With great power comes great responsibility: high-dimensional spectral flow cytometry to support clinical trials.

Flow cytometry is a powerful technology used in research, drug development and clinical sample analysis for cell identification and characterization, allowing for the simultaneous interrogation of multiple targets on various cell subsets from limited samples. Recent advancements in instrumentation and fluorochrome availability have resulted in significant increases in the complexity and dimensionality of flow cytometry panels. Though this increase in panel size allows for detection of a broader range of markers and sub-populations, even in restricted biological samples, it also comes with many challenges in panel design, optimization, and downstream data analysis and interpretation. In the current paper we describe the practices we established for development of high-dimensional panels on the Aurora spectral flow cytometer to aid clinical sample analysis.

2020 White Paper on Recent Issues in Bioanalysis: BAV Guidance, CLSI H62, Biotherapeutics Stability, Parallelism Testing, CyTOF and Regulatory Feedback (Part 2A - Recommendations on Biotherapeutics Stability, PK LBA Regulated Bioanalysis, Biomarkers Assays, Cytometry Validation & Innovation Part 2B - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 2A) BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation and (Part 2B) Regulatory Input. Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 4, and 6 (2021), respectively.

Influenza vaccine-induced human bone marrow plasma cells decline within a year after vaccination.

A universal vaccine against influenza would ideally generate protective immune responses that are not only broadly reactive against multiple influenza strains but also long-lasting. Because long-term serum antibody levels are maintained by bone marrow plasma cells (BMPCs), we investigated the production and maintenance of these cells after influenza vaccination. We found increased numbers of influenza-specific BMPCs 4 weeks after immunization with the seasonal inactivated influenza vaccine, but numbers returned to near their prevaccination levels after 1 year. This decline was driven by the loss of BMPCs induced by the vaccine, whereas preexisting BMPCs were maintained. Our results suggest that most BMPCs generated by influenza vaccination in adults are short-lived. Designing strategies to enhance their persistence will be a key challenge for the next generation of influenza vaccines.

Adjuvanted H5N1 influenza vaccine enhances both cross-reactive memory B cell and strain-specific naive B cell responses in humans.

There is a need for improved influenza vaccines. In this study we compared the antibody responses in humans after vaccination with an AS03-adjuvanted versus nonadjuvanted H5N1 avian influenza virus inactivated vaccine. Healthy young adults received two doses of either formulation 3 wk apart. We found that AS03 significantly enhanced H5 hemagglutinin (HA)-specific plasmablast and antibody responses compared to the nonadjuvanted vaccine. Plasmablast response after the first immunization was exclusively directed to the conserved HA stem region and came from memory B cells. Monoclonal antibodies (mAbs) derived from these plasmablasts had high levels of somatic hypermutation (SHM) and recognized the HA stem region of multiple influenza virus subtypes. Second immunization induced a plasmablast response to the highly variable HA head region. mAbs derived from these plasmablasts exhibited minimal SHM (naive B cell origin) and largely recognized the HA head region of the immunizing H5N1 strain. Interestingly, the antibody response to H5 HA stem region was much lower after the second immunization, and this suppression was most likely due to blocking of these epitopes by stem-specific antibodies induced by the first immunization. Taken together, these findings show that an adjuvanted influenza vaccine can substantially increase antibody responses in humans by effectively recruiting preexisting memory B cells as well as naive B cells into the response. In addition, we show that high levels of preexisting antibody can have a negative effect on boosting. These findings have implications toward the development of a universal influenza vaccine.

Breadth and Functionality of Varicella-Zoster Virus Glycoprotein-Specific Antibodies Identified after Zostavax Vaccination in Humans.

