Frequency and phenotype of B cell subpopulations in young and aged HIV-1 infected patients receiving ART

BACKGROUND: Aged individuals respond poorly to vaccination and have a higher risk of contracting infections in comparison to younger individuals; whether age impacts on the composition and function of B cell subpopulations relevant for immune responses is still controversial. It is also not known whether increased age during HIV-1 infection further synergizes with the virus to alter B cell subpopulations. In view of the increased number of HIV-1 infected patients living to high age as a result of anti-retroviral treatment this is an important issue to clarify. RESULTS: In this work we evaluated the distribution of B cell subpopulations in young and aged healthy individuals and HIV-1 infected patients, treated and naïve to treatment. B cell populations were characterized for the expression of inhibitory molecules (PD-1 and FcRL4) and activation markers (CD25 and CD69); the capacity of B cells to respond to activation signals through up-regulation of IL-6 expression was also evaluated. Increased frequencies of activated and tissue-like memory B cells occurring during HIV-1 infection are corrected by prolonged ART therapy. Our findings also reveal that, in spite of prolonged treatment, resting memory B cells in both young and aged HIV-1 infected patients are reduced in number, and all memory B cell subsets show a low level of expression of the activation marker CD25. CONCLUSIONS: The results of our study show that resting memory B cells in ART-treated young and aged HIV-1 infected patients are reduced in number and memory B cell subsets exhibit low expression of the activation marker CD25. Aging per se in the HIV-1 infected population does not worsen impairments initiated by HIV-1 in the memory B cell populations of young individuals. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12977-014-0076-x) contains supplementary material, which is available to authorized users

The impact of active HIV-1 replication on the physiological age-related decline of immature-transitional B-cells in HIV-1 infected children

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CD27− B-Cells Produce Class Switched and Somatically Hyper-Mutated Antibodies during Chronic HIV-1 Infection

Class switch recombination and somatic hypermutation occur in mature B-cells in response to antigen stimulation. These processes are crucial for the generation of functional antibodies. During HIV-1 infection, loss of memory B-cells, together with an altered differentiation of naïve B-cells result in production of low quality antibodies, which may be due to impaired immunoglobulin affinity maturation. In the current study, we evaluated the effect of HIV-1 infection on class switch recombination and somatic hypermutation by studying the expression of activation-induced cytidine deaminase (AID) in peripheral B-cells from a cohort of chronically HIV-1 infected patients as compared to a group of healthy controls. In parallel, we also characterized the phenotype of B-cells and their ability to produce immunoglobulins in vitro. Cells from HIV-1 infected patients showed higher baseline levels of AID expression and increased IgA production measured ex-vivo and upon CD40 and TLR9 stimulation in vitro. Moreover, the percentage of CD27−IgA+ and CD27−IgG+ B-cells in blood was significantly increased in HIV-1 infected patients as compared to controls. Interestingly, our results showed a significantly increased number of somatic hypermutations in the VH genes in CD27− cells from patients. Taken together, these results show that during HIV-1 infection, CD27− B-cells can also produce class switched and somatically hypermutated antibodies. Our data add important information for the understanding of the mechanisms underlying the loss of specific antibody production observed during HIV-1 infection

Design of Nanoparticulate Group 2 Influenza Virus Hemagglutinin Stem Antigens That Activate Unmutated Ancestor B Cell Receptors of Broadly Neutralizing Antibody Lineages.

Influenza vaccines targeting the highly conserved stem of the hemagglutinin (HA) surface glycoprotein have the potential to protect against pandemic and drifted seasonal influenza viruses not covered by current vaccines. While HA stem-based immunogens derived from group 1 influenza A viruses have been shown to induce intragroup heterosubtypic protection, HA stem-specific antibody lineages originating from group 2 may be more likely to possess broad cross-group reactivity. We report the structure-guided development of mammalian-cell-expressed candidate vaccine immunogens based on influenza A virus group 2 H3 and H7 HA stem trimers displayed on self-assembling ferritin nanoparticles using an iterative, multipronged approach involving helix stabilization, loop optimization, disulfide bond addition, and side-chain repacking. These immunogens were thermostable, formed uniform and symmetric nanoparticles, were recognized by cross-group-reactive broadly neutralizing antibodies (bNAbs) with nanomolar affinity, and elicited protective, homosubtypic antibodies in mice. Importantly, several immunogens were able to activate B cells expressing inferred unmutated common ancestor (UCA) versions of cross-group-reactive human bNAbs from two multidonor classes, suggesting they could initiate elicitation of these bNAbs in humans. Current influenza vaccines are primarily strain specific, requiring annual updates, and offer minimal protection against drifted seasonal or pandemic strains. The highly conserved stem region of hemagglutinin (HA) of group 2 influenza A virus subtypes is a promising target for vaccine elicitation of broad cross-group protection against divergent strains. We used structure-guided protein engineering employing multiple protein stabilization methods simultaneously to develop group 2 HA stem-based candidate influenza A virus immunogens displayed as trimers on self-assembling nanoparticles. Characterization of antigenicity, thermostability, and particle formation confirmed structural integrity. Group 2 HA stem antigen designs were identified that, when displayed on ferritin nanoparticles, activated B cells expressing inferred unmutated common ancestor (UCA) versions of human antibody lineages associated with cross-group-reactive, broadly neutralizing antibodies (bNAbs). Immunization of mice led to protection against a lethal homosubtypic influenza virus challenge. These candidate vaccines are now being manufactured for clinical evaluation

