Infants Harness the Germline against RSV.

In this issue of Immunity, Goodwin et al. (2018) offer hope for an RSV vaccine for young infants by demonstrating that RSV infection in very young infants induces neutralizing antibodies that are close to the germline and have unusual epitope specificity.

Immune imprinting and SARS-CoV-2 vaccine design.

Reformulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines with variant strains is being pursued to combat the global surge in infections. We hypothesize that this may be suboptimal due to immune imprinting from earlier vaccination or infection with the original SARS-CoV-2 strain. New strategies may be needed to improve efficacy of SARS-CoV-2 variant vaccines.

Influenza A(H1N1)pdm09 But Not A(H3N2) Virus Infection Induces Durable Seroprotection: Results From the Ha Nam Cohort.

BACKGROUND: The extent to which influenza recurrence depends upon waning immunity from prior infection is undefined. We used antibody titers of Ha-Nam cohort participants to estimate protection curves and decay trajectories. METHODS: Households (270) participated in influenza-like-illness (ILI) surveillance and provided blood at intervals spanning laboratory-confirmed virus transmission. Sera were tested in hemagglutination inhibition assay. Infection was defined as influenza virus-positive ILI and/or seroconversion. Median protective titers were estimated using scaled-logistic regression to model pretransmission titer against infection status in that season, limiting analysis to households with infection(s). Titers were modelled against month since infection using mixed-effects linear regression to estimate decay and when titers fell below protection thresholds. RESULTS: From December 2008-2012, 295 and 314 participants were infected with H1N1pdm09-like and A/Perth/16/09-like (H3N2Pe09) viruses, respectively. The proportion protected rose more steeply with titer for H1N1pdm09 than for H3N2Pe09, and estimated 50% protection titers were 19.6 and 37.3, respectively. Postinfection titers started higher against H3N2Pe09 but decayed more steeply than against H1N1pdm09. Seroprotection was estimated to be sustained against H1N1pdm09 but to wane by 8-months for H3N2Pe09. CONCLUSIONS: Estimates indicate that infection induces durable seroprotection against H1N1pdm09 but not H3N2Pe09, which could in part account for the younger age of A(H1N1) versus A(H3N2) cases.

Trend in Sensitivity of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Serology One Year After Mild and Asymptomatic Coronavirus Disease 2019 (COVID-19): Unpacking Potential Bias in Seroprevalence Studies.

A key aim of serosurveillance during the coronavirus disease 2019 (COVID-19) pandemic has been to estimate the prevalence of prior infection, by correcting crude seroprevalence against estimated test performance for polymerase chain reaction (PCR)-confirmed COVID-19. We show that poor generalizability of sensitivity estimates to some target populations may lead to substantial underestimation of case numbers.

Limited Phenotypic and Functional Plasticity of Influenza Virus-Specific Memory CD8+ T Cells during Activation in an Alternative Cytokine Environment.

Naive CD8+ T cells show phenotypic, functional, and epigenetic plasticity, enabling differentiation into distinct cellular states. However, whether memory CD8+ T cells demonstrate similar flexibility upon recall is poorly understood. We investigated the potential of influenza A virus (IAV)-specific memory CD8+ T cells from mice to alter their phenotype and function in response to reactivation in the presence of IL-4 and anti-IFN-γ Ab (type 2 conditions). Compared with naive CD8+ T cells, only a small proportion of IAV-specific memory T cells exhibited phenotypic and functional plasticity after clonal activation under type 2 conditions. The potential for modulation of cell-surface phenotype (CD8α expression) was associated with specific epigenetic changes at the Cd8a locus, was greater in central memory T cells than effector memory T cells, and was observed in endogenous memory cells of two TCR specificities. Using a novel technique for intracellular cytokine staining of small clonal populations, we showed that IAV-specific memory CD8+ T cells reactivated under type 2 conditions displayed robust IFN-γ expression and, unlike naive CD8+ T cells activated under type 2 conditions, produced little IL-4 protein. Secondary activation of memory cells under type 2 conditions increased GATA-3 levels with minimal change in T-bet levels. These data suggest that a small population of memory cells, especially central memory T cells, exhibits plasticity; however, most IAV-specific memory CD8+ T cells resist reprogramming upon reactivation and retain the functional state established during priming.

Association between Hemagglutinin Stem-Reactive Antibodies and Influenza A/H1N1 Virus Infection during the 2009 Pandemic.

