Immunogenicity of COVID-19 vaccines in patients with follicular lymphoma receiving frontline chemoimmunotherapy.

Immune responses to primary COVID-19 vaccination were investigated in 58 patients with follicular lymphoma (FL) as part of the PETReA trial of frontline therapy (EudraCT 2016-004010-10). COVID-19 vaccines (BNT162b2 or ChAdOx1) were administered before, during or after cytoreductive treatment comprising rituximab (depletes B cells) and either bendamustine (depletes CD4+ T cells) or cyclophosphamide-based chemotherapy. Blood samples obtained after vaccine doses 1 and 2 (V1, V2) were analysed for antibodies and T cells reactive to the SARS-CoV-2 spike protein using the Abbott Architect and interferon-gamma ELISpot assays respectively. Compared to 149 healthy controls, patients with FL exhibited lower antibody but preserved T-cell responses. Within the FL cohort, multivariable analysis identified low pre-treatment serum IgA levels and V2 administration during induction or maintenance treatment as independent determinants of lower antibody and higher T-cell responses, and bendamustine and high/intermediate FLIPI-2 score as additional determinants of a lower antibody response. Several clinical scenarios were identified where dichotomous immune responses were estimated with >95% confidence based on combinations of predictive variables. In conclusion, the immunogenicity of COVID-19 vaccines in FL patients is influenced by multiple disease- and treatment-related factors, among which B-cell depletion showed differential effects on antibody and T-cell responses.

Obesity Differs from Diabetes Mellitus in Antibody and T Cell Responses Post COVID-19 Recovery.

Obesity and type 2 diabetes (DM) are risk factors for severe COVID-19 outcomes, which disproportionately affect South Asian populations. This study aims to investigate the humoral and cellular immune responses to SARS-CoV-2 in adult COVID-19 survivors with obesity and DM in Bangladesh. In this cross-sectional study, SARS-CoV-2-specific antibody and T cell responses were investigated in 63 healthy and 75 PCR-confirmed COVID-19 recovered individuals in Bangladesh, during the pre-vaccination first wave of the COVID-19 pandemic in 2020. In COVID-19 survivors, SARS-CoV-2 infection induced robust antibody and T cell responses, which correlated with disease severity. After adjusting for age, sex, DM status, disease severity, and time since onset of symptoms, obesity was associated with decreased neutralising antibody titers, and increased SARS-CoV-2 spike-specific IFN-γ response along with increased proliferation and IL-2 production by CD8+ T cells. In contrast, DM was not associated with SARS-CoV-2-specific antibody and T cell responses after adjustment for obesity and other confounders. Obesity is associated with lower neutralising antibody levels and higher T cell responses to SARS-CoV-2 post COVID-19 recovery, while antibody or T cell responses remain unaltered in DM.

Diabetes and risk of hospitalisation due to infection in northeastern Thailand: Retrospective cohort study using population-based healthcare service data.

BACKGROUND: Population-based studies describing the association between diabetes and increased risk of infection have largely been based in high-income countries. There is limited information describing the burden of infectious disease attributable to diabetes in low and middle-income countries. This study aimed to describe the burden and risk of infectious disease hospitalisation in people with diabetes compared to those without diabetes in northeastern Thailand. METHODS: In a retrospective cohort study using electronic health record data for 2012-2018 for 3.8 million people aged ≥20 years in northeastern Thailand, hospitalisation rates for any infectious diseases (ICD-10 codes A00-B99) were estimated and negative binomial regression used to estimate rate ratios (RR) for the association between diabetes and infectious disease hospitalisation adjusted for age, sex and area of residence. RESULTS: In this study, 164,177 people had a diagnosis of diabetes mellitus at any point over the study period. Infectious disease hospitalisation rates per 1000 person-years (95%CI) were 71.8 (70.9, 72.8), 27.7 (27.1, 28.3) and 7.5 (7.5, 7.5) for people with prevalent diabetes, incident diabetes and those without diabetes respectively. Diabetes was associated with a 4.6-fold higher risk of infectious disease hospitalisation (RR (95% CI) 4.59 (4.52, 4.66)). RRs for infectious disease hospitalisation were 3.38 (3.29, 3.47) for people with diabetes managed by lifestyle alone and 5.29 (5.20, 5.39) for people receiving prescriptions for diabetes drugs. CONCLUSIONS: In this Thai population, diabetes was associated with substantially increased risk of hospitalisation due to infectious diseases and people with diabetes who were on pharmacological treatment had a higher risk than those receiving lifestyle modification advice alone.