Herpes zoster (HZ) (shingles) is the clinical manifestation of varicella-zoster virus (VZV) reactivation. HZ typically develops as people age, due to decreased cell-mediated immunity. However, the importance of antibodies for immunity against HZ prevention remains to be understood. The goal of this study was to examine the breadth and functionality of VZV-specific antibodies after vaccination with a live attenuated HZ vaccine (Zostavax). Direct enumeration of VZV-specific antibody-secreting cells (ASCs) via enzyme-linked immunosorbent spot assay (ELISPOT assay) showed that Zostavax can induce both IgG and IgA ASCs 7 days after vaccination but not IgM ASCs. The VZV-specific ASCs range from 33 to 55% of the total IgG ASCs. Twenty-five human VZV-specific monoclonal antibodies (MAbs) were cloned and characterized from single-cell-sorted ASCs of five subjects (>60 years old) who received Zostavax. These MAbs had an average of ∼20 somatic hypermutations per VH gene, similar to those seen after seasonal influenza vaccination. Fifteen of the 25 MAbs were gE specific, whereas the remaining MAbs were gB, gH, or gI specific. The most potent neutralizing antibodies were gH specific and were also able to inhibit cell-to-cell spread of the virus in vitro Most gE-specific MAbs were able to neutralize VZV, but they required the presence of complement and were unable to block cell-to-cell spread. These data indicate that Zostavax induces a memory B cell recall response characterized by anti-gE > anti-gI > anti-gB > anti-gH antibodies. While antibodies to gH could be involved in limiting the spread of VZV upon reactivation, the contribution of anti-gE antibodies toward protective immunity after Zostavax needs further evaluation.IMPORTANCE Varicella-zoster virus (VZV) is the causative agent of chickenpox and shingles. Following infection with VZV, the virus becomes latent and resides in nerve cells. Age-related declines in immunity/immunosuppression can result in reactivation of this latent virus, causing shingles. It has been shown that waning T cell immunity correlates with an increased incidence of VZV reactivation. Interestingly, serum with high levels of VZV-specific antibodies (VariZIG; IV immunoglobulin) has been administered to high-risk populations, e.g., immunocompromised children, newborns, and pregnant women, after exposure to VZV and has shown some protection against chickenpox. However, the relative contribution of antibodies against individual surface glycoproteins toward protection from shingles in elderly/immunocompromised individuals has not been established. Here, we examined the breadth and functionality of VZV-specific antibodies after vaccination with the live attenuated VZV vaccine Zostavax in humans. This study will add to our understanding of the role of antibodies in protection against shingles.

Origin and differentiation of human memory CD8 T cells after vaccination.

The differentiation of human memory CD8 T cells is not well understood. Here we address this issue using the live yellow fever virus (YFV) vaccine, which induces long-term immunity in humans. We used in vivo deuterium labelling to mark CD8 T cells that proliferated in response to the virus and then assessed cellular turnover and longevity by quantifying deuterium dilution kinetics in YFV-specific CD8 T cells using mass spectrometry. This longitudinal analysis showed that the memory pool originates from CD8 T cells that divided extensively during the first two weeks after infection and is maintained by quiescent cells that divide less than once every year (doubling time of over 450 days). Although these long-lived YFV-specific memory CD8 T cells did not express effector molecules, their epigenetic landscape resembled that of effector CD8 T cells. This open chromatin profile at effector genes was maintained in memory CD8 T cells isolated even a decade after vaccination, indicating that these cells retain an epigenetic fingerprint of their effector history and remain poised to respond rapidly upon re-exposure to the pathogen.

Metabolic Phenotypes of Response to Vaccination in Humans.

Herpes zoster (shingles) causes significant morbidity in immune compromised hosts and older adults. Whereas a vaccine is available for prevention of shingles, its efficacy declines with age. To help to understand the mechanisms driving vaccinal responses, we constructed a multiscale, multifactorial response network (MMRN) of immunity in healthy young and older adults immunized with the live attenuated shingles vaccine Zostavax. Vaccination induces robust antigen-specific antibody, plasmablasts, and CD4+ T cells yet limited CD8+ T cell and antiviral responses. The MMRN reveals striking associations between orthogonal datasets, such as transcriptomic and metabolomics signatures, cell populations, and cytokine levels, and identifies immune and metabolic correlates of vaccine immunity. Networks associated with inositol phosphate, glycerophospholipids, and sterol metabolism are tightly coupled with immunity. Critically, the sterol regulatory binding protein 1 and its targets are key integrators of antibody and T follicular cell responses. Our approach is broadly applicable to study human immunity and can help to identify predictors of efficacy as well as mechanisms controlling immunity to vaccination.

Proliferation of PD-1+ CD8 T cells in peripheral blood after PD-1-targeted therapy in lung cancer patients.