UNLABELLED: The discovery of influenza virus broadly neutralizing (BrN) antibodies prompted efforts to develop universal vaccines. Influenza virus stem-reactive (SR) broadly neutralizing antibodies have been detected by screening antibody phage display libraries. However, studies of SR BrN antibodies in human serum, and their association with natural infection, are limited. To address this, pre- and postpandemic sera from a prospective community cohort study in Vietnam were assessed for antibodies that inhibit SR BrN monoclonal antibody (MAb) (C179) binding to H1N1 pandemic 2009 virus (H1N1pdm09). Of 270 households, 33 with at least one confirmed H1N1pdm09 illness or at least two seroconverters were included. The included households comprised 71 infected and 41 noninfected participants. Sera were tested as 2-fold dilutions between 1:5 and 1:40. Fifty percent C179 inhibition (IC50) titers did not exceed 10, although both IC50 titers and percent C179 inhibition by sera diluted 1:5 or 1:10 correlated with hemagglutination inhibition (HI) and microneutralization (MN) titers (all P < 0.001). Thirteen (12%) participants had detectable prepandemic IC50 titers, but only one reached a titer of 10. This proportion increased to 44% after the pandemic, when 39 participants had a titer of 10, and 67% of infected compared to 44% of noninfected had detectable IC50 titers (P < 0.001). The low levels of SR antibodies in prepandemic sera were not associated with subsequent H1N1pdm09 infection (P = 0.241), and the higher levels induced by H1N1pdm09 infection returned to prepandemic levels within 2 years. The findings indicate that natural infection induces only low titers of SR antibodies that are not sustained. IMPORTANCE: Universal influenza vaccines could have substantial health and economic benefits. The focus of universal vaccine research has been to induce antibodies that prevent infection by diverse influenza virus strains. These so-called broadly neutralizing antibodies are readily detected in mice and ferrets after infection with a series of distinct influenza virus strains. The 2009 H1N1 pandemic provided an opportunity to investigate whether infection with a novel strain induced broadly neutralizing antibodies in humans. We found that broadly neutralizing antibodies were induced, but levels were low and poorly maintained. This could represent an obstacle for universal vaccine development and warrants further investigation.

Strongyloides stercoralis seroprevalence in Vietnam.

Strongyloidiasis is a neglected tropical disease caused by the roundworm Strongyloides stercoralis affecting 30-100 million people worldwide. Many Southeast-Asian countries report a high prevalence of S. stercoralis infection, but there are little data from Vietnam. Here, we evaluated the seroprevalence of S. stercoralis related to geography, sex and age in Vietnam through serological testing of anonymized sera. Sera (n = 1710, 1340 adults and 270 children) from an anonymized age-stratified serum bank from four regions in Vietnam between 2012 and 2013 were tested using a commercial Strongyloides ratti immunoglobulin G ELISA. Seroreactivity was found in 29·1% (390/1340) of adults and 5·5% (15/270) of children. Male adults were more frequently seroreactive than females (33·3% vs. 24·9%, P = 0·001). The rural central highlands had the highest seroprevalence (42·4% of adults). Seroreactivity in the other regions was 29·9% (Hue) and 26·0% and 18·2% in the large urban centres of Hanoi and Ho Chi Minh City, respectively. We conclude that seroprevalence of S. stercoralis was high in the Vietnamese adult population, especially in rural areas.

Association of Microvascular Function and Endothelial Biomarkers With Clinical Outcome in Dengue: An Observational Study.

BACKGROUND: The hallmark of severe dengue is increased microvascular permeability, but alterations in the microcirculation and their evolution over the course of dengue are unknown. METHODS: We conducted a prospective observational study to evaluate the sublingual microcirculation using side-stream dark-field imaging in patients presenting early (<72 hours after fever onset) and patients hospitalized with warning signs or severe dengue in Vietnam. Clinical findings, microvascular function, global hemodynamics assessed with echocardiography, and serological markers of endothelial activation were determined at 4 time points. RESULTS: A total of 165 patients were enrolled. No difference was found between the microcirculatory parameters comparing dengue with other febrile illnesses. The proportion of perfused vessels (PPV) and the mean flow index (MFI) were lower in patients with dengue with plasma than those without leakage (PPV, 88.1% vs 90.6% [P = .01]; MFI, 2.1 vs 2.4 [P = .007]), most markedly during the critical phase. PPV and MFI were correlated with the endothelial activation markers vascular cell adhesion molecule 1 (P < .001 for both) and angiopoietin 2 (P < .001 for both), negatively correlated. CONCLUSIONS: Modest microcirculatory alterations occur in dengue, are associated with plasma leakage, and are correlate with molecules of endothelial activation, angiopoietin 2 and vascular cell adhesion molecule 1.

