measles and mumps outbreaks remain a public health issue in the US and elsewhere, even among individuals immunized with 2 doses of the vaccine. Immune correlates of vaccine-elicited protection against disease are typically assessed with serum antibody assays, but in some cases, these correlates fail to predict immunity, with the complexity and heterogeneity of the immune response. We used multicolor flow cytometry to evaluate changes in the frequency of peripheral T and B cell subsets in 82 study participants after receipt of a third dose of the M-M-RII vaccine (Merck & Co, Inc). We assessed correlations between flow cytometry variables and measles virus (MV), mumps virus (MuV), or rubella virus (RV)-specific immune response outcomes. Following a third vaccine dose, major changes were observed in the T-cell compartment. CD4+ T cell subsets were significantly increased from baseline to day 28, whereas CD8+ T cell subsets were predominantly decreased. Changes in regulatory T cells (Tregs) correlated with RV- and MV-specific immune outcomes and with high- and low-RV antibody responder groups, implicating the importance of Tregs in regulating MMR vaccine-induced immune responses. This information may help define additional correlates of protection and aid in the design of improved vaccines.

The envelope glycoproteins of the class I family, which include human immunodeficiency virus (HIV), influenza, and severe acute respiratory syndrome coronavirus (SARS-CoV), mediate viral entry by first binding to their cellular receptors and subsequently inducing fusion of the viral and cellular membranes. In the case of SARS-CoV, heptad repeat domains of the envelope glycoprotein, termed S2-HR1 and S2-HR2, are thought to undergo structural changes from a prefusion state, in which S2-HR1 and S2-HR2 do not interact, to a postfusion state in which S2-HR1 and S2-HR2 associate to form a six-helix bundle. In the present work, the structural and dynamic properties of S2-HR2 have been characterized. Evidence is presented for an equilibrium between a structured trimer thought to represent a prefusion state and an ensemble of unstructured monomers thought to represent a novel transition state. A model for viral entry is presented in which S2-HR2 is in a dynamic equilibrium between an ensemble of unstructured monomers in the transition state and a structured trimer in the prefusion state. Conversion from the prefusion state to the postfusion state requires passage through the transition state, a state that may give insight into the design of structure-based antagonists of SARS-CoV in particular, as well as other enveloped viruses in general.

Topic: 34. Thrombosis and vascular biology - Biology & Translational Research Background: Increasing Von Willebrand factor (VWF) correlates with the development of left atrial blood stasis in non-valvular atrial fibrillation (NVAF). However, the long-term impact of elevated VWF relative to ADAMTS13 activity level, its key regulator, is not well established in patients NVAF. Aims: To assess the long-term impact of VWF and ADAMTS13 activity levels on clinical outcomes of patients with NVAF and normal sinus rhythm controls. Methods: Baseline VWF antigen, VWF activity, ADAMTS13 activity, and echocardiographic measures were obtained for all subjects. Adverse cardiovascular events, major bleeding, clinically relevant non-major bleeding (CRNMB), and all-cause mortality were assessed by clinical follow-up, questionnaire, or telephone communication. Results: During 10 years of follow-up, there were 7 strokes, 2 TIA, 10 other thromboembolic events and 96 deaths in NVAF group compared to 2 TIA events, 4 systemic emboli and 30 deaths in NSR control group. In general, there were more adverse cardiovascular events in NVAF compared to NSR groups (p=0.022, see Figure). Major bleeding and CRNMB happened in 21 and 45 in NVAF and 1 and 12 in NSR patients. VWF antigen concentration and activity were an independent predictor of all-cause mortality (HR: 1.011, 95%CI: 1.008, 1.014, p<0.0001, and HR 1.010 (95%Cl:1.007,1.013, p<0.001) while lower activity of ADAMTS13 was associated with better survival ((HR 0.992, 95%CI 0.987, 0.996, P=0.0004). Presence of left atrial appendage thrombus, spontaneous echo contrast, low left atrial appendage emptying velocity, and increased left atrium volume index were not associated with adverse cardiovascular events or bleeding in NVAF patients. Summary/Conclusion: Higher level of VWF at baseline predicted a higher risk of death while lower activity of its key regulator ADAMTS13, was associated with better survival in NVAF and NSR patients.Keywords: ADAMTS13, Thromboembolism, von Willebrand factor (vWF), Bleeding

As an extremely contagious pathogen, a high rate of vaccine coverage and the durability of vaccine-induced immunity are key factors to control and eliminate measles. Herein, we assessed the seroprevalence of antibodies specific to measles in a cohort of 1393 adults (20–44 years old). ELISA results showed a nontrivial proportion of 37.6% study subjects being negative for measles immunoglobulin G (IgG). We also found significant influences of sex and age of the study cohort on the IgG level. Our findings suggest that even within a highly vaccinated population, a subset of individuals may still have sub-optimal immunity against measles and potentially be susceptible during any future measles outbreaks.

