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There is growing consensus for an urgent need to develop efficient methods to analyze the human T and B cell response in order to understand its regulation, to unravel host-pathogen interactions and to design better vaccines. In this review we will consider different methods that have been developed to analyze human T and B cells and focus in particular on those based on high-throughput cell culture, such as memory B cell immortalization, plasma cell cultures and libraries of naïve or memory T cells. These culture-based methods can be used to interrogate with high efficiency the immune repertoires in order to characterize the frequency, function and specificity of naïve or memory T cells and to isolate potent and broadly neutralizing monoclonal antibodies. [Copyright &y& Elsevier]

Following an immune response two types of differentiated B cells persist in the memory pool: plasma cells, which confer immediate protection by the secretion of specific antibodies; and memory B cells, which confer rapid and enhanced response to secondary challenge. We will review recent advances in understanding the heterogeneity, dynamics, and persistence of human memory B cells and plasma cells as well as new methods to isolate human monoclonal antibodies. These findings offer new insights into the human B cell response, which are relevant for vaccination and therapeutic intervention. [Copyright &y& Elsevier]

The administration of hyper immune sera to prevent or treat life-threatening infections is a remarkable milestone in medicine and biotechnology that has been achieved more than a century ago. Yet, the therapeutic use of monoclonal antibodies in this field has developed slowly over the last decades. Here we compare and contrast current methods to generate human monoclonal antibodies and highlight the advantages of exploiting the human antibody repertoire using a novel method that allows efficient immortalization and cloning of human memory B cells. This method, which has been successfully applied to isolate broadly neutralizing antibodies against SARS and H5N1 influenza viruses, is expected to accelerate the development of therapeutics in the field of infectious diseases not only by providing neutralizing antibodies for passive serotherapy, but also by generating relevant information for vaccine design. [Copyright &y& Elsevier]

Ebola virus disease in humans is highly lethal, with case fatality rates ranging from 25 to 90%. There is no licensed treatment or vaccine against the virus, underscoring the need for efficacious countermeasures. We ascertained that a human survivor of the 1995 Kikwit Ebola virus disease outbreak maintained circulating antibodies against the Ebola virus surface glycoprotein for more than a decade after infection. From this survivor we isolated monoclonal antibodies (mAbs) that neutralize recent and previous outbreak variants of Ebola virus and mediate antibody-dependent cell-mediated cytotoxicity in vitro. Strikingly, monotherapy with mAb114 protected macaques when given as late as 5 days after challenge. Treatment with a single human mAb suggests that a simplified therapeutic strategy for human Ebola infection may be possible. [ABSTRACT FROM AUTHOR]

Melanoma prognosis is dictated by tumor-infiltrating lymphocytes, the migratory and functional behavior of which is guided by chemokine or cytokine gradients. Here, we retrospectively analyzed the expression patterns of 9 homing receptors (CCR/CXCR) in naive and memory CD4+ and CD8+ T lymphocytes in 57 patients with metastatic melanoma (MMel) with various sites of metastases to evaluate whether T cell CCR/CXCR expression correlates with intratumoral accumulation, metastatic progression, and/or overall survival (OS). Homing receptor expression on lymphocytes strongly correlated with MMel dissemination. Loss of CCR6 or CXCR3, but not cutaneous lymphocyte antigen (CLA), on circulating T cell subsets was associated with skin or lymph node metastases, loss of CXCR4, CXCR5, and CCR9 corresponded with lung involvement, and a rise in CCR10 or CD103 was associated with widespread dissemination. High frequencies of CD8+CCR9+ naive T cells correlated with prolonged OS, while neutralizing the CCR9/CCL25 axis in mice stimulated tumor progression. The expansion of CLA-expressing effector memory CD8+ T cells in response to a single administration of CTLA4 blockade predicted disease control at 3 months in 47 patients with MMel. Thus, specific CCR/CXCR expression patterns on circulating T lymphocytes may guide potential diagnostic and therapeutic approaches. [ABSTRACT FROM AUTHOR]

Broadly neutralizing antibodies reactive against most and even all variants of the same viral species have been described for influenza and HIV-1 (ref. 1). However, whether a neutralizing antibody could have the breadth of range to target different viral species was unknown. Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are common pathogens that cause severe disease in premature newborns, hospitalized children and immune-compromised patients, and play a role in asthma exacerbations. Although antisera generated against either HRSV or HMPV are not cross-neutralizing, we speculated that, because of the repeated exposure to these viruses, cross-neutralizing antibodies may be selected in some individuals. Here we describe a human monoclonal antibody (MPE8) that potently cross-neutralizes HRSV and HMPV as well as two animal paramyxoviruses: bovine RSV (BRSV) and pneumonia virus of mice (PVM). In its germline configuration, MPE8 is HRSV-specific and its breadth is achieved by somatic mutations in the light chain variable region. MPE8 did not result in the selection of viral escape mutants that evaded antibody targeting and showed potent prophylactic efficacy in animal models of HRSV and HMPV infection, as well as prophylactic and therapeutic efficacy in the more relevant model of lethal PVM infection. The core epitope of MPE8 was mapped on two highly conserved anti-parallel β-strands on the pre-fusion viral F protein, which are rearranged in the post-fusion F protein conformation. Twenty-six out of the thirty HRSV-specific neutralizing antibodies isolated were also found to be specific for the pre-fusion F protein. Taken together, these results indicate that MPE8 might be used for the prophylaxis and therapy of severe HRSV and HMPV infections and identify the pre-fusion F protein as a candidate HRSV vaccine. [ABSTRACT FROM AUTHOR]