Determinants of influenza transmission in South East Asia: insights from a household cohort study in Vietnam.

To guide control policies, it is important that the determinants of influenza transmission are fully characterized. Such assessment is complex because the risk of influenza infection is multifaceted and depends both on immunity acquired naturally or via vaccination and on the individual level of exposure to influenza in the community or in the household. Here, we analyse a large household cohort study conducted in 2007-2010 in Vietnam using innovative statistical methods to ascertain in an integrative framework the relative contribution of variables that influence the transmission of seasonal (H1N1, H3N2, B) and pandemic H1N1pdm09 influenza. Influenza infection was diagnosed by haemagglutination-inhibition (HI) antibody assay of paired serum samples. We used a Bayesian data augmentation Markov chain Monte Carlo strategy based on digraphs to reconstruct unobserved chains of transmission in households and estimate transmission parameters. The probability of transmission from an infected individual to another household member was 8% (95% CI, 6%, 10%) on average, and varied with pre-season titers, age and household size. Within households of size 3, the probability of transmission from an infected member to a child with low pre-season HI antibody titers was 27% (95% CI 21%-35%). High pre-season HI titers were protective against infection, with a reduction in the hazard of infection of 59% (95% CI, 44%-71%) and 87% (95% CI, 70%-96%) for intermediate (1∶20-1∶40) and high (≥1∶80) HI titers, respectively. Even after correcting for pre-season HI titers, adults had half the infection risk of children. Twenty six percent (95% CI: 21%, 30%) of infections may be attributed to household transmission. Our results highlight the importance of integrated analysis by influenza sub-type, age and pre-season HI titers in order to infer influenza transmission risks in and outside of the household.

Influenza infection rates, measurement errors and the interpretation of paired serology.

Serological studies are the gold standard method to estimate influenza infection attack rates (ARs) in human populations. In a common protocol, blood samples are collected before and after the epidemic in a cohort of individuals; and a rise in haemagglutination-inhibition (HI) antibody titers during the epidemic is considered as a marker of infection. Because of inherent measurement errors, a 2-fold rise is usually considered as insufficient evidence for infection and seroconversion is therefore typically defined as a 4-fold rise or more. Here, we revisit this widely accepted 70-year old criterion. We develop a Markov chain Monte Carlo data augmentation model to quantify measurement errors and reconstruct the distribution of latent true serological status in a Vietnamese 3-year serological cohort, in which replicate measurements were available. We estimate that the 1-sided probability of a 2-fold error is 9.3% (95% Credible Interval, CI: 3.3%, 17.6%) when antibody titer is below 10 but is 20.2% (95% CI: 15.9%, 24.0%) otherwise. After correction for measurement errors, we find that the proportion of individuals with 2-fold rises in antibody titers was too large to be explained by measurement errors alone. Estimates of ARs vary greatly depending on whether those individuals are included in the definition of the infected population. A simulation study shows that our method is unbiased. The 4-fold rise case definition is relevant when aiming at a specific diagnostic for individual cases, but the justification is less obvious when the objective is to estimate ARs. In particular, it may lead to large underestimates of ARs. Determining which biological phenomenon contributes most to 2-fold rises in antibody titers is essential to assess bias with the traditional case definition and offer improved estimates of influenza ARs.

Quantifying the impact of pre-vaccination titre and vaccination history on influenza vaccine immunogenicity.

Epidemiological studies suggest that heterogeneity in influenza vaccine antibody response is associated with host factors, including pre-vaccination immune status, age, gender, and vaccination history. However, the pattern of reported associations varies between studies. To better understand the underlying influences on antibody responses, we combined host factors and vaccine-induced in-host antibody kinetics from a cohort study conducted across multiple seasons with a unified analysis framework. We developed a flexible individual-level Bayesian model to estimate associations and interactions between host factors, including pre-vaccine HAI titre, age, sex, vaccination history and study setting, and vaccine-induced HAI titre antibody boosting and waning. We applied the model to derive population-level and individual effects of post-vaccine antibody kinetics for vaccinating and circulating strains for A(H1N1) and A(H3N2) influenza subtypes. We found that post-vaccine HAI titre dynamics were significantly influenced by pre-vaccination HAI titre and vaccination history and that lower pre-vaccination HAI titre results in longer durations of seroprotection (HAI titre equal to 1:40 or higher). Consequently, for A(H1N1), our inference finds that the expected duration of seroprotection post-vaccination was 171 (95% Posterior Predictive Interval[PPI] 128-220) and 159 (95% PPI 120-200) days longer for those who are infrequently vaccinated (<2 vaccines in last five years) compared to those who are frequently vaccinated (2 or more vaccines in the last five years) at pre-vaccination HAI titre values of 1:10 and 1:20 respectively. In addition, we found significant differences in the empirical distributions that describe the individual-level duration of seroprotection for A(H1N1) circulating strains. In future, studies that rely on serological endpoints should include the impact of pre-vaccine HAI titre and prior vaccination status on seropositivity and seroconversion estimates, as these significantly influence an individual's post-vaccination antibody kinetics.

