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Booster vaccinations are recommended to improve protection against severe disease from SARS-CoV-2 infection. With primary vaccinations involving various adenoviral vector and mRNA-based formulations, it remains unclear if these differentially affect the immune response to booster doses. We examined the effects of homologous (mRNA/mRNA) and heterologous (adenoviral vector/mRNA) vaccination on antibody and memory B cell (Bmem) responses against ancestral and Omicron subvariants. Healthy adults who received primary BNT162b2 (mRNA) or ChAdOx1 (vector) vaccination were sampled 1-month and 6-months after their 2nd and 3rd dose (homologous or heterologous) vaccination. Recombinant spike receptor-binding domain (RBD) proteins from ancestral, Omicron BA.2 and BA.5 variants were produced for ELISA-based serology, and tetramerized for immunophenotyping of RBD-specific Bmem. Dose 3 boosters significantly increased ancestral RBD-specific plasma IgG and Bmem in both cohorts. Up to 80% of ancestral RBD-specific Bmem expressed IgG1+. IgG4+ Bmem were detectable after primary mRNA vaccination, and expanded significantly to 5-20% after dose 3, whereas heterologous boosting did not elicit IgG4+ Bmem. Recognition of Omicron BA.2 and BA.5 by ancestral RBD-specific plasma IgG increased from 20% to 60% after the 3rd dose in both cohorts. Reactivity of ancestral RBD-specific Bmem to Omicron BA.2 and BA.5 increased following a homologous booster from 40% to 60%, but not after a heterologous booster. A 3rd mRNA dose generates similarly robust serological and Bmem responses in homologous and heterologous vaccination groups. The expansion of IgG4+ Bmem after mRNA priming might result from the unique vaccine formulation or dosing schedule affecting the Bmem response duration and antibody maturation.

BACKGROUND: House dust mite (HDM) is the most common allergen trigger globally for allergic rhinitis and atopic asthma. OBJECTIVES: To expedite accurate confirmation of allergen sensitization, we designed fluorescent allergen tetramers to directly stain specific IgE on basophils to detect specific allergen sensitization using the flow cytometric CytoBas assay. METHODS: Recombinant proteins of major HDM allergens (component), Der f 1, Der p 1, and Der p 2 were biotinylated and conjugated with fluorochrome streptavidins as tetramers. Blood samples from 64 patients who are HDM-allergic and 26 controls that are non-HDM-sensitized were incubated with allergen tetramers for evaluation of basophil binding (CytoBas) and activation (BAT) with flow cytometry. RESULTS: The tetramers effectively bound and activated basophils from patients who are allergic but not from controls who are nonsensitized. CytoBas with Der p 1 as a single allergen had comparable sensitivity and specificity (92% and 100%) to BAT (91% and 100%) in detecting allergen sensitization, as did CytoBas with Der p 2 (95% and 96%) to BAT (95% and 87%). A positive staining for Der p 1 and/or Der p 2 in CytoBas was 100% sensitive and 96% specific for HDM allergy. CONCLUSIONS: CytoBas has diagnostic accuracy for group 1 and group 2 HDM allergens that is comparable to BAT, but with additional advantages of multiple allergen components in a single tube and no requirement for in vitro basophil activation. These findings endorse a single, multiplex CytoBas assay for accurate and component-resolved diagnosis of aeroallergen sensitization in patients with allergic asthma and/or rhinitis.