The isolation of broadly neutralizing antibodies against influenza A viruses has been a long-sought goal for therapeutic approaches and vaccine design. Using a single-cell culture method for screening large numbers of human plasma cells, we isolated a neutralizing monoclonal antibody that recognized the hemagglutinin (HA) glycoprotein of all 16 subtypes and neutralized both group 1 and group 2 influenza A viruses. Passive transfer of this antibody conferred protection to mice and ferrets. Complexes with HAs from the group 1 H1 and the group 2 H3 subtypes analyzed by x-ray crystallography showed that the antibody bound to a conserved epitope in the F subdomain. This antibody may be used for passive protection and to inform vaccine design because of its broad specificity and neutralization potency. [ABSTRACT FROM AUTHOR]

Abstract: It remains unclear whether antibody-dependent-enhancement (ADE) of dengue infection merely augments viral attachment and entry through Fcγ receptors or immune complex binding to Fcγ receptors triggers an intrinsic signaling cascade that changes the viral permissiveness of the cell. Using human dengue–immune sera and novel human monoclonal antibodies against dengue in combination with virologic and immunologic techniques, we found that ADE infection increased the proportion of infected primary human monocytes modestly from 0.2%±0.1% (no Ab) to 1.7%±1.6% (with Ab) but the total virus output markedly from 2±2 (×103)FFU to 120±153 (×103)FFU. However, this increased virus production was not associated with a reduced secretion of type I interferon or an elevated secretion of anti-inflammatory cytokine, IL-10. These results demonstrate that the regulation of virus production in ADE infection of primary human monocytes is more complex than previously appreciated. [ABSTRACT FROM AUTHOR]

The target of neutralizing antibodies that protect against influenza virus infection is the viral protein HA. Genetic and antigenic variation in HA has been used to classify influenza viruses into subtypes (H1-H16). The neutralizing antibody response to influenza virus is thought to be specific for a few antigenically related isolates within a given subtype. However, while heterosubtypic antibodies capable of neutralizing multiple influenza virus subtypes have been recently isolated from phage display libraries, it is not known whether such antibodies are produced in the course of an immune response to influenza virus infection or vaccine. Here we report that, following vaccination with seasonal influenza vaccine containing H1 and H3 influenza virus subtypes, some individuals produce antibodies that cross-react with H5 HA. By immortalizing IgG-expressing B cells from 4 individuals, we isolated 20 heterosubtypic mAbs that bound and neutralized viruses belonging to several HA subtypes (H1, H2, H5, H6, and H9), including the pandemic A/California/07/09 H1N1 isolate. The mAbs used different VH genes and carried a high frequency of somatic mutations. With the exception of a mAb that bound to the HA globular head, all heterosubtypic mAbs bound to acid-sensitive epitopes in the HA stem region. Four mAbs were evaluated in vivo and protected mice from challenge with influenza viruses representative of different subtypes. These findings reveal that seasonal influenza vaccination can induce polyclonal heterosubtypic neutralizing antibodies that cross-react with the swine-origin pandemic H1N1 influenza virus and with the highly pathogenic H5N1 virus. [ABSTRACT FROM AUTHOR]

Background: Transmission of highly pathogenic avian H5N1 viruses from poultry to humans have raised fears of an impending influenza pandemic. Concerted efforts are underway to prepare effective vaccines and therapies including polyclonal or monoclonal antibodies against H5N1. Current efforts are hampered by the paucity of information on protective immune responses against avian influenza. Characterizing the B cell responses in convalescent individuals could help in the design of future vaccines and therapeutics. Methods and Findings: To address this need, we generated whole-genome-fragment phage display libraries (GFPDL) expressing fragments of 15-350 amino acids covering all the proteins of A/Vietnam/1203/2004 (H5N1). These GFPDL were used to analyze neutralizing human monoclonal antibodies and sera of five individuals who had recovered from H5N1 infection. This approach led to the mapping of two broadly neutralizing human monoclonal antibodies with conformationdependent epitopes. In H5N1 convalescent sera, we have identified several potentially protective H5N1-specific human antibody epitopes in H5 HA[(-10)-223], neuraminidase catalytic site, and M2 ectodomain. In addition, for the first time to our knowledge in humans, we identified strong reactivity against PB1-F2, a putative virulence factor, following H5N1 infection. Importantly, novel epitopes were identified, which were recognized by H5N1-convalescent sera but did not react with sera from control individuals (H5N1 naïve, H1N1 or H3N2 seropositive). Conclusion: This is the first study, to our knowledge, describing the complete antibody repertoire following H5N1 infection. Collectively, these data will contribute to rational vaccine design and new H5N1-specific serodiagnostic surveillance tools. [ABSTRACT FROM AUTHOR]