Subclinical avian influenza A(H5N1) virus infection in human, Vietnam.

Laboratory-confirmed cases of subclinical infection with avian influenza A(H5N1) virus in humans are rare, and the true number of these cases is unknown. We describe the identification of a laboratory-confirmed subclinical case in a woman during an influenza A(H5N1) contact investigation in northern Vietnam.

Identification of H5N1-specific T-cell responses in a high-risk cohort in vietnam indicates the existence of potential asymptomatic infections.

BACKGROUND: Most reported human H5N1 viral infections have been severe and were detected after hospital admission. A case ascertainment bias may therefore exist, with mild cases or asymptomatic infections going undetected. We sought evidence of mild or asymptomatic H5N1 infection by examining H5N1-specific T-cell and antibody responses in a high-risk cohort in Vietnam. METHODS: Peripheral blood mononuclear cells were tested using interferon-γ enzyme-linked immunospot T assays measuring the response to peptides of influenza H5, H3, and H1 hemagglutinin (HA), N1 and N2 neuraminidase, and the internal proteins of H3N2. Horse erythrocyte hemagglutination inhibition assay was performed to detect antibodies against H5N1. RESULTS: Twenty-four of 747 individuals demonstrated H5-specific T-cell responses but little or no cross-reactivity with H3 or H1 HA peptides. H5N1 peptide-specific T-cell lines that did not cross-react with H1 or H3 influenza virus HA peptides were generated. Four individuals also had antibodies against H5N1. CONCLUSIONS: This is the first report of ex vivo H5 HA-specific T-cell responses in a healthy but H5N1-exposed population. Our results indicate that the presence of H5N1-specific T cells could be an additional diagnostic tool for asymptomatic H5N1 infection.

Drift and shape-new insights into human immunity against influenza virus neuraminidase.

Influenza virus hemagglutinin mediates infection by binding sialic acids, whereas neuraminidase cleaves sialic acids to release progeny virions. Both are targets of protective antibodies, but influenza vaccine strain selection and antigen dose are based on hemagglutinin alone. Virus characterization using first infection ferret sera indicates that escape from hemagglutination inhibiting (HI) antibodies occurs more frequently and is not coordinated with escape from neuraminidase inhibiting (NI) antibodies. A key question addressed by Daulagala et al. (P. Daulagala, B. R. Mann, K. Leung, E. H. Y. Lau, et al., mBio 14:e00084-23, 2023, https://doi.org/10.1128/mbio.00084-23) is how this translates to humans who encounter multiple influenza viruses throughout life. Their cross-sectional study, using sera from a wide age range of participants and H1N1 viruses spanning 1977-2015, indicates that NI antibodies are more broadly cross-reactive than HI antibodies. Both HI and NI titers were highest against strains encountered in childhood indicating that both are shaped by priming exposures. The study further supports the development of NA-optimized vaccines.

Does repeated influenza vaccination attenuate effectiveness? A systematic review and meta-analysis.

