Pre-existing neutralizing antibody provides the first line of defence against pathogens in general. For influenza virus, annual vaccinations are given to maintain protective levels of antibody against the currently circulating strains. Here we report that after booster vaccination there was a rapid and robust influenza-specific IgG+ antibody-secreting plasma cell (ASC) response that peaked at approximately day 7 and accounted for up to 6% of peripheral blood B cells. These ASCs could be distinguished from influenza-specific IgG+ memory B cells that peaked 14-21 days after vaccination and averaged 1% of all B cells. Importantly, as much as 80% of ASCs purified at the peak of the response were influenza specific. This ASC response was characterized by a highly restricted B-cell receptor (BCR) repertoire that in some donors was dominated by only a few B-cell clones. This pauci-clonal response, however, showed extensive intraclonal diversification from accumulated somatic mutations. We used the immunoglobulin variable regions isolated from sorted single ASCs to produce over 50 human monoclonal antibodies (mAbs) that bound to the three influenza vaccine strains with high affinity. This strategy demonstrates that we can generate multiple high-affinity mAbs from humans within a month after vaccination. The panel of influenza-virus-specific human mAbs allowed us to address the issue of original antigenic sin (OAS): the phenomenon where the induced antibody shows higher affinity to a previously encountered influenza virus strain compared with the virus strain present in the vaccine. However, we found that most of the influenza-virus-specific mAbs showed the highest affinity for the current vaccine strain. Thus, OAS does not seem to be a common occurrence in normal, healthy adults receiving influenza vaccination.

We used a systems biological approach to study innate and adaptive responses to influenza vaccination in humans, during 3 consecutive influenza seasons. Healthy adults were vaccinated with inactivated (TIV) or live attenuated (LAIV) influenza vaccines. TIV induced greater antibody titers and enhanced numbers of plasmablasts than LAIV. In TIV vaccinees, early molecular signatures correlated with, and accurately predicted, later antibody titers in two independent trials. Interestingly, the expression of Calcium/calmodulin-dependent kinase IV (CamkIV) at day 3 was inversely correlated with later antibody titers. Vaccination of CamkIV −/− mice with TIV induced enhanced antigen-specific antibody titers, demonstrating an unappreciated role for CaMKIV in the regulation of antibody responses. Thus systems approaches can predict immunogenicity, and reveal new mechanistic insights about vaccines.

Although scarce after annual influenza vaccination, B cells producing antibodies capable of neutralizing multiple influenza strains are abundant in humans infected with pandemic 2009 H1N1 influenza.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus of significant public health concern. ZIKV shares a high degree of sequence and structural homology compared with other flaviviruses, including dengue virus (DENV), resulting in immunological cross-reactivity. Improving our current understanding of the extent and characteristics of this immunological cross-reactivity is important, as ZIKV is presently circulating in areas that are highly endemic for dengue. To assess the magnitude and functional quality of cross-reactive immune responses between these closely related viruses, we tested acute and convalescent sera from nine Thai patients with PCRconfirmed DENV infection against ZIKV. All of the sera tested were cross-reactive with ZIKV, both in binding and in neutralization. To deconstruct the observed serum cross-reactivity in depth, we also characterized a panel of DENV-specific plasmablast-derived monoclonal antibodies (mAbs) for activity against ZIKV. Nearly half of the 47 DENV-reactive mAbs studied bound to both whole ZIKV virion and ZIKV lysate, of which a subset also neutralized ZIKV. In addition, both sera and mAbs from the dengue-infected patients enhanced ZIKV infection of Fc gamma receptor (FcγR)-bearing cells in vitro. Taken together, these findings suggest that preexisting immunity to DENV may impact protective immune responses against ZIKV. In addition, the extensive cross-reactivity may have implications for ZIKV virulence and disease severity in DENV-experienced populations. (2-4). However, the more recent outbreaks have caused severe neurological complications including GuillainBarré Syndrome in adults and an increase in congenital microcephaly and other adverse birth outcomes in Brazil (5-7). The Pan American Health Organization has reported that as of May 2016, local transmission of ZIKV had spread to over 38 countries or territories in the Americas. In addition, a recent WHO report states that 44 new countries are experiencing their first ZIKV outbreak since 2015. Despite the improving surveillance of the virus, accurate diagnosis has been challenging given the similarities in the clinical presentation of ZIKV to other arboviral infections endemic in these regions, among other factors.During the viremic period, ZIKV can be found in patient blood, saliva, urine, and other bodily fluids early after symptom onset (8-10). During the Yap Islands epidemic in 2007, anti-ZIKV IgM ELISAs and ZIKV plaque reduction neutralization titer (PRNT) assays were performed to confirm infection in RT-PCR negative cases (2, 8). However, as these studies showed, the cross-reactivity between ZIKV and other flaviviruses makes confirmation of infection difficult, especially when patients may have had flavivirus exposures before their suspected ZIKV infection (2,8). Given the overlapping presence of DENV and other flaviviruses in a majority of ZIKV epidemic regions (11), there are great challenges in serology-based testing of flavivirus-immune patients (12).The DENV envelope (E) protein, considered a major imun...