Exhausted T cells in chronic infections and cancer have sustained expression of the inhibitory receptor programmed cell death 1 (PD-1). Therapies that block the PD-1 pathway have shown promising clinical results in a significant number of advanced-stage cancer patients. Nonetheless, a better understanding of the immunological responses induced by PD-1 blockade in cancer patients is lacking. Identification of predictive biomarkers is a priority in the field, but whether peripheral blood analysis can provide biomarkers to monitor or predict patients' responses to treatment remains to be resolved. In this study, we analyzed longitudinal blood samples from advanced stage non-small cell lung cancer (NSCLC) patients (n = 29) receiving PD-1-targeted therapies. We detected an increase in Ki-67+ PD-1+ CD8 T cells following therapy in ∼70% of patients, and most responses were induced after the first or second treatment cycle. This T-cell activation was not indiscriminate because we observed only minimal effects on EBV-specific CD8 T cells, suggesting that responding cells may be tumor specific. These proliferating CD8 T cells had an effector-like phenotype (HLA-DR+, CD38+, Bcl-2lo), expressed costimulatory molecules (CD28, CD27, ICOS), and had high levels of PD-1 and coexpression of CTLA-4. We found that 70% of patients with disease progression had either a delayed or absent PD-1+ CD8 T-cell response, whereas 80% of patients with clinical benefit exhibited PD-1+ CD8 T-cell responses within 4 wk of treatment initiation. Our results suggest that peripheral blood analysis may provide valuable insights into NSCLC patients' responses to PD-1-targeted therapies.

Novel multiplex assay platforms to detect influenza A hemagglutinin subtype-specific antibody responses for high-throughput and in-field applications.

BACKGROUND: Detections of influenza A subtype-specific antibody responses are often complicated by the presence of cross-reactive antibodies. We developed two novel multiplex platforms for antibody detection. The multiplexed magnetic fluorescence microsphere immunoassay (MAGPIX) is a high-throughput laboratory-based assay. Chembio Dual Path Platform (DPP) is a portable and rapid test that could be used in the field. METHODS: Twelve recombinant globular head domain hemagglutinin (GH HA1) antigens from A(H1N1)pdm09 (pH1N1), A(H2N2), A(H3N2), A(H5N1), A(H7N9), A(H9N2), A(H13N9), B/Victoria lineage, B/Yamagata lineage viruses, and protein A control were used. Human sera from U.S. residents either vaccinated (with H5N1 or pH1N1) or infected with pH1N1 influenza viruses and sera from live bird market workers in Bangladesh (BDPW) were evaluated. GH HA1 antigens and serum adsorption using full ectodomain recombinant hemagglutinins from A(pH1N1) and A(H3N2) were introduced into the platforms to reduce cross-reactivity. RESULTS: Serum adsorption reduced cross-reactivity to novel subtype HAs. Compared to traditional hemagglutination inhibition or microneutralization assays, when serum adsorption and the highest fold rise in signals were used to determine positivity, the correct subtype-specific responses were identified in 86%-100% of U.S. residents exposed to influenza antigens through vaccination or infection (N=49). For detection of H5N1-specific antibodies in sera collected from BDPW, H5 sensitivity was 100% (six of six) for MAGPIX, 83% (five of six) for DPP, H5 specificity was 100% (15/15), and cross-reactivity against other subtype was 0% (zero of six) for both platforms. CONCLUSION: MAGPIX and DPP platforms can be utilized for high-throughput and in-field detection of novel influenza virus infections.

Evaluation of multiplex assay platforms for detection of influenza hemagglutinin subtype specific antibody responses.

Influenza hemagglutination inhibition (HI) and virus microneutralization assays (MN) are widely used for seroprevalence studies. However, these assays have limited field portability and are difficult to fully automate for high throughput laboratory testing. To address these issues, three multiplex influenza subtype-specific antibody detection assays were developed using recombinant hemagglutinin antigens in combination with Chembio, Luminex®, and ForteBio® platforms. Assay sensitivity, specificity, and subtype cross-reactivity were evaluated using a panel of well characterized human sera. Compared to the traditional HI, assay sensitivity ranged from 87% to 92% and assay specificity in sera collected from unexposed persons ranged from 65% to 100% across the platforms. High assay specificity (86-100%) for A(H5N1) rHA was achieved for sera from exposed or unexposed to hetorosubtype influenza HAs. In contrast, assay specificity for A(H1N1)pdm09 rHA using sera collected from A/Vietnam/1204/2004 (H5N1) vaccinees in 2008 was low (22-30%) in all platforms. Although cross-reactivity against rHA subtype proteins was observed in each assay platform, the correct subtype specific responses were identified 78%-94% of the time when paired samples were available for analysis. These results show that high throughput and portable multiplex assays that incorporate rHA can be used to identify influenza subtype specific infections.