Pregnancy-associated malaria (PAM) is caused by Plasmodium falciparum-infected erythrocytes (IEs) that bind to chondroitin sulphate A (CSA) in the placenta by PAM-associated clonally variant surface antigens (VSA). Pregnancy-specific VSA (VSAPAM), which include the PfEMP1 variant VAR2CSA, are targets of IgG-mediated protective immunity to PAM. Here, we report an investigation of the specificity of naturally acquired immunity to PAM, using eight human monoclonal IgG1 antibodies that react exclusively with intact CSA-adhering IEs expressing VSAPAM. Four reacted in Western blotting with high-molecular-weight (> 200 kDa) proteins, while seven reacted with either the DBL3-X or the DBL5-ε domains of VAR2CSA expressed either as Baculovirus constructs or on the surface of transfected Jurkat cells. We used a panel of recombinant antigens representing DBL3-X domains from P. falciparum field isolates to evaluate B-cell epitope diversity among parasite isolates, and identified the binding site of one monoclonal antibody using a chimeric DBL3-X construct. Our findings show that there is a high-frequency memory response to VSAPAM, indicating that VAR2CSA is a primary target of naturally acquired PAM-specific protective immunity, and demonstrate the value of human monoclonal antibodies and conformationally intact recombinant antigens in VSA characterization. [ABSTRACT FROM AUTHOR]

The work of our laboratory has focused on the study of human memory B cells. Using an in vitro approach we dissected the triggering requirements of B cells and unveiled a distinct role for TLRs in the activation of naive versus memory B cells. Using an ex vivo approach we analyzed the dynamics of memory B cells and ASCs and the kinetics of serum antibodies during secondary immune responses and in steady state conditions and used these quantitative data to build up a model of serological memory. According to this model memory B cells behave as `stem cells' capable of generating plasma cells and antibodies in an antigen-dependent as well as in an antigen-independent fashion. Finally we developed an efficient method to interrogate human memory B cells and to isolate human monoclonal antibodies. This method can be exploited for the production of neutralizing antibodies for serotherapy and for “analytic vaccinology”. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) is a widely circulating pathogen that causes severe disease in immunocompromised patients and infected fetuses. By immortalizing memory B cells from HCMV-immune donors, we isolated a panel of human monoclonal antibodies that neutralized at extremely low concentrations (90% inhibitory concentration [IC90] values ranging from 5 to 200 pM) HCMV infection of endothelial, epithelial, and myeloid cells. With the single exception of an antibody that bound to a conserved epitope in the UL128 gene product, all other antibodies bound to conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex. Antibodies against gB, gH, or gM/gN were also isolated and, albeit less potent, were able to neutralize infection of both endothelial-epithelial cells and fibroblasts. This study describes unusually potent neutralizing antibodies against HCMV that might be used for passive immunotherapy and identifies, through the use of such antibodies, novel antigenic targets in HCMV for the design of immunogens capable of eliciting previously unknown neutralizing antibody responses. [ABSTRACT FROM AUTHOR]

Background: New prophylactic and therapeutic strategies to combat human infections with highly pathogenic avian influenza (HPAI) H5N1 viruses are needed. We generated neutralizing anti-H5N1 human monoclonal antibodies (mAbs) and tested their efficacy for prophylaxis and therapy in a murine model of infection.Methods and Findings: Using Epstein-Barr virus we immortalized memory B cells from Vietnamese adults who had recovered from infections with HPAI H5N1 viruses. Supernatants from B cell lines were screened in a virus neutralization assay. B cell lines secreting neutralizing antibodies were cloned and the mAbs purified. The cross-reactivity of these antibodies for different strains of H5N1 was tested in vitro by neutralization assays, and their prophylactic and therapeutic efficacy in vivo was tested in mice. In vitro, mAbs FLA3.14 and FLD20.19 neutralized both Clade I and Clade II H5N1 viruses, whilst FLA5.10 and FLD21.140 neutralized Clade I viruses only. In vivo, FLA3.14 and FLA5.10 conferred protection from lethality in mice challenged with A/Vietnam/1203/04 (H5N1) in a dose-dependent manner. mAb prophylaxis provided a statistically significant reduction in pulmonary virus titer, reduced associated inflammation in the lungs, and restricted extrapulmonary dissemination of the virus. Therapeutic doses of FLA3.14, FLA5.10, FLD20.19, and FLD21.140 provided robust protection from lethality at least up to 72 h postinfection with A/Vietnam/1203/04 (H5N1). mAbs FLA3.14, FLD21.140 and FLD20.19, but not FLA5.10, were also therapeutically active in vivo against the Clade II virus A/Indonesia/5/2005 (H5N1).Conclusions: These studies provide proof of concept that fully human mAbs with neutralizing activity can be rapidly generated from the peripheral blood of convalescent patients and that these mAbs are effective for the prevention and treatment of H5N1 infection in a mouse model. A panel of neutralizing, cross-reactive mAbs might be useful for prophylaxis or adjunctive treatment of human cases of H5N1 influenza. [ABSTRACT FROM AUTHOR]