Application of B cell immortalization for the isolation of antibodies and B cell clones from vaccine and infection settings

The isolation and characterization of neutralizing antibodies from infection and vaccine settings informs future vaccine design, and methodologies that streamline the isolation of antibodies and the generation of B cell clones are of great interest. Retroviral transduction to express Bcl-6 and Bcl-xL and transform primary B cells has been shown to promote long-term B cell survival and antibody secretion in vitro, and can be used to isolate antibodies from memory B cells. However, application of this methodology to B cell subsets from different tissues and B cells from chronically infected individuals has not been well characterized. Here, we characterize Bcl-6/Bcl-xL B cell immortalization across multiple tissue types and B cell subsets in healthy and HIV-1 infected individuals, as well as individuals recovering from malaria. In healthy individuals, naïve and memory B cell subsets from PBMCs and tonsil tissue transformed with similar efficiencies, and displayed similar characteristics with respect to their longevity and immunoglobulin secretion. In HIV-1-viremic individuals or in individuals with recent malaria infections, the exhausted CD27-CD21- memory B cells transformed with lower efficiency, but the transformed B cells expanded and secreted IgG with similar efficiency. Importantly, we show that this methodology can be used to isolate broadly neutralizing antibodies from HIV-infected individuals. Overall, we demonstrate that Bcl-6/Bcl-xL B cell immortalization can be used to isolate antibodies and generate B cell clones from different B cell populations, albeit with varying efficiencies

HIV-1 infection and loss of serological memory : The role of altered expression of B-cell chemokine receptors, timing of HAART and impaired antibody affinity maturation

Currently, big efforts in HIV-1 research are devoted to the design of a HIV-1 vaccine. However, no successful vaccine is yet available, also due to the lack of basic knowledge in HIV-1 pathogenesis which is still profound. HIV-1 infected patients suffer of several abnormalities related to B-lymphocytes, which function is to produce antibodies (immunoglobulins) against common pathogenic viruses and bacteria, and experience loss of serological memory since early stages of infection. In the absence of a HIV-1 vaccine, ensuring a better protection against common diseases in infected adults and children is fundamental to prevent lethal infections of these patients. The research presented in this thesis aimed at improving the general knowledge on HIV-1 pathogenesis and at better understanding the mechanisms behind the B-cell impairments observed during HIV-1 infection. We found that (paper I), the loss of memory B-cells is a progressive event starting early during primary infection, when a partial but irreversible depletion of specific memory B-cells occurs. A possible explanation for this phenomenon is memory B-cell exhaustion and cell death as a consequence of the general (polyclonal) B-cell activation due to the reactivation of many pathogens typical of HIV-1 infection. In this scenario, the death of specific memory B-cells could be compensated through the activation of naïve B-cells by unspecific stimuli which may lead to the production of low-quality (affinity) antibodies. Through the work presented in paper II, we found that naïve B-cells in fact undergo processes of immunoglobulin affinity maturation in the immunoglobulin genes. Another important aspect which might be impaired during HIV-1 infection is lymphocyte trafficking between the periphery and (within) secondary lymphoid organs, for example lymph-nodes and spleen, where immunoglobulin affinity maturation takes place. In paper III, we observed that altered expression of key molecules for B-cell migration, such as the chemokine receptor CXCR5 and the chemokine CXCL13, occurs in B-cells and alters B-cell migration. This may result in defective reactions in the secondary lymphoid organs. In this respect, the application of the currently available antiretroviral therapy (HAART) early during primary infection might help minimizing these detrimental effects. In paper IV, we found that in the milieu of a developing immune system, such as the one of children infected with HIV-1 in utero, at birth or through breast feeding (vertically infected), an application of therapy within the first year of life in fact preserves both the development and the function of B-cells. This results in effective and long-lasting immunization upon common childhood vaccinations with measles, tetanus and pneumococcus. Summarizing, the work presented in this thesis enlightens the role of altered expression of B-cell chemokine receptors and impaired immunoglobulin affinity maturation for the loss of serological memory observed during HIV-1 infection and suggests the early initiation of HAART in vertically infected children. The understanding of these phenomena, in the absence of a HIV-1 vaccine, might be the key event for ensuring a better life of HIV-1 infected patients and might help solving the problems encountered so far in the development of a HIV-1 vaccine

Immune Responses Induced by mRNA Vaccination in Mice, Monkeys and Humans

In this concise review, we summarize the concepts behind mRNA vaccination. We discuss the innate and adaptive immune response generated by mRNA vaccines in different animal models and in humans. We give examples of viral infections where mRNA vaccines have shown to induce potent responses and we discuss in more detail the recent SARS-CoV-2 mRNA vaccine trials in humans

Immune Responses Induced by mRNA Vaccination in Mice, Monkeys and Humans

In this concise review, we summarize the concepts behind mRNA vaccination. We discuss the innate and adaptive immune response generated by mRNA vaccines in different animal models and in humans. We give examples of viral infections where mRNA vaccines have shown to induce potent responses and we discuss in more detail the recent SARS-CoV-2 mRNA vaccine trials in humans