Induction of broadly cross-reactive antibody responses to the influenza HA stem region following H5N1 vaccination in humans.

The emergence of pandemic influenza viruses poses a major public health threat. Therefore, there is a need for a vaccine that can induce broadly cross-reactive antibodies that protect against seasonal as well as pandemic influenza strains. Human broadly neutralizing antibodies directed against highly conserved epitopes in the stem region of influenza virus HA have been recently characterized. However, it remains unknown what the baseline levels are of antibodies and memory B cells that are directed against these conserved epitopes. More importantly, it is also not known to what extent anti-HA stem B-cell responses get boosted in humans after seasonal influenza vaccination. In this study, we have addressed these two outstanding questions. Our data show that: (i) antibodies and memory B cells directed against the conserved HA stem region are prevalent in humans, but their levels are much lower than B-cell responses directed to variable epitopes in the HA head; (ii) current seasonal influenza vaccines are efficient in inducing B-cell responses to the variable HA head region but they fail to boost responses to the conserved HA stem region; and (iii) in striking contrast, immunization of humans with the avian influenza virus H5N1 induced broadly cross-reactive HA stem-specific antibodies. Taken together, our findings provide a potential vaccination strategy where heterologous influenza immunization could be used for increasing the levels of broadly neutralizing antibodies and for priming the human population to respond quickly to emerging pandemic influenza threats.

Broadly reactive human CD8 T cells that recognize an epitope conserved between VZV, HSV and EBV.

Human herpesviruses are important causes of potentially severe chronic infections for which T cells are believed to be necessary for control. In order to examine the role of virus-specific CD8 T cells against Varicella Zoster Virus (VZV), we generated a comprehensive panel of potential epitopes predicted in silico and screened for T cell responses in healthy VZV seropositive donors. We identified a dominant HLA-A*0201-restricted epitope in the VZV ribonucleotide reductase subunit 2 and used a tetramer to analyze the phenotype and function of epitope-specific CD8 T cells. Interestingly, CD8 T cells responding to this VZV epitope also recognized homologous epitopes, not only in the other α-herpesviruses, HSV-1 and HSV-2, but also the γ-herpesvirus, EBV. Responses against these epitopes did not depend on previous infection with the originating virus, thus indicating the cross-reactive nature of this T cell population. Between individuals, the cells demonstrated marked phenotypic heterogeneity. This was associated with differences in functional capacity related to increased inhibitory receptor expression (including PD-1) along with decreased expression of co-stimulatory molecules that potentially reflected their stimulation history. Vaccination with the live attenuated Zostavax vaccine did not efficiently stimulate a proliferative response in this epitope-specific population. Thus, we identified a human CD8 T cell epitope that is conserved in four clinically important herpesviruses but that was poorly boosted by the current adult VZV vaccine. We discuss the concept of a "pan-herpesvirus" vaccine that this discovery raises and the hurdles that may need to be overcome in order to achieve this.

Identification of novel markers for mouse CD4(+) T follicular helper cells.

CD4(+) T follicular helper (TFH) cells are central for generation of long-term B-cell immunity. A defining phenotypic attribute of TFH cells is the expression of the chemokine R CXCR5, and TFH cells are typically identified by co-expression of CXCR5 together with other markers such as PD-1, ICOS, and Bcl-6. Herein, we report high-level expression of the nutrient transporter folate R 4 (FR4) on TFH cells in acute viral infection. Distinct from the expression profile of conventional TFH markers, FR4 was highly expressed by naive CD4(+) T cells, was downregulated after activation and subsequently re-expressed on TFH cells. Furthermore, FR4 expression was maintained, albeit at lower levels, on memory TFH cells. Comparative gene expression profiling of FR4(hi) versus FR4(lo) Ag-specific CD4(+) effector T cells revealed a molecular signature consistent with TFH and TH1 subsets, respectively. Interestingly, genes involved in the purine metabolic pathway, including the ecto-enzyme CD73, were enriched in TFH cells compared with TH1 cells, and phenotypic analysis confirmed expression of CD73 on TFH cells. As there is now considerable interest in developing vaccines that would induce optimal TFH cell responses, the identification of two novel cell surface markers should be useful in characterization and identification of TFH cells following vaccination and infection.

Cross-reactive humoral responses to influenza and their implications for a universal vaccine.