Anti-SARS-CoV-2 antibody dynamics after primary vaccination with two-dose inactivated whole-virus vaccine, heterologous mRNA-1273 vaccine booster, and Omicron breakthrough infection in Indonesian health care workers.

BACKGROUND: Data on the dynamics and persistence of humoral immunity against SARS-CoV-2 after primary vaccination with two-dose inactivated vaccine (CoronaVac) are limited. This study evaluated the sequential effects of prior infection, heterologous boosting with mRNA-1273 (Moderna), and the occurrence of Omicron vaccine-breakthrough infection (VBI) thereafter. METHODS: We evaluated anti-spike IgG (Abbott) and neutralising (cPASS/GenScript) antibody (nAb) titers up to one year after mRNA-1273 boost in two-dose-CoronaVac-primed Indonesian healthcare workers (August 2021-August 2022). We used linear mixed modeling to estimate the rate of change in antibody levels, and logistic regression to examine associations between antibody levels and VBI. RESULTS: Of 138 participants, 52 (37.7%) had a prior infection and 78 (56.5%) received an mRNA-1273 booster. After two-dose CoronaVac, antibody titers had significantly declined within 180 days, irrespective of prior infection. After mRNA-1273 booster, anti-spike IgG (1.47% decline/day) and Omicron B.1.1.529/BA.2 nAbs declined between day 28-90, and IgG titers plateaued between day 90-360. During the BA.1/BA.2 wave (February-March 2022), 34.6% (27/78) of individuals experienced a VBI (median 181 days after mRNA-1273), although none developed severe illness. VBI was associated with low pre-VBI anti-spike IgG and B.1.1.529/BA.2 nAbs, which were restored post-VBI. CONCLUSIONS: mRNA-1273 booster after two-dose CoronaVac did not prevent BA.1/BA.2 VBI. Periodic vaccine boosters may be warranted against emerging SARS-CoV-2 variants.

Metabolic requirements of CD160 expressing memory-like NK cells in Gram-negative bacterial infection.

Objective: Unique metabolic requirements accompany the development and functional fates of immune cells. How cellular metabolism is important in natural killer (NK) cells and their memory-like differentiation in bacterial infections remains elusive. Methods: Here, we utilise our established NK cell memory assay to investigate the metabolic requirement for memory-like NK cell formation and function in response to the Gram-negative intracellular bacteria Burkholderia pseudomallei (BP), the causative agent of melioidosis. Results: We demonstrate that CD160+ memory-like NK cells upon BP stimulation upregulate glucose and amino acid transporters in a cohort of recovered melioidosis patients which is maintained at least 3-month post-hospital admission. Using an in vitro assay, human BP-specific CD160+ memory-like NK cells show metabolic priming including increased expression of glucose and amino acid transporters with elevated glucose uptake, increased mTOR activation and mitochondrial membrane potential upon BP re-stimulation. Antigen-specific and cytokine-induced IFN-γ production of this memory-like NK cell subset are highly dependent on oxidative phosphorylation (OXPHOS) with some dependency on glycolysis, whereas the formation of CD160+ memory-like NK cells in vitro is dependent on fatty acid oxidation and OXPHOS and further increased by metformin. Conclusion: This study reveals the link between metabolism and cellular function of memory-like NK cells, which can be exploited for vaccine design and for monitoring protection against Gram-negative bacterial infection.

Concerted deletions eliminate a neutralizing supersite in SARS-CoV-2 BA.2.87.1 spike.

BA.2.87.1 represents a major shift in the BA.2 lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is unusual in having two lengthy deletions of polypeptide in the spike (S) protein, one of which removes a beta-strand. Here we investigate its neutralization by a variety of sera from infected and vaccinated individuals and determine its spike (S) ectodomain structure. The BA.2.87.1 receptor binding domain (RBD) is structurally conserved and the RBDs are tightly packed in an "all-down" conformation with a small rotation relative to the trimer axis as compared to the closest previously observed conformation. The N-terminal domain (NTD) maintains a remarkably similar structure overall; however, the rearrangements resulting from the deletions essentially destroy the so-called supersite epitope and eliminate one glycan site, while a mutation creates an additional glycan site, effectively shielding another NTD epitope. BA.2.87.1 is relatively easily neutralized but acquisition of additional mutations in the RBD could increase antibody escape allowing it to become a dominant sub-lineage.