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BACKGROUND: Sublingual immunotherapy (SLIT) for grass pollen allergy can modify the natural history of allergic rhinitis and is associated with increased allergen-specific IgG4 . IgG4 competitively inhibits functional IgE on the surface of effector cells, such as mast cells and basophils, from binding to allergens. To further understand the important role memory B-cell (Bmem) responses play in mediating the beneficial effects of SLIT, we assessed changes in allergen-specific Bmem subsets induced by SLIT for grass pollen allergy. METHODS: Blood samples were collected twice outside the pollen season from twenty-seven patients with sensitization to ryegrass pollen (RGP; Lolium perenne) and seasonal rhinoconjunctivitis. Thirteen received 4-month pre-seasonal SLIT for grass pollen allergy, and 14 received standard pharmacotherapy only. Single-cell RNA sequencing was performed on FACS-purified Lol p 1-specific Bmem before and after SLIT from four patients, and significant genes were validated by flow cytometry on the total cohort. RESULTS: Four months of SLIT increased RGP-specific IgE and IgG4 in serum and induced two Lol p 1-specific Bmem subsets with unique transcriptional profiles. Both subsets had upregulated expression of beta 1 integrin ITGB1 (CD29), whereas IGHE (IgE), IGHG4 (IgG4 ), FCER2 (CD23), and IL13RA1 were upregulated in one subset. There was an increase in the proportion of Lol p 1+ Bmem expressing surface IgG4 , CD23, and CD29 after SLIT. CONCLUSIONS: A clinically successful 4 months course of SLIT for grass pollen allergy induces two transcriptionally unique Bmem fates. Associated changes in surface-expressed proteins on these Bmem subsets can be used as early biomarkers for treatment effects.

BACKGROUND: Protective malarial antibodies are acquired more rapidly in adults than children, independently of cumulative exposure, however the cellular responses mediating these differences are unknown. CD4 T-follicular helper (Tfh) cells have key roles in inducing antibodies, with Th2-Tfh cell activation associated with antibody development in malaria. Whether Tfh cell activation in malaria is age dependent is unknown and no studies have compared Tfh cell activation in children and adults with malaria. METHODS: We undertook a comprehensive study of Tfh cells, along with B cells and antibody induction in children and adults with malaria. Activation and proliferation of circulating Tfh (cTfh) cell subsets was measured ex vivo and parasite-specific Tfh cell frequencies and functions studied with Activation Induced Marker (AIM) assays and intracellular cytokine staining. FINDINGS: During acute malaria, the magnitude of cTfh cell activation was higher in adults than in children and occurred across all cTfh cell subsets in adults but was restricted only to the Th1-cTfh subset in children. Further, adults had higher levels of parasite-specific cTfh cells, and cTfh cells which produced more Th2-Tfh associated cytokine IL-4. Consistent with a role of higher Tfh cell activation in rapid immune development in adults, adults had higher activation of B cells during infection and higher induction of antibodies 7 and 28 days after malaria compared to children. INTERPRETATION: Our data provide evidence that age impacts Tfh cell activation during malaria, and that these differences may influence antibody induction after treatment. Findings have important implications for vaccine development in children. FUNDING: This word was supported by the National Health and Medical Research Council of Australia, Wellcome Trust, Charles Darwin University Menzies School of Health Research, Channel 7 Children's Research Foundation, and National Health Institute.

T-follicular helper (Tfh) cells are key drivers of antibodies that protect from malaria. However, little is known regarding the host and parasite factors that influence Tfh and functional antibody development. Here, we use samples from a large cross-sectional study of children residing in an area of high malaria transmission in Uganda to characterize Tfh cells and functional antibodies to multiple parasites stages. We identify a dramatic re-distribution of the Tfh cell compartment with age that is independent of malaria exposure, with Th2-Tfh cells predominating in early childhood, while Th1-Tfh cell gradually increase to adult levels over the first decade of life. Functional antibody acquisition is age-dependent and hierarchical acquired based on parasite stage, with merozoite responses followed by sporozoite and gametocyte antibodies. Antibodies are boosted in children with current infection, and are higher in females. The children with the very highest antibody levels have increased Tfh cell activation and proliferation, consistent with a key role of Tfh cells in antibody development. Together, these data reveal a complex relationship between the circulating Tfh compartment, antibody development and protection from malaria.