Influenza remains one of the most important causes of respiratory infection despite the widespread availability of vaccines. As effective vaccines against influenza principally rely on the induction of strain-specific neutralizing antibodies for their protective efficacy, drifted escape mutants and genetically reassortant pandemic strains can rapidly overcome them. Several groups have recently described cross-reactive influenza antibodies in humans, some of which bind to the conserved hemagglutinin stem. If such antibodies could be consistently induced at high levels by vaccines, they might protect against both seasonal and pandemic influenza strains. Here we discuss the humoral responses to influenza infection and vaccination, with particular reference to the pandemic H1N1 2009 virus and induction of broadly cross-reactive stem-binding antibodies. Having shown that cross-reactive antibodies are preferentially induced by a pandemic hemagglutinin, the challenge is now to design a vaccine that applies these principles to the induction of heterosubtypic immunity.

Antibody pressure by a human monoclonal antibody targeting the 2009 pandemic H1N1 virus hemagglutinin drives the emergence of a virus with increased virulence in mice.

UNLABELLED: In 2009, a novel H1N1 influenza A virus (2009 pH1N1) emerged and caused a pandemic. A human monoclonal antibody (hMAb; EM4C04), highly specific for the 2009 pH1N1 virus hemagglutinin (HA), was isolated from a severely ill 2009 pH1N1 virus-infected patient. We postulated that under immune pressure with EM4C04, the 2009 pH1N1 virus would undergo antigenic drift and mutate at sites that would identify the antibody binding site. To do so, we infected MDCK cells in the presence of EM4C04 and generated 11 escape mutants, displaying 7 distinct amino acid substitutions in the HA. Six substitutions greatly reduced MAb binding (K123N, D131E, K133T, G134S, K157N, and G158E). Residues 131, 133, and 134 are contiguous with residues 157 and 158 in the globular domain structure and contribute to a novel pH1N1 antibody epitope. One mutation near the receptor binding site, S186P, increased the binding affinity of the HA to the receptor. 186P and 131E are present in the highly virulent 1918 virus HA and were recently identified as virulence determinants in a mouse-passaged pH1N1 virus. We found that pH1N1 escape variants expressing these substitutions enhanced replication and lethality in mice compared to wild-type 2009 pH1N1 virus. The increased virulence of these viruses was associated with an increased affinity for α2,3 sialic acid receptors. Our study demonstrates that antibody pressure by an hMAb targeting a novel epitope in the Sa region of 2009 pH1N1 HA is able to inadvertently drive the development of a more virulent virus with altered receptor binding properties. This broadens our understanding of antigenic drift. IMPORTANCE: Influenza viruses accumulate amino acid substitutions to evade the antibody response in a process known as antigenic drift, making it necessary to vaccinate against influenza annually. Mapping human monoclonal antibody (hMAb) epitopes is a necessary step towards understanding antigenic drift in humans. We defined the specificity of an hMAb that specifically targeted the 2009 pH1N1 virus and describe a novel epitope. In addition, we identified a previously unappreciated potential for antibody escape to enhance the pathogenicity of a virus. The escape mutation that we identified with in vitro immune pressure was independently reported by other investigators using in vivo selection in nonimmune mice. Although in vitro generation of escape mutants is unlikely to recapitulate antigenic drift in its entirety, the data demonstrate that pressure by a human monoclonal antibody targeting a novel epitope in the hemagglutinin of the 2009 pandemic H1N1 virus can inadvertently drive the development of escape mutants, of which a subset have increased virulence and altered receptor binding properties.

Pandemic H1N1 influenza vaccine induces a recall response in humans that favors broadly cross-reactive memory B cells.

We have previously shown that broadly neutralizing antibodies reactive to the conserved stem region of the influenza virus hemagglutinin (HA) were generated in people infected with the 2009 pandemic H1N1 strain. Such antibodies are rarely seen in humans following infection or vaccination with seasonal influenza virus strains. However, the important question remained whether the inactivated 2009 pandemic H1N1 vaccine, like the infection, could also induce these broadly neutralizing antibodies. To address this question, we analyzed B-cell responses in 24 healthy adults immunized with the pandemic vaccine in 2009. In all cases, we found a rapid, predominantly IgG-producing vaccine-specific plasmablast response. Strikingly, the majority (25 of 28) of HA-specific monoclonal antibodies generated from the vaccine-specific plasmablasts neutralized more than one influenza strain and exhibited high levels of somatic hypermutation, suggesting they were derived from recall of B-cell memory. Indeed, memory B cells that recognized the 2009 pandemic H1N1 HA were detectable before vaccination not only in this cohort but also in samples obtained before the emergence of the pandemic strain. Three antibodies demonstrated extremely broad cross-reactivity and were found to bind the HA stem. Furthermore, one stem-reactive antibody recognized not only H1 and H5, but also H3 influenza viruses. This exceptional cross-reactivity indicates that antibodies capable of neutralizing most influenza subtypes might indeed be elicited by vaccination. The challenge now is to improve upon this result and design influenza vaccines that can elicit these broadly cross-reactive antibodies at sufficiently high levels to provide heterosubtypic protection.