Incident dementia risk among patients with type 2 diabetes receiving metformin versus alternative oral glucose-lowering therapy: an observational cohort study using UK primary healthcare records.

INTRODUCTION: 4.2 million individuals in the UK have type 2 diabetes, a known risk factor for dementia and mild cognitive impairment (MCI). Diabetes treatment may modify this association, but existing evidence is conflicting. We therefore aimed to assess the association between metformin therapy and risk of incident all-cause dementia or MCI compared with other oral glucose-lowering therapies (GLTs). RESEARCH DESIGN AND METHODS: We conducted an observational cohort study using the Clinical Practice Research Datalink among UK adults diagnosed with diabetes at ≥40 years between 1990 and 2019. We used an active comparator new user design to compare risks of dementia and MCI among individuals initially prescribed metformin versus an alternative oral GLT using Cox proportional hazards regression controlling for sociodemographic, lifestyle and clinical confounders. We assessed for interaction by age and sex. Sensitivity analyses included an as-treated analysis to mitigate potential exposure misclassification. RESULTS: We included 211 396 individuals (median age 63 years; 42.8% female), of whom 179 333 (84.8%) initiated on metformin therapy. Over median follow-up of 5.4 years, metformin use was associated with a lower risk of dementia (adjusted HR (aHR) 0.86 (95% CI 0.79 to 0.94)) and MCI (aHR 0.92 (95% CI 0.86 to 0.99)). Metformin users aged under 80 years had a lower dementia risk (aHR 0.77 (95% CI 0.68 to 0.85)), which was not observed for those aged ≥80 years (aHR 0.95 (95% CI 0.87 to 1.05)). There was no interaction with sex. The as-treated analysis showed a reduced effect size compared with the main analysis (aHR 0.90 (95% CI 0.83 to 0.98)). CONCLUSIONS: Metformin use was associated with lower risks of incident dementia and MCI compared with alternative GLT among UK adults with diabetes. While our findings are consistent with a neuroprotective effect of metformin against dementia, further research is needed to reduce risks of confounding by indication and assess causality.

Repurposing rapid diagnostic tests to detect falsified vaccines in supply chains.

Substandard (including degraded) and falsified (SF) vaccines are a relatively neglected issue with serious global implications for public health. This has been highlighted during the rapid and widespread rollout of COVID-19 vaccines. There has been increasing interest in devices to screen for SF non-vaccine medicines including tablets and capsules to empower inspectors and standardise surveillance. However, there has been very limited published research focussed on repurposing or developing new devices for screening for SF vaccines. To our knowledge, rapid diagnostic tests (RDTs) have not been used for this purpose but have important potential for detecting falsified vaccines. We performed a proof-in-principle study to investigate their diagnostic accuracy using a diverse range of RDT-vaccine/falsified vaccine surrogate pairs. In an initial assessment, we demonstrated the utility of four RDTs in detecting seven vaccines. Subsequently, the four RDTs were evaluated by three blinded assessors with seven vaccines and four falsified vaccines surrogates. The results provide preliminary data that RDTs could be used by multiple international organisations, national medicines regulators and vaccine manufacturers/distributors to screen for falsified vaccines in supply chains, aligned with the WHO global 'Prevent, Detect and Respond' strategy.

The SARS-CoV-2 neutralizing antibody response to SD1 and its evasion by BA.2.86.

Under pressure from neutralising antibodies induced by vaccination or infection the SARS-CoV-2 spike gene has become a hotspot for evolutionary change, leading to the failure of all mAbs developed for clinical use. Most potent antibodies bind to the receptor binding domain which has become heavily mutated. Here we study responses to a conserved epitope in sub-domain-1 (SD1) of spike which have become more prominent because of mutational escape from antibodies directed to the receptor binding domain. Some SD1 reactive mAbs show potent and broad neutralization of SARS-CoV-2 variants. We structurally map the dominant SD1 epitope and provide a mechanism of action by blocking interaction with ACE2. Mutations in SD1 have not been sustained to date, but one, E554K, leads to escape from mAbs. This mutation has now emerged in several sublineages including BA.2.86, reflecting selection pressure on the virus exerted by the increasing prominence of the anti-SD1 response.

Dysregulated immunologic landscape of early host response in melioidosis.

