A Limited Number of Antibody Specificities Mediate Broad and Potent Serum Neutralization in Selected HIV-1 Infected Individuals

A protective vaccine against HIV-1 will likely require the elicitation of a broadly neutralizing antibody (bNAb) response. Although the development of an immunogen that elicits such antibodies remains elusive, a proportion of HIV-1 infected individuals evolve broadly neutralizing serum responses over time, demonstrating that the human immune system can recognize and generate NAbs to conserved epitopes on the virus. Understanding the specificities that mediate broad neutralization will provide insight into which epitopes should be targeted for immunogen design and aid in the isolation of broadly neutralizing monoclonal antibodies from these donors. Here, we have used a number of new and established technologies to map the bNAb specificities in the sera of 19 donors who exhibit among the most potent cross-clade serum neutralizing activities observed to date. The results suggest that broad and potent serum neutralization arises in most donors through a limited number of specificities (1–2 per donor). The major targets recognized are an epitope defined by the bNAbs PG9 and PG16 that is associated with conserved regions of the V1, V2 and V3 loops, an epitope overlapping the CD4 binding site and possibly the coreceptor binding site, an epitope sensitive to a loss of the glycan at N332 and distinct from that recognized by the bNAb 2G12 and an epitope sensitive to an I165A substitution. In approximately half of the donors, key N-linked glycans were critical for expression of the epitopes recognized by the bNAb specificities in the sera

GM-CSF Production Allows the Identification of Immunoprevalent Antigens Recognized by Human CD4+ T Cells Following Smallpox Vaccination

The threat of bioterrorism with smallpox and the broad use of vaccinia vectors for other vaccines have led to the resurgence in the study of vaccinia immunological memory. The importance of the role of CD4+ T cells in the control of vaccinia infection is well known. However, more CD8+ than CD4+ T cell epitopes recognized by human subjects immunized with vaccinia virus have been reported. This could be, in part, due to the fact that most of the studies that have identified human CD4+ specific protein-derived fragments or peptides have used IFN-γ production to evaluate vaccinia specific T cell responses. Based on these findings, we reasoned that analyzing a large panel of cytokines would permit us to generate a more complete analysis of the CD4 T cell responses. The results presented provide clear evidence that TNF-α is an excellent readout of vaccinia specificity and that other cytokines such as GM-CSF can be used to evaluate the reactivity of CD4+ T cells in response to vaccinia antigens. Furthermore, using these cytokines as readout of vaccinia specificity, we present the identification of novel peptides from immunoprevalent vaccinia proteins recognized by CD4+ T cells derived from smallpox vaccinated human subjects. In conclusion, we describe a “T cell–driven” methodology that can be implemented to determine the specificity of the T cell response upon vaccination or infection. Together, the single pathogen in vitro stimulation, the selection of CD4+ T cells specific to the pathogen by limiting dilution, the evaluation of pathogen specificity by detecting multiple cytokines, and the screening of the clones with synthetic combinatorial libraries, constitutes a novel and valuable approach for the elucidation of human CD4+ T cell specificity in response to large pathogens

H5N1 Influenza Vaccine Formulated with AS03A Induces Strong Cross-Reactive and Polyfunctional CD4 T-Cell Responses

Objective Adjuvantation of an H5N1 split-virion influenza vaccine with AS03(A) substantially reduces the antigen dose required to produce a putatively protective humoral response and promotes cross-clade neutralizing responses. We determined the effect of adjuvantation on antibody persistence and B- and T-cell-mediated immune responses. Methods Two vaccinations with a split-virion A/Vietnam/1194/2004 (H5N1, clade 1) vaccine containing 3.75-30 mu g hemagglutinin and formulated with or without adjuvant were administered to groups of 50 volunteers aged 18-60 years. Results Adjuvantation of the vaccine led to better persistence of neutralizing and hemagglutination-inhibiting antibodies and higher frequencies of antigen-specific memory B cells. Cross-reactive and polyfunctional H5N1-specific CD4 T cells were detected at baseline and were amplified by vaccination. Expansion of CD4 T cells was enhanced by adjuvantation. Conclusion Formulation of the H5N1 vaccine with AS03(A) enhances antibody persistence and induces stronger T- and B-cell responses. The cross-clade T-cell immunity indicates that the adjuvanted vaccine primes individuals to respond to either infection and/or subsequent vaccination with strains drifted from the primary vaccine strain