Plasma ACE2 activity is persistently elevated in patients after COVID-19 infection. Larger studies are needed to determine if this identifies people at risk of prolonged illness following COVID-19. https://bit.ly/2XQlrYF

Morbidity and mortality rates from seasonal and pandemic influenza occur disproportionately in high-risk groups, including Indigenous people globally. Although vaccination against influenza is recommended for those most at risk, studies on immune responses elicited by seasonal vaccines in Indigenous populations are largely missing, with no data available for Indigenous Australians and only one report published on antibody responses in Indigenous Canadians. We recruited 78 Indigenous and 84 non-Indigenous Australians vaccinated with the quadrivalent influenza vaccine into the Looking into InFluenza T cell immunity - Vaccination cohort study and collected blood to define baseline, early (day 7), and memory (day 28) immune responses. We performed in-depth analyses of T and B cell activation, formation of memory B cells, and antibody profiles and investigated host factors that could contribute to vaccine responses. We found activation profiles of circulating T follicular helper type-1 cells at the early stage correlated strongly with the total change in antibody titers induced by vaccination. Formation of influenza-specific hemagglutinin-binding memory B cells was significantly higher in seroconverters compared with nonseroconverters. In-depth antibody characterization revealed a reduction in immunoglobulin G3 before and after vaccination in the Indigenous Australian population, potentially linked to the increased frequency of the G3m21* allotype. Overall, our data provide evidence that Indigenous populations elicit robust, broad, and prototypical immune responses following immunization with seasonal inactivated influenza vaccines. Our work strongly supports the recommendation of influenza vaccination to protect Indigenous populations from severe seasonal influenza virus infections and their subsequent complications.

BACKGROUND: As vaccines against SARS-CoV-2 are now being rolled out, a better understanding of immunity to the virus, whether from infection, or passive or active immunisation, and the durability of this protection is required. This will benefit from the ability to measure antibody-based protection to SARS-CoV-2, ideally with rapid turnaround and without the need for laboratory-based testing. METHODS: We have developed a lateral flow POC test that can measure levels of RBD-ACE2 neutralising antibody (NAb) from whole blood, with a result that can be determined by eye or quantitatively on a small instrument. We compared our lateral flow test with the gold-standard microneutralisation assay, using samples from convalescent and vaccinated donors, as well as immunised macaques. FINDINGS: We show a high correlation between our lateral flow test with conventional neutralisation and that this test is applicable with animal samples. We also show that this assay is readily adaptable to test for protection to newly emerging SARS-CoV-2 variants, including the beta variant which revealed a marked reduction in NAb activity. Lastly, using a cohort of vaccinated humans, we demonstrate that our whole-blood test correlates closely with microneutralisation assay data (specificity 100% and sensitivity 96% at a microneutralisation cutoff of 1:40) and that fingerprick whole blood samples are sufficient for this test. INTERPRETATION: Taken together, the COVID-19 NAb-testTM device described here provides a rapid readout of NAb based protection to SARS-CoV-2 at the point of care. FUNDING: Support was received from the Victorian Operational Infrastructure Support Program and the Australian Government Department of Health. This work was supported by grants from the Department of Health and Human Services of the Victorian State Government; the ARC (CE140100011, CE140100036), the NHMRC (1113293, 2002317 and 1116530), and Medical Research Future Fund Awards (2005544, 2002073, 2002132). Individua

BACKGROUND: Diagnostic tests for allergy rely on detecting allergen-specific IgE. Component-resolved diagnostics incorporate multiple defined allergen components to improve the quality of diagnosis and patient care. OBJECTIVE: To develop a new approach for determining sensitization to specific allergen components that utilizes fluorescent protein tetramers for direct staining of IgE on blood basophils by flow cytometry. METHODS: Recombinant forms of Lol p 1 and Lol p 5 proteins from ryegrass pollen (RGP) and Api m 1 from honeybee venom (BV) were produced, biotinylated, and tetramerized with streptavidin-fluorochrome conjugates. Blood samples from 50 RGP-allergic, 41 BV-allergic, and 26 controls were incubated with fluorescent protein tetramers for flow cytometric evaluation of basophil allergen binding and activation. RESULTS: Allergen tetramers bound to and activated basophils from relevant allergic patients but not controls. Direct fluorescence staining of Api m 1 and Lol p 1 tetramers had greater positive predictive values than basophil activation for BV and RGP allergy, respectively, as defined with receiver operator characteristics (ROC) curves. Staining intensities of allergen tetramers correlated with allergen-specific IgE levels in serum. Inclusion of multiple allergens coupled with distinct fluorochromes in a single-tube assay enabled rapid detection of sensitization to both Lol p 1 and Lol p 5 in RGP-allergic patients and discriminated between controls, BV-allergic, and RGP-allergic patients. CONCLUSION: Our novel flow cytometric assay, termed CytoBas, enables rapid and reliable detection of clinically relevant allergic sensitization. The intensity of fluorescent allergen tetramer staining of basophils has a high positive predictive value for disease, and the assay can be multiplexed for a component-resolved and differential diagnostic test for allergy.