Melioidosis, a neglected tropical infection caused by Burkholderia pseudomallei, commonly presents as pneumonia or sepsis with mortality rates up to 50% despite appropriate treatment. A better understanding of the early host immune response to melioidosis may lead to new therapeutic interventions and prognostication strategies to reduce disease burden. Whole blood transcriptomic signatures in 164 melioidosis patients and 70 patients with other infections hospitalized in northeastern Thailand enrolled within 24 hours following hospital admission were studied. Key findings were validated in an independent melioidosis cohort. Melioidosis was characterized by upregulation of interferon signaling responses compared to other infections. Mortality in melioidosis was associated with excessive inflammation, up-regulated type 2 immune responses and a dramatic decrease in T cell-mediated immunity compared to survivors. We identified and independently confirmed a five-gene predictive set classifying fatal melioidosis (validation cohort: an area under the receiver operating characteristic curve 0.83, 95% CI: 0.67-0.99). In conclusion, this study highlights the intricate balance between innate and adaptive immunity during fatal melioidosis and can inform future precision medicine strategies for targeted therapies and prognostication in this severe infection.

Emerging variants develop total escape from potent monoclonal antibodies induced by BA.4/5 infection.

The rapid evolution of SARS-CoV-2 is driven in part by a need to evade the antibody response in the face of high levels of immunity. Here, we isolate spike (S) binding monoclonal antibodies (mAbs) from vaccinees who suffered vaccine break-through infections with Omicron sub lineages BA.4 or BA.5. Twenty eight potent antibodies are isolated and characterised functionally, and in some cases structurally. Since the emergence of BA.4/5, SARS-CoV-2 has continued to accrue mutations in the S protein, to understand this we characterize neutralization of a large panel of variants and demonstrate a steady attrition of neutralization by the panel of BA.4/5 mAbs culminating in total loss of function with recent XBB.1.5.70 variants containing the so-called 'FLip' mutations at positions 455 and 456. Interestingly, activity of some mAbs is regained on the recently reported variant BA.2.86.

Southeast Asia initiative to combat SARS-CoV-2 variants (SEACOVARIANTS) consortium.

A strong and effective COVID-19 and future pandemic responses rely on global efforts to carry out surveillance of infections and emerging SARS-CoV-2 variants and to act accordingly in real time. Many countries in Southeast Asia lack capacity to determine the potential threat of new variants, or other emerging infections. Funded by Wellcome, the Southeast Asia initiative to combat SARS-CoV-2 variants (SEACOVARIANTS) consortium aims to develop and apply a multidisciplinary research platform in Southeast Asia (SEA) for rapid assessment of the biological significance of SARS-CoV-2 variants, thereby informing coordinated local, regional and global responses to the COVID-19 pandemic. Our proposal is delivered by the Vietnam and Thailand Wellcome Africa Asia Programmes, bringing together a multidisciplinary team in Indonesia, Thailand and Vietnam with partners in Singapore, the UK and the USA. Herein we outline five work packages to deliver strengthened regional scientific capacity that can be rapidly deployed for future outbreak responses.

Our project strengthens local scientific capacity in South East Asia (SEA) and therefore enables the rapid assessment of SARS-CoV-2 variants as they emerge within the region. While COVID-19 remains a global pandemic, future emerging infections caused by a novel virus is an inevitable event, with SEA being a global hot-spot for pathogen emergence. Consequently, the research capacity built, the scientists trained and the research network formed as part of this project will lay the foundation for future locally-led outbreak responses. Our project will demonstrate that novel research platforms can be set up in other low and middle income countries to address the unprecedented challenges presented by emerging infections.

A structure-function analysis shows SARS-CoV-2 BA.2.86 balances antibody escape and ACE2 affinity.

BA.2.86, a recently described sublineage of SARS-CoV-2 Omicron, contains many mutations in the spike gene. It appears to have originated from BA.2 and is distinct from the XBB variants responsible for many infections in 2023. The global spread and plethora of mutations in BA.2.86 has caused concern that it may possess greater immune-evasive potential, leading to a new wave of infection. Here, we examine the ability of BA.2.86 to evade the antibody response to infection using a panel of vaccinated or naturally infected sera and find that it shows marginally less immune evasion than XBB.1.5. We locate BA.2.86 in the antigenic landscape of recent variants and look at its ability to escape panels of potent monoclonal antibodies generated against contemporary SARS-CoV-2 infections. We demonstrate, and provide a structural explanation for, increased affinity of BA.2.86 to ACE2, which may increase transmissibility.