The availability of effective smallpox vaccines was a critical element of the successful eradication of smallpox in 1980. Antibody responses play a primary role in protective immunity and neutralizing antibody is an established correlate of protection against smallpox. In this study we used a poxvirus proteome array to assess the antibody response to individual viral proteins in a cohort of 1,037 smallpox vaccine recipients. Several statistically significant differences were observed in the antibody response to immunodominant proteins between men and women, including B5R-a major target of neutralizing antibody in vaccinia immune globulin, and the membrane proteins D8L and A27L, both of which have been used as vaccine antigens providing protection in animal models. We also noted differences across racial/ethnic groups. In this cohort, which consisted of both ACAM2000 and Dryvax recipients, we noted minute differences in the antibody responses to a restricted number of viral proteins, providing additional support for the use of ACAM2000 as a replacement smallpox vaccine. Furthermore, our data indicate that poxvirus proteome microarrays can be valuable for screening and monitoring smallpox vaccine-induced humoral immune responses in large-scale serologic surveillance studies and prove useful in the guidance of developing novel smallpox candidate vaccines.

Background A third dose of measles-mumps-rubella vaccine (MMR3) is recommended in mumps outbreak scenarios, but the immune response and the need for widespread use of MMR3 remain uncertain. Herein, we characterized measles-specific immune responses to MMR3 in a cohort of 232 healthy subjects. Methods Serum and peripheral blood mononuclear cells (PBMCs) were sampled at day 0 and day 28 after MMR3. Measles-specific binding and neutralizing antibodies were quantified in sera by enzyme-linked immunosorbent assay and a microneutralization assay, respectively. PBMCs were stimulated with inactivated measles virus, and the release of cytokines/chemokines was assessed by a multiplex assay. Demographic variables of subjects were examined for potential correlations with immune outcomes. Results Of the study participants, 95.69% and 100% were seropositive at day 0 and day 28, respectively. Antibody avidity significantly increased from 38.08% at day 0 to 42.8% at day 28 (P = .00026). Neutralizing antibodies were significantly enhanced, from 928.7 at day 0 to 1289.64 mIU/mL at day 28 (P = .0001). Meanwhile, cytokine/chemokine responses remained largely unchanged. Body mass index was significantly correlated with the levels of inflammatory cytokines/chemokines. Conclusions Measles-specific humoral immune responses, but not cellular responses, were enhanced after MMR3 receipt, extending current understanding of immune responses to MMR3 and supporting MMR3 administration to seronegative or high-risk individuals. A third dose of measles-mumps-rubella vaccine (MMR) boosted humoral immune responses, but not cellular immune responses, specific to measles in a cohort of 232 healthy subjects immunized with 2 doses of MMR, supporting its administration to seronegative or high-risk individuals.

Rare cases of COVID-19 vaccinated individuals develop anti-platelet factor 4 (PF4) antibodies that cause thrombocytopenia and thrombotic complications, a syndrome referred to as vaccine-induced immune thrombotic thrombocytopenia (VITT). Currently, information on the characteristics and persistence of anti-PF4 antibodies that cause VITT after Ad26.COV2.S vaccination is limited, and available diagnostic assays fail to differentiate Ad26.COV2.S and ChAdOx1 nCoV-19-associated VITT from similar clinical disorders, namely heparin-induced thrombocytopenia (HIT) and spontaneous HIT. Here we demonstrate that while Ad26.COV2.S-associated VITT patients are uniformly strongly positive in PF4-polyanion enzyme-linked immunosorbent assays (ELISAs); they are frequently negative in the serotonin release assay (SRA). The PF4-dependent p-selectin expression assay (PEA) that uses platelets treated with PF4 rather than heparin consistently diagnosed Ad26.COV2.S-associated VITT. Most Ad26.COV2.S-associated VITT antibodies persisted for >5 months in PF4-polyanion ELISAs, while the PEA became negative earlier. Two patients had otherwise unexplained mild persistent thrombocytopenia (140-150 x 103 /µL) 6 months after acute presentation. From an epidemiological perspective, differentiating VITT from spontaneous HIT, another entity that develops in the absence of proximate heparin exposure, and HIT is important, but currently available PF4-polyanion ELISAs and functional assay are non-specific and detect all three conditions. Here, we report that a novel un-complexed PF4 ELISA specifically differentiates VITT, secondary to both Ad26.COV2.S and ChAdOx1 nCoV-19, from both spontaneous HIT, HIT and commonly-encountered HIT-suspected patients who are PF4/polyanion ELISA-positive but negative in functional assays. In summary, Ad26.COV2.S-associated VITT antibodies are persistent, and the un-complexed PF4 ELISA appears to be both sensitive and specific for VITT diagnosis.