BACKGROUND: Plasmodium falciparum causes placental malaria, which results in adverse outcomes for mother and child. P. falciparum-infected erythrocytes that express the parasite protein VAR2CSA on their surface can bind to placental chondroitin sulfate A. It has been hypothesized that naturally acquired antibodies towards VAR2CSA protect against placental infection, but it has proven difficult to identify robust antibody correlates of protection from disease. The objective of this study was to develop a prediction model using antibody features that could identify women protected from placental malaria. METHODS: We used a systems serology approach with elastic net-regularized logistic regression, partial least squares discriminant analysis, and a case-control study design to identify naturally acquired antibody features mid-pregnancy that were associated with protection from placental malaria at delivery in a cohort of 77 pregnant women from Madang, Papua New Guinea. RESULTS: The machine learning techniques selected 6 out of 169 measured antibody features towards VAR2CSA that could predict (with 86% accuracy) whether a woman would subsequently have active placental malaria infection at delivery. Selected features included previously described associations with inhibition of placental binding and/or opsonic phagocytosis of infected erythrocytes, and network analysis indicated that there are not one but multiple pathways to protection from placental malaria. CONCLUSIONS: We have identified candidate antibody features that could accurately identify malaria-infected women as protected from placental infection. It is likely that there are multiple pathways to protection against placental malaria. FUNDING: This study was supported by the National Health and Medical Research Council (Nos. APP1143946, GNT1145303, APP1092789, APP1140509, and APP1104975).

Lasting immunity following SARS-CoV-2 infection is questioned because serum antibodies decline in convalescence. However, functional immunity is mediated by long-lived memory T and B (Bmem) cells. Therefore, we generated fluorescently-labeled tetramers of the spike receptor binding domain (RBD) and nucleocapsid protein (NCP) to determine the longevity and immunophenotype of SARS-CoV-2-specific Bmem cells in COVID-19 patients. A total of 36 blood samples were obtained from 25 COVID-19 patients between 4 and 242 days post-symptom onset including 11 paired samples. While serum IgG to RBD and NCP was identified in all patients, antibody levels began declining at 20 days post-symptom onset. RBD- and NCP-specific Bmem cells predominantly expressed IgM+ or IgG1+ and continued to rise until 150 days. RBD-specific IgG+ Bmem were predominantly CD27+, and numbers significantly correlated with circulating follicular helper T cell numbers. Thus, the SARS-CoV-2 antibody response contracts in convalescence with persistence of RBD- and NCP-specific Bmem cells. Flow cytometric detection of SARS-CoV-2-specific Bmem cells enables detection of long-term immune memory following infection or vaccination for COVID-19.