Immunogenicity of Abdala COVID-19 vaccine in Vietnamese people after primary and booster vaccinations: a prospective observational study in Vietnam.

OBJECTIVES: We studied the immunogenicity after primary and booster vaccinations of Abdala COVID-19 vaccine, a receptor binding domain protein subunit vaccine, in Vietnamese people by determining the level of neutralization and cross-neutralization activities against the ancestral SARS-CoV-2 and its variants, and SARS-CoV-1. METHODS: We performed a prospective observational study, enrolling adults aged 19-59 years in Dong Thap province, southern Vietnam, and collected blood samples from baseline until 4 weeks post booster dose. We measured anti-nucleocapsid, anti-spike and neutralizing antibodies against SARS-CoV-2, and assessed the cross-neutralization against 14 SARS-CoV-2 variants, and SARS-CoV-1. Complementary antibody data came from Vietnamese healthcare workers fully vaccinated with ChAdOx1-S. RESULTS: After primary vaccination, anti-spike antibody and neutralizing antibodies were detectable in 98.4% and 87% of 251 study participants, respectively, with neutralizing antibody titers similar to that induced by ChAdOx1-S vaccine. Antibody responses after a homologous (Abdala COVID-19) or heterologous (mRNA BNT162b2) booster could neutralize 14 SARS-CoV-2 variants (including Omicron), and SARS-CoV-1. CONCLUSIONS: Abdala COVID-19 vaccine is immunogenic in Vietnamese people. Enhanced antibody response after a booster dose could cross-neutralize 14 SARS-CoV-2 variants and SARS-CoV-1. Our results have added to the growing body of knowledge about the contribution of protein subunit vaccine platforms to pandemic control.

Immunogenicity of third dose COVID-19 vaccine strategies in patients who are immunocompromised with suboptimal immunity following two doses (OCTAVE-DUO): an open-label, multicentre, randomised, controlled, phase 3 trial.

BACKGROUND: The humoral and T-cell responses to booster COVID-19 vaccine types in multidisease immunocompromised individuals who do not generate adequate antibody responses to two COVID-19 vaccine doses, is not fully understood. The OCTAVE DUO trial aimed to determine the value of third vaccinations in a wide range of patients with primary and secondary immunodeficiencies. METHODS: OCTAVE-DUO was a prospective, open-label, multicentre, randomised, controlled, phase 3 trial investigating humoral and T-cell responses in patients who are immunocompromised following a third vaccine dose with BNT162b2 or mRNA-1273, and of NVX-CoV2373 for those with lymphoid malignancies. We recruited patients who were immunocompromised from 11 UK hospitals, aged at least 18 years, with previous sub-optimal responses to two doses of SARS-CoV-2 vaccine. Participants were randomly assigned 1:1 (1:1:1 for those with lymphoid malignancies), stratified by disease, previous vaccination type, and anti-spike antibody response following two doses. Individuals with lived experience of immune susceptibility were involved in the study design and implementation. The primary outcome was vaccine-specific immunity defined by anti-SARS-CoV-2 spike antibodies (Roche Diagnostics UK and Ireland, Burgess Hill, UK) and T-cell responses (Oxford Immunotec, Abingdon, UK) before and 21 days after the third vaccine dose analysed by a modified intention-to-treat analysis. The trial is registered with the ISRCTN registry, ISRCTN 15354495, and the EU Clinical Trials Register, EudraCT 2021-003632-87, and is complete. FINDINGS: Between Aug 4, 2021 and Mar 31, 2022, 804 participants across nine disease cohorts were randomly assigned to receive BNT162b2 (n=377), mRNA-1273 (n=374), or NVX-CoV2373 (n=53). 356 (45%) of 789 participants were women, 433 (55%) were men, and 659 (85%) of 775 were White. Anti-SARS-CoV-2 spike antibodies measured 21 days after the third vaccine dose were significantly higher than baseline pre-third dose titres in the modified intention-to-treat analysis (median 1384 arbitrary units [AU]/mL [IQR 4·3-7990·0] compared with median 11·5 AU/mL [0·4-63·1]; p<0·001). Of participants who were baseline low responders, 380 (90%) of 423 increased their antibody concentrations to more than 400 AU/mL. Conversely, 166 (54%) of 308 baseline non-responders had no response after the third dose. Detectable T-cell responses following the third vaccine dose were seen in 494 (80%) of 616 participants. There were 24 serious adverse events (BNT612b2 eight [33%] of 24, mRNA-1273 12 [50%], NVX-CoV2373 four [17%]), two (8%) of which were categorised as vaccine-related. There were seven deaths (1%) during the trial, none of which were vaccine-related. INTERPRETATION: A third vaccine dose improved the serological and T-cell response in the majority of patients who are immunocompromised. Individuals with chronic renal disease, lymphoid malignancy, on B-cell targeted therapies, or with no serological response after two vaccine doses are at higher risk of poor response to a third vaccine dose. FUNDING: Medical Research Council, Blood Cancer UK.