BACKGROUND: Influenza vaccines require annual readministration; however, several reports have suggested that repeated vaccination might attenuate the vaccine's effectiveness. We aimed to estimate the reduction in vaccine effectiveness associated with repeated influenza vaccination. METHODS: In this systematic review and meta-analysis, we searched MEDLINE, EMBASE, and CINAHL Complete databases for articles published from Jan 1, 2016, to June 13, 2022, and Web of Science for studies published from database inception to June 13, 2022. For studies published before Jan 1, 2016, we consulted published systematic reviews. Two reviewers (EJ-G and EJR) independently screened, extracted data using a data collection form, assessed studies' risk of bias using the Risk Of Bias In Non-Randomized Studies of Interventions (ROBINS-I) and evaluated the weight of evidence by Grading of Recommendations Assessment, Development, and Evaluation (GRADE). We included observational studies and randomised controlled trials that reported vaccine effectiveness against influenza A(H1N1)pdm09, influenza A(H3N2), or influenza B using four vaccination groups: current season; previous season; current and previous seasons; and neither season (reference). For each study, we calculated the absolute difference in vaccine effectiveness (ΔVE) for current season only and previous season only versus current and previous season vaccination to estimate attenuation associated with repeated vaccination. Pooled vaccine effectiveness and ∆VE were calculated by season, age group, and overall. This study is registered with PROSPERO, CRD42021260242. FINDINGS: We identified 4979 publications, selected 681 for full review, and included 83 in the systematic review and 41 in meta-analyses. ΔVE for vaccination in both seasons compared with the current season was -9% (95% CI -16 to -1, I2=0%; low certainty) for influenza A(H1N1)pdm09, -18% (-26 to -11, I2=7%; low certainty) for influenza A(H3N2), and -7% (-14 to 0, I2=0%; low certainty) for influenza B, indicating lower protection with consecutive vaccination. However, for all types, A subtypes and B lineages, vaccination in both seasons afforded better protection than not being vaccinated. INTERPRETATION: Our estimates suggest that, although vaccination in the previous year attenuates vaccine effectiveness, vaccination in two consecutive years provides better protection than does no vaccination. The estimated effects of vaccination in the previous year are concerning and warrant additional investigation, but are not consistent or severe enough to support an alternative vaccination regimen at this time. FUNDING: WHO and the US National Institutes of Health.

Comparative B cell and antibody responses induced by adenoviral vectored and mRNA vaccines against COVID-19.

Both vector and mRNA vaccines were an important part of the response to the COVID-19 pandemic and may be required in future outbreaks and pandemics. However, adenoviral vectored (AdV) vaccines may be less immunogenic than mRNA vaccines against SARS-CoV-2. We assessed anti-spike and anti-vector immunity among infection-naïve Health Care Workers (HCW) following two doses of AdV (AZD1222) versus mRNA (BNT162b2) vaccine. 183 AdV and 274 mRNA vaccinees enrolled between April and October 2021. Median ages were 42 and 39 years, respectively. Blood was collected at least once, 10-48 days after vaccine dose 2. Surrogate virus neutralization test (sVNT) and spike binding antibody titres were a median of 4.2 and 2.2 times lower, respectively, for AdV compared to mRNA vaccinees (p<0.001). Median percentages of memory B cells that recognized fluorescent-tagged spike and RBD were 2.9 and 8.3 times lower, respectively for AdV compared to mRNA vaccinees. Titres of IgG reactive with human Adenovirus type 5 hexon protein rose a median of 2.2-fold after AdV vaccination but were not correlated with anti-spike antibody titres. Together the results show that mRNA induced substantially more sVNT antibody than AdV vaccine due to greater B cell expansion and targeting of the RBD. Pre-existing AdV vector cross-reactive antibodies were boosted following AdV vaccination but had no detectable effect on immunogenicity.

An external quality assessment feasibility study; cross laboratory comparison of haemagglutination inhibition assay and microneutralization assay performance for seasonal influenza serology testing: A FLUCOP study.

Introduction: External Quality Assessment (EQA) schemes are designed to provide a snapshot of laboratory proficiency, identifying issues and providing feedback to improve laboratory performance and inter-laboratory agreement in testing. Currently there are no international EQA schemes for seasonal influenza serology testing. Here we present a feasibility study for conducting an EQA scheme for influenza serology methods. Methods: We invited participant laboratories from industry, contract research organizations (CROs), academia and public health institutions who regularly conduct hemagglutination inhibition (HAI) and microneutralization (MN) assays and have an interest in serology standardization. In total 16 laboratories returned data including 19 data sets for HAI assays and 9 data sets for MN assays. Results: Within run analysis demonstrated good laboratory performance for HAI, with intrinsically higher levels of intra-assay variation for MN assays. Between run analysis showed laboratory and strain specific issues, particularly with B strains for HAI, whilst MN testing was consistently good across labs and strains. Inter-laboratory variability was higher for MN assays than HAI, however both assays showed a significant reduction in inter-laboratory variation when a human sera pool is used as a standard for normalization. Discussion: This study has received positive feedback from participants, highlighting the benefit such an EQA scheme would have on improving laboratory performance, reducing inter laboratory variation and raising awareness of both harmonized protocol use and the benefit of biological standards for seasonal influenza serology testing.