Influence of Prior Influenza Vaccination on Antibody and B-Cell Responses

Currently two vaccines, trivalent inactivated influenza vaccine (TIV) and live attenuated influenza vaccine (LAIV), are licensed in the USA. Despite previous studies on immune responses induced by these two vaccines, a comparative study of the influence of prior influenza vaccination on serum antibody and B-cell responses to new LAIV or TIV vaccination has not been reported. During the 2005/6 influenza season, we quantified the serum antibody and B-cell responses to LAIV or TIV in adults with differing influenza vaccination histories in the prior year: LAIV, TIV, or neither. Blood samples were collected on days 0, 7–9 and 21–35 after immunization and used for serum HAI assay and B-cell assays. Total and influenza-specific circulating IgG and IgA antibody secreting cells (ASC) in PBMC were detected by direct ELISPOT assay. Memory B cells were also tested by ELISPOT after polyclonal stimulation of PBMC in vitro. Serum antibody, effector, and memory B-cell responses were greater in TIV recipients than LAIV recipients. Prior year TIV recipients had significantly higher baseline HAI titers, but lower HAI response after vaccination with either TIV or LAIV, and lower IgA ASC response after vaccination with TIV than prior year LAIV or no vaccination recipients. Lower levels of baseline HAI titer were associated with a greater fold-increase of HAI titer and ASC number after vaccination, which also differed by type of vaccine. Our findings suggest that the type of vaccine received in the prior year affects the serum antibody and the B-cell responses to subsequent vaccination. In particular, prior year TIV vaccination is associated with sustained higher HAI titer one year later but lower antibody response to new LAIV or TIV vaccination, and a lower effector B-cell response to new TIV but not LAIV vaccination

A Novel High-Throughput Vaccinia Virus Neutralization Assay and Preexisting Immunity in Populations from Different Geographic Regions in China

Background: Pre-existing immunity to Vaccinia Tian Tan virus (VTT) resulting from a large vaccination campaign against smallpox prior to the early 1980s in China, has been a major issue for application of VTT-vector based vaccines. It is essential to establish a sensitive and high-throughput neutralization assay to understand the epidemiology of Vaccinia-specific immunity in current populations in China. Methodology/Principal Findings: A new anti-Vaccinia virus (VACV) neutralization assay that used the attenuated replication-competent VTT carrying the firefly luciferase gene of Photinus pyralis (rTV-Fluc) was established and standardized for critical parameters that included the choice of cell line, viral infection dose, and the infection time. The current study evaluated the maintenance of virus-specific immunity after smallpox vaccination by conducting a non-randomized, crosssectional analysis of antiviral antibody-mediated immune responses in volunteers examined 30–55 years after vaccination. The rTV-Fluc neutralization assay was able to detect neutralizing antibodies (NAbs) against Vaccinia virus without the ability to differentiate strains of Vaccinia virus. We showed that the neutralizing titers measured by our assay were similar to those obtained by the traditional plaque reduction neutralization test (PRNT). Using this assay, we found a low prevalence of NAb to VTT (7.6%) in individuals born before 1980 from Beijing and Anhui provinces in China, and when present, anti-VTT NAb titers were low. No NAbs were detected in all 222 samples from individuals born after 1980. There was no significan

Impact of Aetiological Treatment on Conventional and Multiplex Serology in Chronic Chagas Disease

The main criterion for treatment effectiveness in Chagas Disease has been the seronegative conversion of previously reactive serology, generally achieved many years post-treatment. The lack of reliable tests to ensure parasite clearance and to examine the effect of treatment is the main difficulty in evaluating treatment for chronic Chagas disease. Decreases of conventional and non-conventional serological titers can be useful tools to monitor the early impact of treatment. We serially measured changes in antibody levels, including seronegative conversion as well as declines in titers in 53 benznidazole-treated and 89 untreated chronically T. cruzi-infected subjects. Seronegative conversion as well as decreases of titers was significantly higher in treated compared with untreated patients. A strong concordance was found between decreases of titers of conventional and non-conventional serologic tests post-treatment, reaffirming the findings. When seronegative conversion plus decreases of titers were considered altogether, the impact of treatment was higher, in a shorter follow-up period than previously considered. New tools for monitoring the effectiveness of treatment of chronic Chagas disease are necessary, and the results showed in this study is a contribution to researchers and physicians who assist patients suffering from this disease

Antigen-Specific B Memory Cell Responses to Plasmodium falciparum Malaria Antigens and Schistosoma haematobium Antigens in Co-Infected Malian Children