Impact of shipping temperature on cell viability and T cell responses to bacterial antigens.

Background: Interferon-γ (IFN-γ) secretion by T cells is a key correlate of immune protection against many pathogens including tuberculosis and the neglected tropical disease melioidosis. Clinical studies in tropical regions of immune responses to pathogens and vaccine monitoring studies require the collection of samples in resource-limited rural areas and subsequent shipment to central laboratories for downstream assays and long-term storage. Here, we studied the impact of two different shipping temperatures on the viability, composition and function of peripheral blood mononuclear cells (PBMC) using multi-colour flow cytometry and IFN-γ enzyme-linked immunospot assay (IFN-γ ELISpot), in order to provide guidance on sample shipment conditions for future clinical studies. Methods: Paired peripheral blood mononuclear cell (PBMC) samples from recovered melioidosis patients were stored in liquid nitrogen (-196°C) and then shipped from Bangkok, Thailand to Oxford, UK at either -80°C (dry ice) or -196°C (dry shipper). After thawing, cell viability and composition were assessed by flow cytometry and antigen specific responses to Burkholderia pseudomallei (BP) were measured using IFN-γ ELISpot. Results: We observed modest lowering of viability in the majority of samples and a reduction in IFN-γ responses to BP which correlated to a decrease of monocytes and natural killer cells in samples shipped at -80°C compared to -196°C. Despite being lower in magnitude antigen-specific responses remained detectable in the majority of samples. Conclusions: Here we demonstrate that shipment of cryopreserved PBMC at -196°C has a benefit on cell viability, recovery and T cell responses to bacterial antigens, although useful information can still be obtained from samples shipped at -80°C, thus providing important guidance for sample management in future clinical trials.

Deciphering the human antibody response against Burkholderia pseudomallei during melioidosis using a comprehensive immunoproteome approach.

Introduction: The environmental bacterium Burkholderia pseudomallei causes the often fatal and massively underreported infectious disease melioidosis. Antigens inducing protective immunity in experimental models have recently been identified and serodiagnostic tools have been improved. However, further elucidation of the antigenic repertoire of B. pseudomallei during human infection for diagnostic and vaccine purposes is required. The adaptation of B. pseudomallei to very different habitats is reflected by a huge genome and a selective transcriptional response to a variety of conditions. We, therefore, hypothesized that exposure of B. pseudomallei to culture conditions mimicking habitats encountered in the human host might unravel novel antigens that are recognized by melioidosis patients. Methods and results: In this study, B. pseudomallei was exposed to various stress and growth conditions, including anaerobiosis, acid stress, oxidative stress, iron starvation and osmotic stress. Immunogenic proteins were identified by probing two-dimensional Western blots of B. pseudomallei intracellular and extracellular protein extracts with sera from melioidosis patients and controls and subsequent MALDI-TOF MS. Among B. pseudomallei specific immunogenic signals, 90 % (55/61) of extracellular immunogenic proteins were identified by acid, osmotic or oxidative stress. A total of 84 % (44/52) of intracellular antigens originated from the stationary growth phase, acidic, oxidative and anaerobic conditions. The majority of the extracellular and intracellular protein antigens were identified in only one of the various stress conditions. Sixty-three immunoreactive proteins and an additional 38 candidates from a literature screening were heterologously expressed and subjected to dot blot analysis using melioidosis sera and controls. Our experiments confirmed melioidosis-specific signals in 58 of our immunoproteome candidates. These include 15 antigens with average signal ratios (melioidosis:controls) greater than 10 and another 26 with average ratios greater than 5, including new promising serodiagnostic candidates with a very high signal-to-noise ratio. Conclusion: Our study shows that a comprehensive B. pseudomallei immunoproteomics approach, using conditions which are likely to be encountered during infection, can identify novel antibody targets previously unrecognized in human melioidosis.