BACKGROUND: Annual influenza vaccination is recommended to all individuals over 6 months of age, including predominantly antibody deficiency (PAD) patients. Vaccination responses are typically evaluated by serology, and because PAD patients are by definition impaired in generating IgG and receive immunoglobulin replacement therapy (IgRT), it remains unclear whether they can mount an antigen-specific response. OBJECTIVE: To quantify and characterise the antigen-specific memory B (Bmem) cell compartment in healthy controls and PAD patients following an influenza booster vaccination. METHODS: Recombinant hemagglutinin (HA) from the A/Michigan/2015 H1N1 (AM15) strain with an AviTag was generated in a mammalian cell line, and following targeted biotinylation, was tetramerised with BUV395 or BUV737 streptavidin conjugates. Multicolour flow cytometry was applied on blood samples before and 28 days after booster influenza vaccination in 16 healthy controls and five PAD patients with circulating Bmem cells. RESULTS: Recombinant HA tetramers were specifically recognised by 0.5-1% of B cells in previously vaccinated healthy adults. HA-specific Bmem cell numbers were significantly increased following booster vaccination and predominantly expressed IgG1. Similarly, PAD patients carried HA-specific Bmem cells, predominantly expressing IgG1. However, these numbers were lower than in controls and did not increase following booster vaccination. CONCLUSION: We have successfully identified AM15-specific Bmem cells in healthy controls and PAD patients. The presence of antigen-specific Bmem cells could offer an additional diagnostic tool to aid in the clinical diagnosis of PAD. Furthermore, alterations in the number or immunophenotype of HA-specific Bmem cells post-booster vaccination could assist in the evaluation of immune responses in individuals receiving IgRT.

CD4+ T follicular helper cells (Tfh) are key drivers of antibody development. During Plasmodium falciparum malaria in children, the activation of Tfh is restricted to the Th1 subset and not associated with antibody levels. To identify Tfh subsets that are associated with antibody development in malaria, we assess Tfh and antibodies longitudinally in human volunteers with experimental P. falciparum infection. Tfh cells activate during infection, with distinct dynamics in different Tfh subsets. Th2-Tfh cells activate early, during peak infection, while Th1-Tfh cells activate 1 week after peak infection and treatment. Th2-Tfh cell activation is associated with the functional breadth and magnitude of parasite antibodies. In contrast, Th1-Tfh activation is not associated with antibody development but instead with plasma cells, which have previously been shown to play a detrimental role in the development of long-lived immunity. Thus, our study identifies the contrasting roles of Th2 and Th1-Tfh cells during experimental P. falciparum malaria.

A considerable body of evidence suggests that Fc-dependent functions improve the capacity of broadly neutralizing antibodies (BnAbs) to protect against and control HIV-1 infection. This phenomenon, however, has not been formally tested in robust cell-associated macaque simian-human immunodeficiency virus (SHIV) models with newer-generation BnAbs. We studied both the WT BnAb PGT121 and a LALA mutant of PGT121 (which has impaired Fc-dependent functions) for their ability to protect pigtail macaques from an i.v. high-dose cell-associated SHIVSF162P3 challenge. We found that both WT and LALA PGT121 completely protected all 12 macaques studied. Further, partial depletion of NK cells, key mediators of Fc-dependent functions, did not abrogate the protective efficacy of PGT121 in 6 macaques. Additionally, in animals with established SHIVSF162P3 infection, SHIV viremia levels were equally rapidly reduced by LALA and WT PGT121. Our studies suggest that the potent neutralizing capacity of PGT121 renders the Fc-dependent functions of the Ab at least partially redundant. These findings have implications for Ab-mediated protection from and control of HIV-1 infection.

Antibody-based therapy in acute myeloid leukemia (AML) has been marred by significant hematologic toxicity due to targeting of both hematopoietic stem and progenitor cells (HSPCs). Achieving greater success with therapeutic antibodies requires careful characterization of the potential target molecules on AML. One potential target is CD300f, which is an immunoregulatory molecule expressed predominantly on myeloid lineage cells. To confirm the value of CD300f as a leukemic target, we showed that CD300f antibodies bind to AML from 85% of patient samples. While one CD300f monoclonal antibody (mAb) reportedly did not bind healthy hematopoietic stem cells, transcriptomic analysis found that CD300f transcripts are expressed by healthy HSPC. Several CD300f protein isoforms exist as a result of alternative splicing. Importantly for antibody targeting, the extracellular region of CD300f can be present with or without the exon 4-encoded sequence. This results in CD300f isoforms that are differentially bound by CD300f-specific antibodies. Furthermore, binding of one mAb, DCR-2, to CD300f exposes a structural epitope recognized by a second CD300f mAb, UP-D2. Detailed analysis of publicly available transcriptomic data indicated that CD34+ HSPC expressed fewer CD300f transcripts that lacked exon 4 compared to AML with monocytic differentiation. Analysis of a small cohort of AML cells revealed that the UP-D2 conformational binding site could be induced in cells from AML patients with monocytic differentiation but not those from other AML or HSPC. This provides the opportunity to develop an antibody-based strategy to target AMLs with monocytic differentiation but not healthy CD34+ HSPCs. This would be a major step forward in developing effective anti-AML therapeutic antibodies with reduced hematologic toxicity.