Six years have passed since the outbreak of severe acute respiratory syndrome (SARS). Previous studies indicated that specific Abs to SARS-related coronavirus (SARS-CoV) waned over time in recovered SARS patients. It is critical to find out whether a potential anamnestic response, as seen with other viral infections, exists to protect a person from reinfection in case of another SARS outbreak. Recovered SARS patients were followed up to 6 y to estimate the longevity of specific Ab. The specific memory B cell and T cell responses to SARS-CoV Ags were measured by means of ELISPOT assay. Factors in relation to humoral and cellular immunity were investigated. Six years postinfection, specific IgG Ab to SARS-CoV became undetectable in 21 of the 23 former patients. No SARS-CoV Ag-specific memory B cell response was detected in either 23 former SARS patients or 22 close contacts of SARS patients. Memory T cell responses to a pool of SARS-CoV S peptides were identified in 14 of 23 (60.9%) recovered SARS patients, whereas there was no such specific response in either close contacts or healthy controls. Patients with more severe clinical manifestations seemed to present a higher level of Ag-specific memory T cell response. SARS-specific IgG Ab may eventually vanish and peripheral memory B cell responses are undetectable in recovered SARS patients. In contrast, specific T cell anamnestic responses can be maintained for at least 6 y. These findings have applications in preparation for the possible reemergence of SARS.

We describe herein a protocol for the production of antigen-specific human monoclonal antibodies (hmAbs). Antibody-secreting cells (ASCs) are isolated from whole blood collected 7 d after vaccination and sorted by flow cytometry into single cell plates. The antibody genes of the ASCs are then amplified by RT-PCR and nested PCR, cloned into expression vectors and transfected into a human cell line. The expressed antibodies can then be purified and assayed for binding and neutralization. This method uses established techniques but is novel in their combination and application. This protocol can be completed with as little as 20 ml of human blood and in as little as 28 d when optimal. Although previous methodologies to produce hmAbs, including B-cell immortalization or phage display, can be used to isolate the rare specific antibody even years after immunization, in comparison, these approaches are inefficient, resulting in few relevant antibodies. Although dependent on having an ongoing immune response, the approach described herein can be used to rapidly generate numerous antigen-specific hmAbs in a short time.

Generating a broadly protective influenza vaccine is critical to global health. Understanding how immune memory influences influenza immunity is central to this goal. We undertook an in-depth study of the B cell response to the pandemic 2009 H1N1 vaccine over consecutive years. Analysis of monoclonal Abs generated from vaccine-induced plasmablasts demonstrated that individuals with low preexisting serological titers to the vaccinating strain generated a broadly reactive, HA stalk-biased, response. Higher preexisting serum antibody levels correlated with a strain-specific HA head-dominated response. We demonstrate that this HA head immunodominance encompasses poor accessibility of the HA stalk epitopes. Further, we show polyreactivity of HA stalk-reactive antibodies that could cause counterselection of these cells. Thus, preexisting memory against HA head epitopes predominate, inhibiting a broadly protective response against the HA stalk upon revaccination with similar strains. Consideration of influenza exposure history is critical for new vaccine strategies designed to elicit broadly neutralizing antibodies.

HIGHLIGHTSd Cross-sectional study of 44 hospitalized COVID-19 patients d RBD-specific IgG responses detectable in all patients 6 days after PCR confirmation d Neutralizing titers are detectable in all patients 6 days after PCR confirmation d RBD-specific IgG titers correlate with the neutralizing potency