The quest for down scale representativeness: how to exploit CFD to design a shear study for vaccines.

In this work, we exploit computational fluid dynamics (CFD) to evaluate stirred tank reactor (STR) process engineer parameters (PEP) and design a scale-down system (SDS) to be representative of the formulation and filling process steps for an Aluminum adjuvanted vaccine drug product (DP). To study the shear history in the SDS we used the concept of number of passages, combined with an appropriate stirring speed down scale strategy comprising of either (i) tip speed equivalence, widely used as a scale-up criterion for a shear-sensitive product, or (ii) rotating shear, a shear metric introduced by Metz and Otto in 1957 but never used as scaling criterion. The outcome of the CFD simulations shows that the tip equivalence generates a worst-case SDS in terms of shear, whereas the rotating shear scaling approach could be used to design a more representative SDS. We monitored the trend over time for "In Vitro Relative Potency" as DP Critical Quality Attribute for both scaling approaches, which highlighted the crucial role of choosing the appropriate scaling-down approach to be representative of the manufacturing scale during process characterization studies.

Analytical Insights into Protein-Alum Interactions and Their Impact on Conformational Epitope.

Several alum-adjuvanted vaccines have been licensed in the past 40 years. Despite its extensive and continuous use, the immune mechanism of action of alum adjuvants is not yet completely understood. Many different variables during the formulation process have been assessed as critical for alum-adjuvanted vaccines, although most of them are still not yet fully understood. The absence of a clear understanding of all the possible variables regulating the mechanism of action and the behavior that alum adjuvant imposes on the protein antigen may also be related to analytical challenges. For this reason, there is an urgent need for a fast and simple tool that is possible without a preliminary sample manipulation and is able to control the amount and the degree of antigen adsorption levels and their consistency across different production processes. This work attempts to develop new analytical tools with the aim of directly quantifying and assessing both the content and/or the purity of formulated alum-adsorbed antigens, without any preliminary sample manipulation (e.g., antigen desorption) being reported. In addition, the different confirmation/behavior in terms of the response to specific monoclonal antibodies in the presence of different ratios of alum-OH adsorbent antigens have been investigated. As a proxy to develop new analytical tools, three recombinant protein adsorbed models were used as follows: Neisseria adhesin A (NadA), Neisserial Heparin Binding Antigen (NHBA), and factor H binding protein (fHbp) as antigens, as well as aluminum hydroxide (AH) as an adjuvant system. The selection of the adjuvanted system model was dictated due to the substantial quantity of the literature regarding the protein structure and immunological activities, meaning that they are well characterized, including their adhesion rate to alum. In conclusion, three different analytical tools were explored to quantify, detect, and study the behavior of antigens in the presence of the alum adjuvant.

Vaccine adjuvant MF59 promotes retention of unprocessed antigen in lymph node macrophage compartments and follicular dendritic cells.

Ag retention within lymph nodes (LNs) upon vaccination is critical for the development of adaptive immune responses, because it facilitates the encounter of the Ag with cognate lymphocytes. During a secondary exposure of the immune system to an Ag, immune complexes (ICs) that contain the unprocessed Ag are captured by subcapsular sinus macrophages and are transferred onto follicular dendritic cells, where they persist for weeks, facilitating Ag presentation to cognate memory B cells. The impact of adjuvants on Ag retention within the draining LNs is unknown. In this article, we provide the first evidence, to our knowledge, that the oil-in-water emulsion adjuvant MF59 localizes in subcapsular sinus and medullary macrophage compartments of mouse draining LNs, where it persists for at least 2 wk. In addition, we demonstrate that MF59 promotes accumulation of the unprocessed Ag within these LN compartments and facilitates the consequent deposition of the IC-trapped Ag onto activated follicular dendritic cells. These findings correlate with the ability of MF59 to boost germinal center generation and Ag-specific Ab titers. Our data suggest that the adjuvant effect of MF59 is, at least in part, due to an enhancement of IC-bound Ag retention within the LN and offer insights to improve the efficacy of new vaccine adjuvants.

Impact of preexisting memory to seasonal A/H1N1 influenza virus on the immune response following vaccination against avian A/H5N1 virus.

Cross-protection against divergent strains of influenza virus is an objective of various vaccination approaches. B cells cross-neutralizing several influenza A heterosubtypes have been isolated from cultured human memory B cells (MBCs) and plasmablasts early after influenza vaccination or infection. However, a systematic assessment of the frequency of MBCs and plasmablasts in the blood of healthy individuals is lacking. Here, we show that under resting conditions about 45% of human adults never vaccinated nor exposed to avian A/H5N1 influenza have detectable circulating MBCs cross-reacting with H5N1. This proportion rises to 63.3% among subjects with a large pool of MBCs specific for seasonal H1N1 (i.e. frequency ≥1% of total IgG MBCs). Moreover, subjects with high baseline frequencies of H1N1-specific MBCs had an expansion of H5N1-specific MBCs producing H5-neutralizing antibodies already after the first dose of an MF59-adjuvanted H5N1 vaccine. These results suggest that H1N1-specific MBCs contain a subset of cells cross-reacting to H5. We propose that a proportion of human adults have a pool of H5/H1 cross-reactive MBCs that contribute to the rapid rise of the antibody response to divergent influenza strains. This may have implications on vaccination strategies aimed at counteracting future influenza pandemics.