Rationale: Platelets are increasingly recognized as mediators of tumor growth and metastasis. Hypothesizing that activated platelets in the tumor microenvironment provide a targeting epitope for tumor-directed chemotherapy, we developed an antibody-drug conjugate (ADC), comprised of a single-chain antibody (scFv) against the platelet integrin GPIIb/IIIa (scFvGPIIb/IIIa) linked to the potent chemotherapeutic microtubule inhibitor, monomethyl auristatin E (MMAE). Methods: We developed an ADC comprised of three components: 1) A scFv which specifically binds to the high affinity, activated integrin GPIIb/IIIa on activated platelets. 2) A highly potent microtubule inhibitor, monomethyl auristatin E. 3) A drug activation/release mechanism using a linker cleavable by cathepsin B, which we demonstrate to be abundant in the tumor microenvironment. The scFvGPIIb/IIIa-MMAE was first conjugated with Cyanine7 for in vivo imaging. The therapeutic efficacy of the scFvGPIIb/IIIa-MMAE was then tested in a mouse metastasis model of triple negative breast cancer. Results: In vitro studies confirmed that this ADC specifically binds to activated GPIIb/IIIa, and cathepsin B-mediated drug release/activation resulted in tumor cytotoxicity. In vivo fluorescence imaging demonstrated that the newly generated ADC localized to primary tumors and metastases in a mouse xenograft model of triple negative breast cancer, a difficult to treat tumor for which a selective tumor-targeting therapy remains to be clinically established. Importantly, we demonstrated that the scFvGPIIb/IIIa-MMAE displays marked efficacy as an anti-cancer agent, reducing tumor growth and preventing metastatic disease, without any discernible toxic effects. Conclusion: Here, we demonstrate the utility of a novel ADC that targets a potent cytotoxic drug to activated platelets and specifically releases the cytotoxic agent within the confines of the tumor. This unique targeting mechanism, specific to the tumor microenvironment, h

Background: There is growing interest in immune therapies to clear the latent HIV-1 after combination antiretroviral therapy (cART). There is limited information on the effect of cART on antibody-dependent cellular cytotoxicity (ADCC), and no studies have directly compared ADCC in HIV-1 subtype B- and subtype C-infected subjects. The effect of improving immunocompetence on ADCC to influenza also remains unexplored. Methods: The effect of cART on HIV-1- and influenza-specific ADCC was analyzed in 2 cohorts (39 subtype B- and 47 subtype C-infected subjects) before and after 2 years of cART. ADCC analyses included an enzyme-linked immunosorbent assay-based dimeric recombinant soluble (rs) FcγRIIIa-binding assay, antibody-dependent natural killer cell activation assay, and ADCC-mediated killing assays. Results: HIV-1 subtype B and C Env-specific antibody binding to dimeric rsFcγRIIIa were reduced in subtypes B- and C-infected cohorts after 2 years of cART (both P < 0.05). Reduced ADCC-mediated killing of target cells expressing subtype B Env in the subtype B-infected cohort (P = 0.003) was observed after 96 weeks of cART, but not of subtype C Env in the subtype C-infected cohort. A greater reduction in ADCC was detected in subjects with baseline CD4 counts >300 cells/L (P < 0.05). The resolving immunodeficiency after 96 weeks of cART resulted in improved HA-specific ADCC to 6 strains of influenza (all P < 0.01). Conclusions: cART results in HIV-1 antigen loss and reductions in HIV-1 Env-specific antibodies with Fc functionality in both subtype B- and C-infected subjects, particularly in immunocompetent subjects. Simultaneously, cART improves ADCC to diverse strains of influenza, suggesting reduction in influenza disease after cART.