Systems biology of vaccination for seasonal influenza in humans.

Here we have used a systems biology approach to study innate and adaptive responses to vaccination against influenza in humans during three consecutive influenza seasons. We studied healthy adults vaccinated with trivalent inactivated influenza vaccine (TIV) or live attenuated influenza vaccine (LAIV). TIV induced higher antibody titers and more plasmablasts than LAIV did. In subjects vaccinated with TIV, early molecular signatures correlated with and could be used to accurately predict later antibody titers in two independent trials. Notably, expression of the kinase CaMKIV at day 3 was inversely correlated with later antibody titers. Vaccination of CaMKIV-deficient mice with TIV induced enhanced antigen-specific antibody titers, which demonstrated an unappreciated role for CaMKIV in the regulation of antibody responses. Thus, systems approaches can be used to predict immunogenicity and provide new mechanistic insights about vaccines.

Protective murine and human monoclonal antibodies against eczema vaccinatum.

BACKGROUND: Eczema vaccinatum is the most common severe pathology associated with smallpox vaccination (vaccinia virus), occurring at high rates among individuals with a previous history of atopic dermatitis (atopic eczema). METHODS: Monoclonal antibodies capable of neutralizing vaccinia virus, anti-H3 and anti-B5, were developed as a potential therapy for treatment of human eczema vaccinatum. RESULTS: Using a small animal model of eczema vaccinatum, we demonstrated that both murine and fully human monoclonal antibodies effectively limited eczema vaccinatum disease, foreshortening both the disease kinetics and the severity of the erosive viral skin lesions. CONCLUSIONS: These neutralizing antibodies would likely be effective at reducing or eliminating clinical disease in people with eczema vaccinatum or other severe side effects of the smallpox vaccine.

Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection.

The 2009 pandemic H1N1 influenza pandemic demonstrated the global health threat of reassortant influenza strains. Herein, we report a detailed analysis of plasmablast and monoclonal antibody responses induced by pandemic H1N1 infection in humans. Unlike antibodies elicited by annual influenza vaccinations, most neutralizing antibodies induced by pandemic H1N1 infection were broadly cross-reactive against epitopes in the hemagglutinin (HA) stalk and head domain of multiple influenza strains. The antibodies were from cells that had undergone extensive affinity maturation. Based on these observations, we postulate that the plasmablasts producing these broadly neutralizing antibodies were predominantly derived from activated memory B cells specific for epitopes conserved in several influenza strains. Consequently, most neutralizing antibodies were broadly reactive against divergent H1N1 and H5N1 influenza strains. This suggests that a pan-influenza vaccine may be possible, given the right immunogen. Antibodies generated potently protected and rescued mice from lethal challenge with pandemic H1N1 or antigenically distinct influenza strains, making them excellent therapeutic candidates.

Combination therapy of vaccinia virus infection with human anti-H3 and anti-B5 monoclonal antibodies in a small animal model.

BACKGROUND: Treatment of rare severe side effects of vaccinia virus (VACV) immunization in humans is currently very challenging. VACV possesses two immunologically distinct virion forms in vivo - intracellular mature virion (MV, IMV) and extracellular virion (EV, EEV). METHODS: Antibody-mediated therapeutic efficacy was determined against VACV infection in a small animal model of progressive vaccinia. The model consisted of severe combined immunodeficiency mice infected with VACV New York City Board of Health vaccine strain and treated with monoclonal antibodies (mAbs). RESULTS: Here, we show that combination therapy with two fully human mAbs against an immunodominant MV antigen, H3 (H3L), and an EV antigen, B5 (B5R), provides significantly better protection against disease and death than either single human monoclonal or human vaccinia immune globulin, the currently licensed therapeutic for side effects of smallpox vaccination. CONCLUSIONS: The preclinical studies validate that this combination of mAbs against H3 and B5 is a promising approach as a poxvirus infection treatment for use in humans.