Superior immunogenicity of mRNA over adenoviral vectored COVID-19 vaccines reflects B cell dynamics independent of anti-vector immunity: Implications for future pandemic vaccines.

Both vector and mRNA vaccines were an important part of the response to the COVID-19 pandemic and may be required in future outbreaks and pandemics. The aim of this study was to validate whether immunogenicity differs for adenoviral vectored (AdV) versus mRNA vaccines against SARS-CoV-2, and to investigate how anti-vector immunity and B cell dynamics modulate immunogenicity. We enrolled SARS-CoV-2 infection-naïve health care workers who had received two doses of either AdV AZD1222 (n = 184) or mRNA BNT162b2 vaccine (n = 274) between April and October 2021. Blood was collected at least once, 10-48 days after vaccine dose 2 for antibody and B cell analyses. Median ages were 42 and 39 years, for AdV and mRNA vaccinees, respectively. Surrogate virus neutralization test (sVNT) and spike binding antibody titres were a median of 4.2 and 2.2 times lower, respectively, for AdV compared to mRNA vaccinees (p < 0.001). Median percentages of memory B cells that recognized fluorescent-tagged spike and RBD were 2.9 and 8.3 times lower, respectively for AdV compared to mRNA vaccinees. Titres of IgG reactive with human adenovirus type 5 hexon protein rose a median of 2.2-fold after AdV vaccination but were not correlated with anti-spike antibody titres. Together the results show that mRNA induced substantially more sVNT antibody than AdV vaccine, which reflected greater B cell expansion and targeting of the RBD rather than an attenuating effect of anti-vector antibodies. ClinicalTrials.gov Identifier: NCT05110911.

The epidemiology of interpandemic and pandemic influenza in Vietnam, 2007-2010: the Ha Nam household cohort study I.

Prospective community-based studies have provided fundamental insights into the epidemiology of influenza in temperate regions, but few comparable studies have been undertaken in the tropics. The authors conducted prospective influenza surveillance and intermittent seroprevalence surveys in a household-based cohort in Vietnam between December 2007 and April 2010, resulting in 1,793 person-seasons of influenza surveillance. Age- and sex-standardized estimates of the risk of acquiring any influenza infection per season in persons 5 years of age or older were 21.1% (95% confidence interval: 17.4, 24.7) in season 1, 26.4% (95% confidence interval: 22.6, 30.2) in season 2, and 17.0% (95% confidence interval: 13.6, 20.4) in season 3. Some individuals experienced multiple episodes of infection with different influenza types/subtypes in the same season (n = 27) or reinfection with the same subtype in different seasons (n = 22). The highest risk of influenza infection was in persons 5-9 years old, in whom the risk of influenza infection per season was 41.8%. Although the highest infection risk was in school-aged children, there were important heterogeneities in the age of infection by subtype and season. These heterogeneities could influence the impact of school closure and childhood vaccination on influenza transmission in tropical areas, such as Vietnam.

H5N1-SeroDetect EIA and rapid test: a novel differential diagnostic assay for serodiagnosis of H5N1 infections and surveillance.

Continuing evolution of highly pathogenic (HP) H5N1 influenza viruses in wild birds with transmission to domestic poultry and humans poses a pandemic threat. There is an urgent need for a simple and rapid serological diagnostic assay which can differentiate between antibodies to seasonal and H5N1 strains and that could provide surveillance tools not dependent on virus isolation and nucleic acid technologies. Here we describe the establishment of H5N1 SeroDetect enzyme-linked immunosorbent assay (ELISA) and rapid test assays based on three peptides in HA2 (488-516), PB1-F2 (2-75), and M2e (2-24) that are highly conserved within H5N1 strains. These peptides were identified by antibody repertoire analyses of H5N1 influenza survivors in Vietnam using whole-genome-fragment phage display libraries (GFPDLs). To date, both platforms have demonstrated high levels of sensitivity and specificity in detecting H5N1 infections (clade 1 and clade 2.3.4) in Vietnamese patients as early as 7 days and up to several years postinfection. H5N1 virus-uninfected individuals in Vietnam and the United States, including subjects vaccinated with seasonal influenza vaccines or with confirmed seasonal virus infections, did not react in the H5N1-SeroDetect assays. Moreover, sera from individuals vaccinated with H5N1 subunit vaccine with moderate anti-H5N1 neutralizing antibody titers did not react positively in the H5N1-SeroDetect ELISA or rapid test assays. The simple H5N1-SeroDetect ELISA and rapid tests could provide an important tool for large-scale surveillance for potential exposure to HP H5N1 strains in both humans and birds.