Broadly neutralizing antibodies (BnAbs) protect macaques from cell-free simian/human immunodeficiency virus (SHIV) challenge, but their efficacy against cell-associated SHIV is unclear. Virus in cell-associated format is highly infectious, present in transmission-competent bodily fluids, and potentially capable of evading antibody-mediated neutralization. The PGT121 BnAb, which recognizes an epitope consisting of the V3 loop and envelope glycans, mediates antibody-dependent cellular cytotoxicity and neutralization of cell-to-cell HIV-1 transmission. To evaluate whether a BnAb can prevent infection after cell-associated viral challenge, we infused pigtail macaques with PGT121 or an isotype control and challenged animals 1 hour later intravenously with SHIVSF162P3-infected splenocytes. All five controls had high viremia 1week after challenge. Three of six PGT121-infused animals were completely protected, two of six animals had a 1-week delay in onset of high viremia, and one animal had a 7-week delay in onset of viremia. The infused antibody had decayed on average to 2.0 mg/ml by 1 week after infusion and was well below 1 mg/ml (range, <0.1 to 0.8 mg/ml) by 8 weeks. The animals with a 1-week delay before high viremia had relatively lower plasma concentrations of PGT121. Transfer of 22 million peripheral bloodmononuclear cells (PBMCs) stored at weeks 1 to 4 fromthe animal with the 7-week delayed onset of viremia into uninfected macaques did not initiate infection. Our results show that HIV-1-specific neutralizing antibodies have partial efficacy against cell-associated virus exposure in macaques. We conclude that sustaining high concentrations of bioavailable BnAb is important for protecting against cellassociated virus.

Rationale The early detection of primary tumours and metastatic disease is vital for successful therapy and is contingent upon highly specific molecular markers and sensitive, non-invasive imaging techniques. We hypothesized that the accumulation of activated platelets within tumours is a general phenomenon and thus represents a novel means for the molecular imaging of cancer. Here we investigate a unique single chain antibody (scFv), which specifically targets activated platelets, as a novel biotechnological tool for molecular imaging of cancer. Methods The scFvGPIIb/IIIa, which binds specifically to the activated form of the platelet integrin receptor GPIIb/IIIa present on activated platelets, was conjugated to either Cy7, 64Cu or ultrasound-enhancing microbubbles. Using the Cy7 labelled scFvGPIIb/IIIa, fluorescence imaging was performed in mice bearing four different human tumour xenograft models; SKBr3, MDA-MB-231, Ramos and HT-1080 cells. Molecular imaging via PET and ultrasound was performed using the scFvGPIIb/IIIa-64Cu and scFvGPIIb/IIIa-microbubbles, respectively, to further confirm specific targeting of scFvGPIIb/IIIa to activated platelets in the tumour stroma. Results Using scFvGPIIb/IIIa we successfully showed specific targeting of activated platelets within the microenvironment of human tumour xenografts models via three different molecular imaging modalities. The presence of platelets within the tumour microenvironment, and as such their relevance as a molecular target epitope in cancer was further confirmed via immunofluorescence of human tumour sections of various cancer types, thus validating the translational importance of our novel approach to human disease. Conclusion Our study provides proof of concept for imaging and localization of tumours by molecular targeting activated platelets. We illustrate the utility of a unique scFv as a versatile biotechnological tool which can be conjugated to various contrast agents for molecular imaging of cancer