Polyparasitism is common in the developing world. We have previously demonstrated that schistosomiasis-positive (SP) Malian children have age-dependent protection from malaria compared to matched schistosomiasis-negative (SN) children. Evidence of durable immunologic memory to malaria antigens is conflicting, particularly in young children and the effect of concomitant schistomiasis upon acquisition of memory is unknown. We examined antigen-specific B memory cell (MBC) frequencies (expressed as percentage of total number of IgG-secreting cells) in 84 Malian children aged 4–14 to malaria blood-stage antigens, apical membrane antigen 1 (AMA-1) and merozoite surface protein 1 (MSP-1) and to schistosomal antigens, Soluble Worm Antigenic Preparation (SWAP) and Schistosoma Egg Antigen (SEA), at a time point during the malaria transmission season and a follow-up dry season visit. We demonstrate, for the first time, MBC responses to S. haematobium antigens in Malian children with urinary egg excretion and provide evidence of seasonal acquisition of immunologic memory, age-associated differences in MBC acquisition, and correlation with circulating S. haematobium antibody. Moreover, the presence of a parasitic co-infection resulted in older children, aged 9–14 years, with underlying S. haematobium infection having significantly more MBC response to malaria antigens (AMA1 and MSP1) than their age-matched SN counterparts. We conclude that detectable MBC response can be measured against both malaria and schistosomal antigens and that the presence of S. haematobium may be associated with enhanced MBC induction in an age-specific manner

Combined loss of the BH3-only proteins Bim and Bmf restores B-cell development and function in TACI-Ig transgenic mice.

Terminal differentiation of B cells depends on two interconnected survival pathways, elicited by the B-cell receptor (BCR) and the BAFF receptor (BAFF-R), respectively. Loss of either signaling pathway arrests B-cell development. Although BCR-dependent survival depends mainly on the activation of the v-AKT murine thymoma viral oncogene homolog 1 (AKT)/PI3-kinase network, BAFF/BAFF-R-mediated survival engages non-canonical NF-κB signaling as well as MAPK/extracellular-signal regulated kinase and AKT/PI3-kinase modules to allow proper B-cell development. Plasma cell survival, however, is independent of BAFF-R and regulated by APRIL that signals NF-κB activation via alternative receptors, that is, transmembrane activator and CAML interactor (TACI) or B-cell maturation (BCMA). All these complex signaling events are believed to secure survival by increased expression of anti-apoptotic B-cell lymphoma 2 (Bcl2) family proteins in developing and mature B cells. Curiously, how lack of BAFF- or APRIL-mediated signaling triggers B-cell apoptosis remains largely unexplored. Here, we show that two pro-apoptotic members of the 'Bcl2 homology domain 3-only' subgroup of the Bcl2 family, Bcl2 interacting mediator of cell death (Bim) and Bcl2 modifying factor (Bmf), mediate apoptosis in the context of TACI-Ig overexpression that effectively neutralizes BAFF as well as APRIL. Surprisingly, although Bcl2 overexpression triggers B-cell hyperplasia exceeding the one observed in Bim(-/-)Bmf(-/-) mice, Bcl2 transgenic B cells remain susceptible to the effects of TACI-Ig expression in vivo, leading to ameliorated pathology in Vav-Bcl2 transgenic mice. Together, our findings shed new light on the molecular machinery restricting B-cell survival during development, normal homeostasis and under pathological conditions. Our data further suggest that Bcl2 antagonists might improve the potency of BAFF/APRIL-depletion strategies in B-cell-driven pathologies

Genomic Expression Libraries for the Identification of Cross-Reactive Orthopoxvirus Antigens

Increasing numbers of human cowpox virus infections that are being observed and that particularly affect young non-vaccinated persons have renewed interest in this zoonotic disease. Usually causing a self-limiting local infection, human cowpox can in fact be fatal for immunocompromised individuals. Conventional smallpox vaccination presumably protects an individual from infections with other Orthopoxviruses, including cowpox virus. However, available live vaccines are causing severe adverse reactions especially in individuals with impaired immunity. Because of a decrease in protective immunity against Orthopoxviruses and a coincident increase in the proportion of immunodeficient individuals in today's population, safer vaccines need to be developed. Recombinant subunit vaccines containing cross-reactive antigens are promising candidates, which avoid the application of infectious virus. However, subunit vaccines should contain carefully selected antigens to confer a solid cross-protection against different Orthopoxvirus species. Little is known about the cross-reactivity of antibodies elicited to cowpox virus proteins. Here, we first identified 21 immunogenic proteins of cowpox and vaccinia virus by serological screenings of genomic Orthopoxvirus expression libraries. Screenings were performed using sera from vaccinated humans and animals as well as clinical sera from patients and animals with a naturally acquired cowpox virus infection. We further analyzed the cross-reactivity of the identified immunogenic proteins. Out of 21 identified proteins 16 were found to be cross-reactive between cowpox and vaccinia virus. The presented findings provide important indications for the design of new-generation recombinant subunit vaccines