Fast rise of broadly cross-reactive antibodies after boosting long-lived human memory B cells primed by an MF59 adjuvanted prepandemic vaccine.

Proactive priming before the next pandemic could induce immune memory responses to novel influenza antigens. In an open-label study, we analyzed B cell memory and antibody responses of 54 adults who received 2 7.5-microg doses of MF59-adjuvanted A/Vietnam/1194/2004 clade 1 (H5N1) vaccine. Twenty-four subjects had been previously primed with MF59-adjuvanted or plain clade 0-like A/duck/Singapore/1997 (H5N3) vaccine during 1999-2001. The prevaccination frequency of circulating memory B cells reactive to A/Vietnam/1194/2004 was low in both primed and unprimed individuals. However, at day 21 after boosting, MF59-adjuvanted primed subjects displayed a higher frequency of H5N1-specific memory B cells than plain-primed or unprimed subjects. The immune memory was rapidly mobilized by a single vaccine administration and resulted in high titers of neutralizing antibodies to antigenically diverse clade 0, 1, and 2 H5N1 viruses already at day 7. In general, postvaccination antibody titers were significantly higher in primed subjects than in unprimed subjects. Subjects primed with MF59-adjuvanted vaccine responded significantly better than those primed with plain vaccine, most notably in early induction and duration of cross-reacting antibody responses. After 6 months, high titers of cross-reactive antibody remained detectable among MF59-primed subjects. We conclude that distant priming with clade 0-like H5N3 induces a pool of cross-reactive memory B cells that can be boosted rapidly years afterward by a mismatched MF59-adjuvanted vaccine to generate high titers of cross-reactive neutralizing antibodies rapidly. These results suggest that pre-pandemic vaccination strategies should be considered.

Adjuvanted H5N1 vaccine induces early CD4+ T cell response that predicts long-term persistence of protective antibody levels.

Immune responses to vaccination are tested in clinical trials. This process usually requires years especially when immune memory and persistence are analyzed. Markers able to quickly predict the immune response would be very useful, particularly when dealing with emerging diseases that require a rapid response, such as avian influenza. To address this question we vaccinated healthy adults at days 1, 22, and 202 with plain or MF59-adjuvanted H5N1 subunit vaccines and tested both cell-mediated and antibody responses up to day 382. Only the MF59-H5N1 vaccine induced high titers of neutralizing antibodies, a large pool of memory H5N1-specific B lymphocytes, and H5-CD4(+) T cells broadly reactive with drifted H5. The CD4(+) response was dominated by IL-2(+) IFN-gamma(-) IL-13(-) T cells. Remarkably, a 3-fold increase in the frequency of virus-specific total CD4(+) T cells, measurable after 1 dose, accurately predicted the rise of neutralizing antibodies after booster immunization and their maintenance 6 months later. We suggest that CD4(+) T cell priming might be used as an early predictor of the immunogenicity of prepandemic vaccines.

NKT-cell help to B lymphocytes can occur independently of cognate interaction.

CD4(+) T (Th)-cell help to B lymphocytes requires cognate interaction and CD40 engagement. Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that recognize alphagalactosylceramide (alphaGalCer) presented by CD1d, and can help B-cell responses. We asked whether alphaGalCer-activated iNKT cells help B lymphocytes through cognate interaction, or indirectly, via enhancement of Th-B-cell interaction. After immunization with protein Ags and alphaGalCer, antibody titers were assessed in wild-type or splenectomized mice, and in bone marrow radiation chimeras lacking CD1d or CD40 expression on B lymphocytes, or expressing CD1d or MHC II disjointly on antigen-presenting cells (APCs). We find that alphaGalCer-dependent enhancement of B-cell response (1) can occur when B cells do not express CD1d but express CD40; (2) requires that iNKT and Th cells interact with the same APCs that coexpress both CD1d and MHC-II; and (3) takes place without spleen. These findings demonstrate alphaGalCer-induced help for antibody responses can occur without cognate iNKT/B-cell interaction, and suggest this help entails activation of APCs by iNKT cells, which in turn activate Th cells and their helper functions for B cells. Thus, the alphaGalCer-induced help recapitulates the function of classical adjuvants that stimulate the innate immune system to support adaptive immune responses.

Invariant NKT cells sustain specific B cell responses and memory.

Invariant natural killer T (iNKT) cells are innate-like lymphocytes recognizing CD1d-restricted glycolipid antigens, such as alpha-galactosylceramide (alphaGC). We assessed whether iNKT cells help B lymphocyte responses and found that mice immunized with proteins and alphaGC develop antibody titers 1-2 logs higher than those induced by proteins alone. Activation of iNKT cells enhances protection against infections such as influenza and elicits higher frequencies of memory B cells and higher antibody responses to booster immunizations. Protein vaccination with alphaGC, but not with conventional adjuvants, elicits IgG responses in mice lacking MHC class II molecules, demonstrating that iNKT cells can substitute for CD4(+) T cell help to B cells. Interestingly, the decay of circulating antibodies is faster in mice lacking iNKT cells. These findings point to a homeostatic role for iNKT cells on critical features of the antibody response such as immunity and B cell memory.