OBJECTIVE: The objective of the study was to profile leukocyte markers modulated during intravenous immunoglobulin (IVIg) treatment, and to identify markers and immune pathways associated with clinical efficacy of IVIg for chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) with potential for monitoring treatment efficacy. METHODS: Response to IVIg treatment in newly diagnosed IVIg-naïve and established IVIg-experienced patients was assessed by changes in expression of inflammatory leukocyte markers by flow cytometry. The adjusted INCAT disability and Medical Research Council sum scores defined clinical response. RESULTS: Intravenous immunoglobulin modulated immunopathogenic pathways associated with inflammatory disease in CIDP. Leukocyte markers of clinical efficacy included reduced CD185+ follicular helper T cells, increased regulatory markers (CD23 and CD72) on B cells, and reduction in the circulating inflammatory CD16+ myeloid dendritic cell (mDC) population and concomitant increase in CD62L and CD195 defining a less inflammatory lymphoid homing mDC phenotype. A decline in inflammatory CD16+ dendritic cells was associated with clinical improvement or stability, and correlated with magnitude of improvement in neurological assessment scores, but did not predict relapse. IVIg also induced a nonspecific improvement in regulatory and reduced inflammatory markers not associated with clinical response. CONCLUSIONS: Clinically effective IVIg modulated inflammatory and regulatory pathways associated with ongoing control or resolution of CIDP disease. Some of these markers have potential for monitoring outcome.

A recombinant soluble form of human Fc gamma RII (rsFc gamma RII) was genetically engineered by the insertion of a termination codon 5' of sequences encoding the transmembrane domain of a human Fc gamma RII cDNA. Chinese hamster ovary cells were transfected with the modified cDNA and the secreted rsFc gamma RII purified from the tissue culture supernatant (to > 95%, assessed by SDS-PAGE) using heat aggregated human immunoglobulin G (IgG) immunoaffinity chromatography. The IgG-purified rsFc gamma RII was relatively homogeneous (approximately 31,000 M(r)) whereas the total unpurified rsFc gamma RII secreted into the tissue culture supernatant was heterogeneous relating to N-linked glycosylation differences. Functional in vitro activity of the rsFc gamma RII was demonstrated by: (a) ability to bind via the Fc portion of human IgG and mouse IgG (IgG2a > IgG1 > > IgG2b); (b) complete inhibition of binding of erythrocytes sensitized with rabbit IgG to membrane-bound Fc gamma RII on K562 cells; and (c) inhibition of the anti-Leu4-induced T cell proliferation assay. Blood clearance and biodistribution studies show the rsFc gamma RII was excreted predominantly through the kidney in a biphasic manner, with an alpha-phase (t1/2 approximately 25 min) and a beta-phase (t1/2 approximately 4.6 h); the kidneys were the only organs noted with tissue-specific accumulation. In vivo, the administration of rsFc gamma RII significantly inhibited the immune complex-mediated inflammatory response induced by the reversed passive Arthus reaction model in rats. There was a specific and dose-dependent relationship between the amount of rsFc gamma RII administered, and the reduction in the size and severity of the macroscopic inflammatory lesion. Histological analysis of the skin showed a diffuse neutrophil infiltrate in both control and rsFc gamma RII-treated rats, however the perivascular infiltrate and the red cell extravasation was less intense in the rsFc gamma RII-treated group. It is lik

The Ly phenotype of cells mediating skin graft rejection was determined using monoclonal anti-Lyt-1.1 and Lyt-2.1 antibodies in CBA mice that received CBA lymphoid cells from mice sensitized to C57BL/6; i.e., alloantigenic differences arising from the H-2 and non-H-2 loci. It was clear that graft rejection was due wholly to the presence of Lyt-1 cells in the inoculum and that Lyt-123 or Lyt-23 cells had no effect. Furthermore, no synergism was noted between Lyt-1 and Lyt-2 cells. In this model, both the cytotoxic T cell and cytotoxic lymphocyte precursors were shown to be Lyt-123 and these could be depleted from sensitized Lyt-1 populations that mediated graft rejection. Thus cytotoxic T cells are not responsible for skin graft rejection, but rather, this is mediated by an Lyt-1 cell. Whether this T cell is distinct from other Lyt-1 cells (T helper, T cells mediating delayed hypersensitivity) is not clear at present, but other evidence, and traditional concepts, link graft rejection and delayed type hypersensitivity as being different manifestations of the same mechanism.